Design of precision therapeutics for a CKD risk allele by targeting Shroom3-Rock interaction

Domain: biology
Published: 2025-12-30
Editorial outcome: major_revision

Abstract
Enhancer variants in Shroom3 associate with renal fibrosis (TIF), but with reduced albuminuria. Detailed mechanisms for these pleiotropic effects are unclear. Here, we focus on identifying the specific profibrotic Shroom3 motif and separating this from its anti-proteinuric function. Given the role for Rho-kinases (Rock) in TIF, and the interaction of Rock with Shroom3 ASD2-domain, we hypothesized that Shroom3-mediated Rock-activation is crucial for profibrotic function. To test this, we develop transgenic tools that overexpress wild-type-(WT-Sh3) or ASD2-domain deletion-Shroom3 (ASD2Δ-Sh3). During TIF, Shroom3 and Rock co-expression occur in injured tubular cells and fibroblasts. In tubular- & fibroblast-lines, ASD2Δ-Sh3 overexpression reduce Rock activation, and pro-fibrotic/pro-inflammatory transcripts downstream of TGFβ1/Wnt/Ctnnb1-signaling vs WT-Sh3. In vivo, inducible global-, or tubular-specific-, but not fibroblast-specific-, ASD2Δ-Sh3 overexpression mitigate TIF, vs WT-Sh3 overexpression. Importantly, ASD2Δ-Sh3 mice do not develop albuminuria, while overexpression of a distinct Fyn-binding deficient mutant Shroom3 (FBDM-Sh3) induces albuminuria. We then develop small molecule inhibitors of Shroom3-Rock interaction (P2Is) and confirm Rock inhibition with these agents in WT-Sh3 cell lines. Our lead P2I from these studies, BT1137, mitigates Rock-activation, profibrotic signaling and TIF in WT-Sh3 mice. Hence, we delineate the profibrotic Shroom3 motif and develop therapeutics for kidney disease from Shroom3 excess.

Introduction
In native and allograft kidneys, chronic kidney disease (CKD) is characterized by progressive renal interstitial fibrosis and tubular atrophy (TIF) reflecting accruing damage. In humans, while genome-wide association studies (GWAS) have identified candidate CKD susceptibility loci1,2, the detailed mechanistic basis of single-nucleotide polymorphisms (SNPs)-variant associations with renal histology/function in CKD remains limited3. This has hindered the development of therapeutics based on GWAS. Specifically, targeting fibrosis mechanisms in the native or allograft CKD context has not successfully translated to the bedside in clinical trials, despite promising preclinical data4. As a result, a significant percentage of patients with susceptibility loci for CKD or allograft injury will eventually progress to end-stage renal disease (ESRD) requiring dialysis or transplantation5,6.

Within the Shroom3 gene locus, multiple intronic SNPs, including rs17319721, have been associated with CKD in GWAS1,2,7. We and others identified that the A-allele at rs17319721 has TCF7L2-dependent enhancer function on Shroom3 in renal epithelial cells8,9. In renal allograft biopsies, enhanced SHROOM3 expression with the donor A-allele was associated with increased TIF8 and reduced glomerular filtration rate (eGFR) by 1-year post transplant10. We showed that A-allele cells increased SHROOM3 expression in a Tcf7l2-dependent manner after TGFβ1 treatment, and more SHROOM3 promoted profibrotic marker production, creating a cross-talk between TGFβ1 and Wnt/Ctnnb1 pathways. In vivo, inducible global or renal-tubular specific Shroom3 knockdown in mice significantly inhibited TIF8. These data suggested a pathogenic role for SHROOM3 in TIF, and potential for testing the role of SHROOM3-antagonism as a therapeutic strategy for TIF in CKD in A-allele kidneys. However, subsequent data from our/other groups showed a protective role for Shroom3 in glomerular development11–13, and indicated the development of proteinuria when Shroom3 was knocked down in adult podocytes14,15. Together, these data suggest an adverse association for rs17319721 & elevated Shroom3 with renal TIF in CKD, but also caution that globally antagonizing Shroom3 would incur the risk of proteinuria. These dichotomous responses to Shroom3 loss led us to carefully analyze the protein structure of Shroom3 and investigate specific domains that could independently regulate cytoskeletal organization or profibrotic signaling.

Shroom3 belongs to the SHROOM family of proteins16 containing a PDZ domain and two Apx/Shrm-specific domains called ASD1 and ASD217. The ASD1 domain directly binds F-Actin11, while the ASD2 domain binds and activates Rho-associated coiled-coil kinases (ROCKs)18. We also reported a previously unidentified -PxxP- domain located between PDZ and ASD1-domains, which was critical for Fyn-binding and activation in podocytes, and that adult mice with Shroom3 knockdown phenocopied Fyn-KO mice, with inhibited Nphs1 phosphorylation and Actin cytoskeletal organization demonstrating that this motif was crucial in adult podocyte homeostasis14. Subsequently, we reported that Shroom3 knockdown in glomerular podocytes induced a minimal change-like injury (MCD), due to Fyn inactivation with collateral activation of AMPK15, and inhibition of AMPK in this context induced podocyte loss and focal sclerosis. Notably, impaired Fyn activation in podocytes has also been specifically associated with human MCD19. These suggested that the Fyn-binding motif of Shroom3 mediated its anti-proteinuric effects in adult animals, distinct from the developmental role in glomerulogenesis reported before11,12.

In this work, we performed a series of experiments that focused on identifying and specifically targeting the profibrotic motif of Shroom3. We first developed deletional variants of each of the consensus domains of Shroom3 and screened them in vitro in multiple cell lines (based on single cell transcriptome data), to identify a key role for the Rock-binding, ASD2-domain in profibrotic signaling, facilitating Tgfβ1 and Wnt/Ctnnb1 pathways. These data were consistent with prior antifibrotic roles for the Rock-inhibitor, Hydroxy-Fasudil20–22. To then study the in vivo role played by Shroom3 motifs in TIF, we developed inducible, cell-type-specific overexpression mice to induce either intact Shroom3 excess (analogous to the risk-allele carriage with high expressor kidneys) or overexpression of a variant of Shroom3 without the ASD2-motif. We compared these in multiple TIF models and demonstrated that the ASD2-motif is the profibrotic motif during TIF. Based on in vivo findings with genetic deletion studies of the ASD2-domain, we designed, synthesized and screened small molecules that bind to Rho-kinases and prevent interaction with SHROOM3 (P2Is), and demonstrated efficacy of a lead P2I against TIF in vivo. Hence, we unravel underlying pathophysiologic mechanisms associating Shroom3 SNPs with CKD and allograft injury and show proof-of-principle for designing Shroom3-targeted precision therapeutics.

Results
ASD2Δ-Sh3-overexpression reduces ROCK activation and signaling

We previously reported that tubular-specific Shroom3 knockdown alone could mitigate TIF in a UUO model8. Recent functional genomics studies evaluating CKD-associated SNPs using single-cell ATAC-seq also concluded that single-cell, cis-eQTL effects of Shroom3 intronic variants are identifiable in proximal tubular cells (PTs) in human CKD23. These pointed to injured PTs (iPTs) as key players for TIF from excess Shroom3. We also considered that fibroblasts, which are key cells for matrix production during TIF, express Shroom3 and show high Rock1, Rock2 expression in published data from murine TIF models24–27. Podocytes show high expression of Shroom3 at rest and injury, but are not expected to play key roles in these TIF models after PT cell injury24–26. Hence, we focused on tubular and fibroblast cell lines, which were potential sites of Shroom3 interaction with Rock1 and/or Rock2 during TIF for subsequent mechanistic experiments.

To screen for the profibrotic domain in Shroom328, we first generated deletional mutants of Shroom3, each without one of the three known consensus domains (PDZ, ASD1, or ASD2) [1a]. The ASD2-domain is known to bind and activate ROCK1 and ROCK229. We confirmed that ASD2-domain deletion in SHROOM3 (ASD2Δ-Sh3) abolished ROCK1- [Fig. 1b] and ROCK2 binding [Fig. S1a] in HEK-293T tubular cell lines vs other deletional mutants. We then screened each domain-deletion variant against WT-Sh3 (whole Shroom3) using TGFβ1/Smad reporter HEK-293T blueTM cells. In this assay, which detects secreted alkaline phosphatase in response to Smad3/4 signaling, ASD2Δ-Sh3 inhibited TGFβ1 signaling reporter at baseline and in the presence of TGFβ1 [S1b]. In HEK-293T cells, we co-transfected SMAD3/4-reporter luciferase constructs with either WT- or ASD2Δ-Sh3 plasmids and analyzed TGFβ1-signaling with/without TGFβ1. Again, the ASD2Δ-Sh3 construct significantly reduced TGFβ1-signaling vs WT-Sh3 [Fig. S1c]. We also evaluated other tubular lines- (mouse Inner medullary collecting duct [mIMCD3]) and fibroblasts (NIH/3T3) stably overexpressing either WT-Sh3- or ASD2Δ-Sh3 and studied Rock-activation and pro-fibrotic signaling. ASD2Δ-Sh3 overexpression reduced phospho-Mypt (pMypt) & total Rock1 as compared to WT-Sh3 and/or control vector [1c-d], altered the actin cytoskeleton [S1d-e] and reduced cell migration of IMCD cells [1e-f] vs WT-Sh3. In the presence of Rock-inhibitor HF (10μM), the difference in migration between ASD2Δ-Sh3 cells vs. WT-Sh3 significantly narrowed [S1f] (although it was not abolished) [Figs. 1f vs S1g]. Transcripts downstream of Wnt/Ctnnb1- signaling, Tgfβ1-related profibrotic markers and pro-inflammatory chemokines were all significantly reduced in IMCD cells overexpressing ASD2Δ-Sh3, while WT-Sh3 overexpression resulted in increases of these transcripts vs. control-vector cells [1g]. pMypt levels and gene-expression changes demonstrated that ASD2Δ-Sh3-cells slightly inhibited Rock-activation even when compared to the Control vector, suggesting a dominant negative effect of the ASD2Δ-Sh3 variant on endogenous Shroom3. WT-Sh3-induced pMypt excess and transcript changes were reduced by HF, suggesting these effects were mediated by Rock-activation [Fig. S1h, i]. Next, in 3T3 fibroblasts overexpressing ASD2Δ-Sh3 or WT-Sh3, ASD2Δ-Sh3 also inhibited pMypt [Fig. S1j], profibrotic marker production (albeit to a lesser extent) [1h], and proliferation [1i] vs WT-Sh3-overexpression. Hence, we observed a key role of the ASD2 domain of Shroom3 on Rock binding/activation, impacting the cytoskeleton, cell migration, and profibrotic signaling in tubular-/fibroblast cells.

Generation of inducible, global, ASD2Δ-Sh3 overexpression mice

To evaluate the role of the ASD2-domain of Shroom3 in vivo, we generated Doxycycline (DOX)-inducible (reverse tetracycline transactivator [rtTA] system), transgenic mice with Flag-tagged, ASD2Δ-Sh3, that could be mated with global (CAGS-rtTA- CMV early enhancer/chicken β-actin) or tissue specific-rtTA, and compared these to intact Shroom3-overexpressing mice (WT-Sh3 mice) [shown in Fig. 2a]. Both WT-Sh3 and ASD2Δ-Sh3 constructs were downstream of tetracycline response elements (TRE).

We induced overexpression of Shroom3 variants in these lines by DOX feeding [See “methods”]. Among multiple transgenic lines initially generated, a founder line each for ASD2Δ-Sh3 and WT-Sh3 was selected for inducible- and optimal- (2–3-fold) overexpression of Flag-tagged Shroom3 variants in kidney cortex by 3 weeks DOX [Fig. S2a, b].

By immunofluorescence (IF) staining [Fig. 2b] in CAGS-rtTA mice, overexpression in glomerular, tubular and interstitial kidney cells was observed for each of these lines vs non-DOX fed littermates, with apical localization of Shroom3 in tubular cells (near Lotus tetragonolobus lectin (LTL) positivity), as described before30. By qPCR for exon-specific primers for the human ASD2-domain, only WT-Sh3 mice showed overexpression of this motif as expected [Fig. S2c]. These mice were born at normal rates, and inducible overexpression of ASD2Δ-Sh3 or WT-Sh3 by itself was not associated with weight loss up to 4-wks DOX [2c]. Both transgenic lines had similar blood urea nitrogen (BUN) and Creatinine levels [2d, S2d], and neither line showed increased albuminuria vs non-transgenic littermates [Fig. 2e]. We isolated and cultured primary renal cells (PCRCs) of ASD2Δ-Sh3 and WT-Sh3 after DOX-induction31, and observed significantly reduced migration of ASD2Δ-Sh3 PCRCs vs WT-Sh3 PCRCs [2f–g], as seen in vitro with stably overexpressing IMCD-lines.

In parallel, we developed transgenic Fyn-binding domain mutant-Shroom3 overexpressing mice (FBDM-Sh3; with -AxxA- in place of -PxxP- at positions 442-445 between the PDZ- and ASD1-domains [see Figs. 1b14, 2a], which were also mated with CAGS-rtTA animals. We confirmed overexpression of flag-tagged Shroom3 mutant in kidney cortex upon DOX [Fig. 2h], with overexpression in glomerular cells on IF [Fig. S2e]. While DOX feeding of adult CAGS-rtTA/ASD2Δ-Sh3 or CAGS-rtTA/WT-Sh3 mice did not induce albuminuria [Fig. 2e] or podocyte injury, inducible overexpression of FBDM-Sh3 did induce albuminuria [Fig. S2f] & FPE [2i–j]. Together, these data showed a role for the Shroom3 Fyn-binding domain in mediating the anti-proteinuric function of Shroom3 in podocytes and explain why global overexpression of WT- or ASD2Δ-Sh3 (both of which have the intact FBD) does not induce proteinuria or podocyte injury.

Global ASD2Δ-Sh3 overexpression mice show reduced TIF vs WT-Sh3 in AAN

To study the role of the ASD2-domain of Shroom3 in TIF development, we induced Aristolochic acid nephropathy model (AAN) in ASD2Δ-Sh3 and WT-Sh3. AAN was induced after transgene induction using a multi-dose regimen [see “methods”; Fig. 3a]. Mice were evaluated at 9-weeks, allowing for post-injury remodeling and TIF. At 9-weeks, CAGS-rtTA/ASD2Δ-Sh3 showed reduced azotemia vs CAGS-rtTA/WT-Sh3 mice [Figs. 3b, S3a]. Both groups showed increased albuminuria after AAN induction vs baseline, but without significant differences between groups [Fig. S3b]. CAGS-rtTA/WT-Sh3 mice showed significantly increased TIF by Masson-trichrome stain [Fig. 3c, d], increased Collagen-I [3e–f], Collagen-III [3g–h], and Fibronectin [S3c–d] staining by IF, vs CAGS-rtTA/ASD2Δ-Sh3. In both mouse lines, kidney expression of Shroom3 was confirmed by qPCR (including ASD2-domain specific SHROOM3 and mouse Shroom3 primers) [Fig. S3e] and by IF [S3f]. Interestingly, WT-Sh3 mice showed reduced LTL-positive tubules with (a) more pronounced Shroom3 staining in tubular cell bases (instead of LTL-adjacent pattern seen in uninjured animals), and (b) greater interstitial Shroom3 expression, while ASD2Δ-Sh3 showed better preserved LTL-positivity and apical Shroom3 staining in tubules, consistent with reduced tubular injury in ASD2Δ-Sh3 at 9-weeks [S3f]. Consistent with in vitro data, lysates from injured and uninjured kidneys in ASD2Δ-Sh3 mice showed reduced pMypt and Rock1 vs WT-Sh3 lysates [Figure S3g].

To evaluate differentially expressed genes (DEG) between the kidneys of CAGS-rtTA/ASD2Δ-Sh3 &-WT-Sh3 during TIF, RNAseq was performed on total RNA from CAGS-rtTA/ASD2Δ-Sh3 &-WT-Sh3 kidneys at 9 weeks [see “methods”]. A principal component analyses plot showed clustering of ASD2Δ-Sh3 &-WT-Sh3 transcriptomes, demonstrating transcriptome-wide differences in TIF kidneys between these genotypes [Fig. 3i]. Differential gene expression was analyzed after aligning reads with both mouse (mm39) as well as aligning human orthologs of transcripts with the human (GRCh38) transcriptome to facilitate downstream enrichment analyses [see “methods”]. Significant DEGs identified by DESeq from both alignments are tabulated in Supplementary dataset S1a, b, respectively. In GRCh38 alignment, we identified 534 DEGs with a p-value threshold p<0.01 [Fig. S3h, Supplementary dataset S1a, b]. Significantly upregulated DEGs in WT-Sh3 transcriptome (downregulated in ASD2Δ-Sh3) demonstrated enrichment of signals related to small GTPase/Rho-kinase activation, along with enhanced TGFβ1/Wnt-βCatenin signaling, extracellular matrix (ECM) production and fibrosis [3j]. Consistent signals of Rho-kinase activation and fibrogenesis were also identified when the top 500 upregulated DEGS in WT-Sh3 mice by fold change were evaluated [p<0.05; Fig. S3k]. Conversely, upregulated DEGs in ASD2Δ-Sh3 demonstrated signals of preserved proximal tubular cell homeostasis and metabolism, suggesting reduced injury [Figs. 3j, S3h]. We also validated key mRNA signals by qPCR. Profibrotic gene transcripts related to Tgfβ1 or Wnt/Ctnnb1 signaling were significantly elevated in WT-Sh3 kidneys, while markers of tubular homeostasis were reduced [3k], a finding orthogonally confirmed by IF for Cdh16 (Figure S3i, j). Proinflammatory transcripts (chemokines, immune cell-related) also tended to be increased in WT-Sh3 vs ASD2Δ-Sh3 kidneys, similar to data in WT-Sh3-IMCD cell lines both by RNAseq and qPCR [Figs. S3h, 3k]. Phosphorylated Smad3 was significantly increased in lysates from WT-Sh3 kidneys vs ASD2Δ-Sh3 by immunoblotting [Fig. 3l, m].

To compare these gene expression changes in WT-Sh3 overexpression with human CKD, we performed co-expression analyses to identify genes that significantly correlated with Shroom3 from tubulo-interstitial transcriptomes of three human CKD cohorts within Nephroseq (R≥0.4; 429±83 genes)32–34. Enrichment analyses of co-expressed genes revealed significant enrichment of small-GTPase Rho-/TGFβ1-/Wnt-βCatenin-signaling consistent with data from WT-Sh3 overexpressing mice with TIF in our AAN model [Figs. 3n, S3l vs 3j, S3k] and showed clear translational relevance of our murine model.

Global ASD2Δ-Sh3 overexpression mice show reduced TIF vs WT-Sh3 in UUO

We evaluated a second model of TIF in CAGS-rtTA/ WT-Sh3 and -ASD2Δ-Sh3 mice – UUO as shown in Fig. 4a. UUO was induced after transgene induction with DOX as described before (see “methods”)35, and obstructed kidneys were evaluated for TIF at 7 days. qPCR confirmed increases in human ASD2-domain specific primers in WT-Sh3 UUO kidneys [Fig. 4Sa], while mouse Shroom3-specific primers remained similar between UUO kidneys of both transgenic animals [Fig. S4b]. In the UUO model again, WT-Sh3 mice showed increased TIF by Trichrome staining [Fig. 4b-c], Picrosirius red staining for Collagens [4d–e] and Fibronectin by IF [4f–g, S4c]; see “methods”] vs ASD2Δ-Sh3 mice. Immunoblotting of cortical lysates confirmed increased phosphorylation of Smad3 in WT-Sh3 animals [Fig. S4d, e]. Analogously, profibrotic, EMT-related, Wnt-signals and pro-inflammatory signaling gene transcripts were significantly increased in WT-Sh3 UUO kidneys vs ASD2Δ-Sh3 UUO kidneys [4h]. These data demonstrated mitigated TIF in a second model by global over-expression of ASD2Δ-Sh3 compared to conditions of WT-Sh3 excess.

Tubular-specific ASD2Δ-Sh3 mice show reduced TIF vs WT-Sh3

Based on the expression of Shroom3-Rock in tubular cells [Fig. 1a], and the reduced TIF in post-tubular injury models, we crossed our Shroom3 transgenic mice with Pax8-rtTA (Paired box 8-rtTA) lines for tubular-specific over-expression. The mRNA expression analysis of the kidney cortex of these Pax8-rtTA-Shroom3 mice confirmed overexpression of ASD2-domain specific sequences in WT-Sh3 mice vs other lines [Fig. S5a] after DOX-induction. Dox-feeding did not induce increased creatinine/BUN or albuminuria in Pax8-rtTA/WT-Sh3 mice vs -ASD2Δ-Sh3 or non-transgenic (NTG) mice [Fig. S5b–d].

We then induced AAN after 4 weeks of transgene induction with Dox and compared Pax8-rtTA/WT-Sh3 mice vs ASD2Δ-Sh3 as well as Dox-fed non-transgenic littermates [Fig. 5a]. Immunostaining of AAN kidneys (9-wk) from Pax8-rtTA/WT-Sh3 mice & -ASD2Δ-Sh3 animals confirmed increased Shroom3 staining in tubular cells [Fig. S5e]. WT-Sh3 mice had greater azotemia [Figs. 5b, S5f] than ASD2Δ-Sh3 or NTG animals. Similar to CAGS-rtTA animals, Pax8-rtTA/WT-Sh3 with greater injury showed more cytoplasmic Shroom3 staining, while Pax8-rtTA/ASD2Δ-Sh3 tubules with reduced injury showed maintained apical localization of Shroom3 (in LTL-positive areas) [S5e], and preserved Cdh16 staining [Fig. 5c–f]. Pax8-rtTA/WT-Sh3 mice showed increased Collagen-I staining by IF [Fig. 5d–g; see “methods”], and more TIF by Trichrome staining [Fig. 5e–h] vs ASD2Δ-Sh3 mice or NTG. By qPCR, profibrotic gene transcripts related to Tgfβ1/Wnt/Ctnnb1 signaling and injury/inflammation were consistently elevated in Pax8-rtTA/WT-Sh3 kidneys, while markers of tubular homeostasis were higher in Pax8-rtTA/ASD2Δ-Sh3 kidneys [5i]. Pax8-rtTA/WT-Sh3 mice with greater overall injury & TIF also tended to have greater albuminuria [Fig. S5g].

In a second TIF model of UUO, we compared Pax8-rtTA/WT-Sh3 mice vs -ASD2Δ-Sh3 mice [Fig. 5j]. Here again, Pax8-rtTA/ASD2Δ-Sh3 kidneys showed significantly reduced Collagen-I and Fibronectin by IF [Fig. 5k–n], and reduced TIF by trichrome staining [Figs. 5o, S5h]. Pro-fibrotic and pro-inflammatory gene signals were significantly reduced in Pax8-rtTA/ASD2Δ-Sh3 kidneys, while tubular homeostasis markers were better preserved [5p]. Hence, deletion of the ASD2-domain during Shroom3 excess in injured tubular cells provided protection from increased TIF observed in WT-Sh3 mice using either model of fibrosis.

Fibroblast-specific ASD2Δ-Sh3 mice show similar TIF to WT-Sh3

To study the role of excess Shroom3 ASD2 domain during TIF development in fibroblasts, Shroom3 transgenic mice were crossed with Pdgfrb-rtTA (platelet-derived growth factor receptor beta-rtTA) lines to generate fibroblast-specific overexpression mice [Fig. 6a]. Pdgfrβ(+) cells isolated (by magnet-assisted cell sorting (see “methods”)) were tested for enrichment by qPCR for Pdgfrb [Fig. S6a]. The mRNA expression of the ASD2-domain sequence was higher in the Pdgfrβ(+) cells obtained from DOX-induced, Pdgfrb-rtTA/WT-Sh3 kidneys vs -ASD2Δ-Sh3 [Fig. S6b]. Mice from both lines, after inducing transgene expression, were subjected to right UUO to study TIF [Fig. 6a].

The obstructed kidneys showed no significant differences between the two lines in TIF as demonstrated by trichrome staining [Fig. 6b, c], Collagen-I [6d–e] and Collagen-III [6f–g] immunostaining. The gene transcripts of profibrotic and pro-inflammatory markers, and Wnt/Ctnnb1 signaling were also similar between WT-Sh3 vs ASD2Δ-Sh3 [Fig. 6h]. Hence, these data suggested that enhanced Shroom3-Rock interaction within injured tubular cells in global- or tubular-specific- WT-Sh3 mice is the main source of increased TIF in conditions of Shroom3 excess, while the contribution from fibroblasts alone is insufficient to significantly impact TIF after UUO.

Synthesis and screening of Shroom3-Rock interaction inhibitors

Based on these experiments showing that inhibiting Shroom3-Rock interaction in conditions of global- or tubular-Shroom3 excess could ameliorate TIF, we designed and synthesized Shroom3-Rock interaction inhibitors (P2Is) [named D1–D7; see “methods” & supplementary materials36]. We developed these initial seven P2Is using computational docking with existing protein data bank (PDB) ID: 4L2W of ROCK1, selecting the lowest energy poses for the most favorable interactions between the ligands and the protein. Figure S7a shows the structure of our hit compound BT-584 from initial screening (D4, among D1-D7). As an example, the docking of our lead compound BT-1137 (D4G) and its interaction sites with the Shroom binding domain (SBD) of ROCK1 are depicted in Fig. 7a, b. [D4G = potassium trifluoro (4-(2-thioxo-1,2,3,4,5,6-hexahydrobenzo [h] quinazolin-4-yl) phenyl) borate].

D1–D7 were initially screened using Rock activity assay (see “methods”), and pMypt1 levels in HEK-293T cells overexpressing WT-Sh3 and compared with Rock-inhibitor Fasudil (HF as a positive control for ROCK inhibition). ROCK activity assay on Wt-Sh3-overexpressing HEK-293T cells with the compounds D1–D7 (50μM) showed that compounds D3–5 & D7 had more potent ROCK inhibition [Fig. 7c]. D4 (BT584; mol wt=337.35) showed potent inhibition of Rock activity (65% inhibition vs DMSO, & similar to HF (30μM), respectively). In IMCD cells, the pMypt1 levels were dose dependently reduced by D4 (at 50- & 100μM), compared to D5–D7 [Fig. S7b].

Using structure-activity relationship studies (SAR) [shown in Fig. S7c], we next developed 7 derivative compounds of our hit compound D4 (called D4A-D4G) with the goal of improving potency. These compounds were tested for efficacy- and toxicity at 50μM in both WT-Sh3-IMCD and WT-Sh3-3T3 cells and compared with HF (30μM). WT-Sh3-IMCD cells treated with D4D-D4G inhibited pMypt1 levels, similar to HF-treated, WT-Sh3-IMCD cells, while Rock1 levels were significantly reduced by D4G alone [Fig. 7d–f]. Only D4F and D4G showed significant inhibition of profibrotic marker and cytokine transcripts (similar to studies with ASD2Δ-Sh3) in IMCD cells [Fig. S7d]. As Rock inhibition has been associated with suppression of cell cycle37, cytokinesis38,39 and cell proliferation40 we checked the effect of P2Is on WT-Sh3-3T3 proliferation. All compounds significantly inhibited 3T3 proliferation (vs DMSO), although D4G tended to have a greater effect [Fig. 7g]. In toxicity studies, D4D showed the greatest toxicity in IMCD cells and in fibroblasts (Cell-glo titer ATP assay) [Figs. 7h, S7e, respectively]. D4E, F & G were similar to HF in ATP assays. These studies at a 50μM concentration of D4A-G are summarized in the heatmap in Fig. 7i.

Based on these, we carried forward D4E, F & G for both efficacy and toxicity testing at progressively lower concentrations (1–10μM). Here, D4G showed more potent reduction in pMypt1 [Fig. S7f] and reduced markers of Wnt-signaling by qPCR in WT-Sh3-IMCDs [Fig. 7j]. Similarly, in WT-Sh3-3T3, D4G showed consistently reduced pMypt1 levels [Fig. S7g], cell proliferation and profibrotic marker production [S7h, i, respectively]. Toxicity studies showed that D4E, -F, &-G had significantly lower toxicity at 10μM compared to HF-10μM in WT-Sh3 IMCD [Fig. 7k] & 3T3 (7l). Hence, from these studies, which are summarized in Fig. 7l, D4G emerged as our lead compound.

We confirmed the ability of D4G to inhibit Shroom3-Rock interaction. Here, lysates from HEK-293T cells overexpressing WT-Sh3 treated with vehicle- or D4G (10μM), were immunoprecipitated with anti-V5 antibody. These were compared with ASD2Δ-Sh3 overexpressing HEK-293T lysates, similarly immunoprecipitated with anti-V5. As shown in Fig. 7m, the interaction of SHROOM3 with ROCK1 was diminished with D4G-treatment vs DMSO-treatment in WT-Sh3 overexpressing HEK-293T and absent with ASD2Δ-Sh3. Notably, the interaction of SHROOM3 with ACTIN—an ASD1-domain mediated effect, was not affected by D4G or ASD2Δ-Sh3 [Fig. 7m]. To evaluate any paralog-specific effects of D4G, we knocked down either Rock1 or Rock2 in WT-Sh3 HEK-293 cells. After confirming ROCK1 and ROCK2 knockdown [Fig. 7n], we performed the ROCK activity assay using D4G. In these experiments, D4G inhibited ROCK1 and ROCK2 nearly identically [7o], suggesting the lack of any preferential inhibition of either paralog by our lead P2I. Finally, using a kinase hotspot assay done in the absence of SHROOM3, to compare D4G with HF, we evaluated in a targeted manner (a) Rock1, Rock2 inhibition, (b) inhibition of off-target kinases, i.e., serine-threonine kinases previously reported inhibited with HF41,42, (c) Fyn (src-family tyrosine kinases related to podocyte function14,43. HF inhibited Rock1, Rock2 (as expected), but showed significant off-target kinase inhibition (except Fyn), while D4G did not inhibit any kinase in the absence of Shroom3 [Fig. S7j], suggesting specificity for inhibition of P2I.

D4G (P2I) ameliorated TIF progression in Tubular-Specific WT-Sh3 mice

Based on in vitro Rock activity assay, using D4G on WT-Sh3 IMCD cells demonstrating EC50 (half maximal efficacy concentration) of 57μM [Fig S8a], and IC50 (half maximal inhibitory concentration) of 613.2μM [Fig. S8b], we next performed pilot UUO experiments to test the in vivo anti-TIF effect of D4G using an empiric initial D4G dose of 50mg/kg (n=6 each), which corresponded to 2.5 times EC50, but was well below IC50. Dox-induced tubular-specific overexpression, Pax8-rtTA/WT-Sh3 mice were given D4G, 50mg/kg intra-peritoneally (IP), or Vehicle after the induction of UUO, and TIF was studied [Fig. 8a]. No weight loss or humane concern was seen with D4G treatment [Fig. S8c]. No increase in albuminuria was seen with D4G vs vehicle [Fig. S8d]. UUO kidney lysates from mice given D4G showed reduced pMypt1, pSmad3, Rock2 [Fig. 8b–e], and Rock1 [Fig. S8e, f] in kidney lysates, with significantly reduced fibrosis shown by trichrome staining [Fig. 8f, g], and IF for Collagen-I, Collagen-III and Fibronectin deposition [Fig. 8h–m] vs vehicle-treated UUO kidneys. Similar to UUO kidneys of ASD2Δ-Sh3 mice, D4G-treatment reduced profibrotic Tgfb1/Wnt/Ctnnb1 signals, and proinflammatory transcripts, and relatively preserved markers of tubular homeostasis [Fig. 8n]. Analogously, Cdh16 protein levels were also greater with D4G by IF [Fig. S8g, h]. Non-obstructed left kidneys from D4G-mice did not demonstrate increases in mRNA expression of injury markers, vs vehicle [Fig. S8i], nor did they exhibit overt injury in PAS-stained sections [Fig. S8j]. In summary, based on our sequential screening, we successfully showed anti-fibrotic efficacy of Shroom3-Rock P2Is in vivo for TIF in mice with WT-Sh3 overexpression.

Methods
The research complies with the ethical regulations for biomedical research enforced by Yale University. All animal protocols were approved by the Yale University Animal Care and Use Committee (IACUC protocol number-20363).

Cell culture
HEK293T (CRL-3216) cell line was obtained from Dr Cijiang John He (Icahn School of Medicine, Mount Sinai), mIMCD-3 (CRL-2123) cell line, and NIH/3T3 (CRL-1658) cell line were gifted by Dr Stefan Somlo (Yale) and Dr Lloyd Cantley (Yale), respectively. All cell lines were originally obtained from ATCC. The mIMCD-3 cells and the HEK293T/NIH/3T3 cells were grown in DMEM/F-12 and DMEM, respectively, supplemented with 10% FBS and 1% penicillin-streptomycin and maintained at 37°C in a humidified atmosphere containing 5% CO2.

Transient overexpression studies
Human SHROOM3 mutant constructs (GenScript) with deletions of PDZ, ASD1 or ASD2 domains, as well as WT, were transfected onto HEK-293T cells using PolyJet transfection (SignaGen Laboratories, Rockville, MD)8. All constructs used had a V5-tag, and the plasmids (5μg) were mixed in culture media (500μL) and added to a microfuge tube containing 5μL transfection reagent mixed in culture media (500μL). This mixture was vortexed and incubated for 10min at room temperature. Then the mixture was added to cultured dishes plated with the target cells and incubated for 48h for the transfection to complete.

Luciferase assay
HEK-293T cells were co-transfected with SBE4-Luc (0.5μg) and pRL plasmids (0.2μg) as 5μg of either of the SHROOM3 mutant constructs, using the PolyJet Transfection Kit according to the manufacturer’s instructions (SignaGen) as described above. The transfected cells were treated with 5ng/mL TGFβ1 or vehicle for 15min, and Luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (E1910; Promega).

Generation of stably transfected cell lines
HEK-293T cells were used as producer cells for lentiviruses with 2nd generation lentiviral system, using one transfer plasmid, one packaging plasmid (psPAX2, Addgene: #12260) and one envelope plasmid (pVSVG, Addgene: #8454). The cells were transfected with 5μg each of the GFP, WT- or ASD2Δ- Shroom3 lentiviral plasmid (pLenti CMV/TO Puro DEST; GenScript), packaging plasmid (pPACK), and envelope plasmid (pVSV) using PolyJet transfection reagent (#SL100688, SignaGen Labs) to generate mammalian VSV-pseudotyped lentiviral expression constructs8. The media containing lentiviral constructs were collected, filtered and concentrated using Lenti-X Concentrator (#631231; Takara) following the manufacturer’s protocol. The lentiviral supernatants were used to infect IMCD and 3T3 cells at a 1:50 ratio with culture media supplemented with 8μg/mL polybrene and incubated in a CO2 incubator for 48h. The stably overexpressing cells were selected by puromycin (#ant-pr-1, InvivoGen) treatment (1μg/mL) for 1–2 weeks before using them for experiments. The overexpression was confirmed by qPCR and western blot.

Western blot
Mouse kidney tissues, cell lines and primary renal cells were lysed using RIPA buffer added with Halt Protease and Phosphatase Inhibitor (78440, Thermo Scientific) as mentioned elsewhere (24300173). Briefly, the tissue or cells were homogenized in the lysis buffer using mechanical digestion (Precellys homogenizer, Bertin technologies) and incubated on a rotator at 4°C for 30min. The lysate was centrifuged at 12000xg at 4°C for pelleting down the undigested cell debris. The protein concentrations of the supernatant lysates were determined by BCA assay (23225, Pierce-Thermofisher). Loading buffer was then added, and the mixtures were denatured at 95°C. The proteins were separated on 4–15% or 10% gels (Bio-Rad) by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore), followed by blocking with 5% BSA or 5% skim milk. The primary antibodies used were: anti-MYPT (D6C1) (1:2000; Rabbit mAb #8574, Cell Signaling), anti-phosphoMYPT-T696 (polyclonal) (1:1000; #5163, Cell Signaling), anti-ROCK1 (C8F7) (1:2000; 4035S, Cell Signaling), anti-ROCK2 (D1B1) (1:2000; #9029, Cell Signaling), anti-HSP90 (C45G5) (1:2000; #4877, Cell Signaling), anti-SHROOM3 (polyclonal) (1:500; #SAB3500818, MilliporeSigma), anti-SHROOM3 (polyclonal) (1:500; #LS-C679459, LSBio), anti-V5(1:1000; #V8012, MilliporeSigma), anti-FLAG (1:500; #A8592, MilliporeSigma), anti-Gapdh (6C5)(1:2000; #AM4300, Invitrogen), anti-β-Actin (AC-15) (1:2000; #A5441, MilliporeSigma), anti-phospho-SMAD3-Ser423/425(C25A9)(1:1000;#9520, Cell Signaling), anti-SMAD3 (C67H9) (1:1000; #9523, Cell Signaling), anti-Vinculin (7F9)(1:5000;#sc-73614, SCBT). The secondary antibodies used for detection included horseradish peroxidase (HRP)-conjugated anti-rabbit (#AP307P, MilliporeSigma) and anti-mouse (#AP308P, MilliporeSigma) polyclonal antibodies, with a dilution ranging from 1:8000 to 1:12000. Licor Odyssey Fc imager (Licor Bio, Lincoln, NE) with Image Studio Lite Ver5.2 was used for western blot image acquisition. Densitometry was performed on images of Western blots using ImageJ software.

Immunoprecipitation
PCDESTSHROOM3 (PDZΔ, ASD1Δ, ASD2Δ and WT) and GFP (control vector) overexpressing HEK-293T cells were generated in 15cm culture dishes. Protein lysates post 36-h of transfection were immunoprecipitated with Anti-V5-tag mAb-Magnetic Beads (MBL #M167-11) and run on PAGE gels as described elsewhere73, and immunoblotted for anti-Shroom3 (polyclonal)(1:500; #SAB3500818, MilliporeSigma), anti-Rock1 and anti-Rock2 (1:2000; #4035 (C8F7);1:2000; #9029 (D1B1) Cell Signaling). For testing the lead compound D4G for Shroom3-Rock interaction inhibition, similarly, HEK-293T cells were transfected with WT-Sh3- or ASD2Δ-Sh3 constructs. Either DMSO or D4G at 50mM was added after 12h to plates with WT-Sh3-IMCD. Protein lysates were collected at 36h post-transfection, run on PAGE gels, and immunoblotted as above.

HEK-293T SMAD reporter (blue) cell studies
PCDESTSHROOM3 (PDZΔ, ASD1Δ, ASD2Δ and WT) and GFP (control vector) overexpressing Smad reporter HEK-Blue™ cells (InvivoGen) were generated in 6-well plates by transient transfection (n=4 replicates each). After 24h, cells were serum starved overnight, and either DMSO or TGFβ1 (5ng/mL). Supernatant was removed and assayed for secreted alkaline phosphatase activity (SEAP) using Quanti blue solution (per manufacturer). Cells were lysed, and lysates immunoblotted for assaying level of transfection.

Immunocytochemistry
WT-Sh3 and ASD2Δ-Sh3 overexpressing IMCD cells were plated in 12-cm wells on coverslips, serum-deprived for 8h and treated with DMSO or Fasudil (#S1573, Selleckchem) for 24h. The treated cells were fixed and permeabilized (36% HCHO, 0.1% Triton X-100 in PBS). The cells were blocked with 3% BSA/10% normal goat serum, followed by incubation with primary antibody (1:100) against anti-ROCK1 (polyclonal) (PA5-22262; Thermo Scientific). F-Actin staining for observing cytoskeletal changes in SHROOM3-transfected cells was done using 100nM Acti-stain 488 phalloidin (#PHDG1, Cytoskeleton Inc.) along with anti-rabbit Alexafluor-568 secondary antibody (1:300; #A-11036; Invitrogen) in 1% BSA-PBS. The fluoromicrographs were analyzed using ImageJ v2.1.0 (NIH, MD).

Cell proliferation assays
Cell lines overexpressing the SHROOM3 variants were plated onto 96 well plates and BrdU incorporation was performed according to the manufacturer’s protocol (#QIA58, MilliporeSigma). For drug screening experiments, different concentrations of drugs were added to the cells and incubated for 24h. The BrdU reagent was added to each well 12h before the completion of incubation. The colorimetric measurements corresponding to the BrdU incorporation were done at 450nm using BioTek Synergy Multimode Reader (Agilent Technologies Inc., Santa Clara, CA).

Cell viability/toxicity assays
Cell lines overexpressing the intact SHROOM3 were plated at a density of 5×105 cells/ml in 96-well white opaque plates (Corning). After 24h of treatments with Fasudil or drugs, 100μL of Cell Titer-Glo 2.0 (Promega) reagent was added to each well, mixed for 2min on an orbital shaker and further incubated for 10min. The luminescence measurements were done using a BioTek Synergy Multimode Reader.

Primary cultured renal cells
The primary renal cells were isolated from mouse kidneys and cultured for 5 days27. Briefly, kidneys were harvested, minced, and digested in Liberase enzyme (0.5mg/mL) with DNase I (100mg/mL) (#5401151001, Roche Diagnostics) and 0.5mM MgCl₂ for 30min. The resulting cell suspension was filtered, washed, and incubated with RBC lysis buffer (#00-4333-57, Invitrogen) for 3min, followed by washing with cold HBSS. The resultant cell pellet was resuspended in DMEM with 10% FBS and 1% penicillin-streptomycin and seeded into culture dishes. After 5 days of expansion and media changes, primary renal cell cultures (P0 PCRCs), which are 75% proximal tubules, were harvested and cryopreserved.

Scratch wound assay
PCRCs or IMCD cells expressing Shroom3 constructs were seeded in a 12-well culture plate at a density of approximately 70–80% confluence and incubated overnight at 37°C in a 5% CO₂ atmosphere. A sterile 10μl pipette tip was used to create perpendicular-line scratches across the cell layer, ensuring uniform width and depth. The plates were gently washed with sterile PBS to remove non-adherent cells. Fresh culture medium without FBS was added to each well, with or without experimental treatments. Images of the scratch area were captured at time zero (T0) using a microscope. Plates were then incubated as before. At 16h (IMCD) and 24h (PCRC), images were taken of the scratch area to observe cell migration. The scratch areas were analyzed using ImageJ software to determine the percentage of wound closure over time.

Murine inducible global WT-Shroom3, ASD2Δ-Shroom3 and FBDM-Shroom3 overexpression models
Plasmid constructs of WT, ASD2Δ and FBDM Shroom3 (under TRE) tagged with FLAG were co-transfected with a plasmid construct with rtTA under a CAGS promoter (MGI:6451722) to generate inducible expression models. The resultant 3 fused plasmids were purified and used for transfecting mouse embryonic stem cells, followed by the generation of founder mouse lines by the mouse core facility. For each construct, 5–7 founder lines were generated harboring random insertions in the genome in the FVB/NJ (Strain: #: 001800) background. The mice from each founder line were tested for optimal (2–3 fold) and inducible overexpression in kidney- and tail-tissue post DOX induction. Founder mice of either gender (6–8 weeks old) were provided DOX chow (#TD.08541; Inotiv [Envigo]) and DOX (#D3447; Millipore Sigma) water (200mg/L) for 4 weeks and tested for mRNA by RT-qPCR and protein overexpression by Western blot. One founder line with the optimal overexpression for each of the Shroom3 mutants was used for in vivo experiments.

Tubular specific WT-Shroom3 and ASD2Δ-Shroom3 overexpression models: The WT-Sh3 and ASD2Δ-Sh3 mice (CAGS-rtTA null) were each crossed to mice expressing Pax8-rtTA (Strain #007176; C57BL/6) for tubular-specific overexpression under DOX induction8.

Fibroblast specific WT-Shroom3 and ASD2Δ-Shroom3 overexpression models: The WT-Sh3 and ASD2Δ-Sh3 mice (CAGS-rtTA null) were each crossed to mice expressing Pdgfrb-rtTA (Strain #: 028570; C57BL/6) for fibroblast-specific overexpression under DOX induction74.

All mice were maintained in individually ventilated cages (IVC) on a 12-h light and 12-h dark cycle at a temperature between 68–79°F and humidity between 30–70% with free access to standard chow until used for experiments.

Injury models
All injury models were initiated in 10–12 week-old mice post-induction of overexpression by DOX. In the Aristolochic acid nephropathy (AAN) model, all mice received intraperitoneal injections (3mg/kg) of aristolochic acid (AAI, #A9451; Millipore Sigma) every third day for 3 weeks for a total of 8 injections and were euthanized 6 weeks after the last injection75,76. In the Unilateral ureteral obstruction (UUO) model77, mice were anesthetized with ketamine/xylazine and maintained on a 37°C heat pad during surgery. Both genders were used for UUO experiments, but only male mice were used for the AAN model owing to the resistance in female mice to AAN. The right kidney was exposed via a flank incision, the ureter was ligated, and the mice were euthanized after 1 week. The kidney tissues were collected for histology, immunostaining for fibrosis markers, RNA isolation for RT-qPCR, and protein extraction for Westerns. Mouse cartoon images in Figs. 3–6, 8 depicting the schema of injury models were created in BioRender. Caplan, M. (2026) https://BioRender.com/nxzeqir.

Reverse transcription qPCR
Intron-spanning primer sets were designed for all assayed genes using Primer-BLAST (NCBI), and PCR amplicons were confirmed by both melting curve analysis and agarose gel electrophoresis and obtained from Integrated DNA Technologies (Coralville, IA). Gene expression was assayed by qPCR using Applied Biosystems 7500 Real-Time PCR System (Thermo Scientific, Waltham, MA). Amplification curves were analyzed using the automated StepOne software v2.3 via the ΔΔCt method. Gapdh was used as an endogenous control. See Supplementary dataset S1c for primer sequences.

Bulk RNA-seq analyses
DNase treated RNA extracted from AAN-kidneys of WT-Sh3 and ASD2Δ-Sh3 (n=4) were depleted of rRNA and underwent quality control (QC) analysis at the Yale Center for Genome Analysis (YCGA) followed by library prep and Poly(A) RNA sequencing (NovaSeq, Illumina). The raw Illumina RNAseq paired-end data were quality controlled and adapter-trimmed using FastQC version 0.12.178,79 and BBDuk version 39.1180,81. We mapped the RNAseq data to the reference genome using STAR version 2.7.11a82. For read alignment, we used both the mouse reference genome (GRCm39) and the human reference (GRCh38). By mapping to human reference, we obtained the gene expression profiles in human orthologs. We computed the read counts in gene sets from mapped data using the Subread R package, FeatureCounts (version 2.0.0)83. To calculate differential gene expressions, we used DESeq2 version 1.44.084. The principal component analysis (PCA) was computed using the DESeq2 plotPCA function.

Immunohistochemistry/immunofluorescence
For immunostaining, 5μm sections of formalin-fixed paraffin-embedded kidney tissues (processed at Yale Pathology Tissue Services (YPTS) facility) were deparaffinized and subjected to antigen retrieval (Retrievagen (pH 6), BD Biosciences), to unmask the antigens, followed by incubation with overnight primary antibodies: anti-SHROOM3 (polyclonal)(1:500; #LS-C679459, LSBio), anti-Collagen-I (polyclonal) (1:200; #1310-01) and anti-Collagen-III polyclonal)(1:200; 1330-01) from Southern Biotech; anti-FLAG (D6W5B)(1:100; #14793, Cell Signaling), anti-Fibronectin (polyclonal)(1:100; #SAB5700724, MilliporeSigma); rabbit polyclonal antibody against mouse Megalin (1:1000; anti-MC220 (PMID: 15180987)) and mouse monoclonal antibody against KSP-Cadherin (clone: 4F6/F6)(1:1000) kindly provided by Dr Robert Brent Thomson at the Yale Nephrology. Anti-rabbit Alexafluor-568 secondary antibody (polyclonal)(1:300; #A-11036; Invitrogen) and Alexafluor-594 rabbit anti-Goat (polyclonal)(1:300; #A-11080; Invitrogen) secondary antibodies in 0.1% BSA (in PBS) were used. FITC labeled Lotus Tetragonolobus Lectin (1:100; #L32480, Invitrogen) was used to stain the brush border of proximal tubules for co-localization with Shroom3. DAPI (1μg/mL; #D9542; MilliporeSigma) was used to stain nuclei. Images were captured using STELLARIS Confocal Microscope (Leica Microsystems, IL) using Leica LASX v1.44. Fluorescence intensities or percent positive area were measured from 8–10 high-power fields (hpf) per slide using ImageJ v2.1.0 (NIH, MD).

Fibrosis score determination
For visualizing the collagen fibers, 5μm sections of formalin-fixed kidney tissues were deparaffinized and stained with Masson’s Trichrome stain (MTS) and/or picrosirius red stain (both stained at YPTS facility). The slides were scanned using a digital slides scanner (Motic Digital Pathology) and the images captured using Aperio ImageScope v12.4.6.5003 (Leica Biosystems). The blue area (MTS) or the plane polarized light Sirius red-stained area was assessed from 10 hpf using ImageJ v2.1.0.

Electron microscopy
Glutaraldehyde-fixed samples were processed, embedded in epoxy resin and underwent ultramicrotomy85. The electron micrographs were captured at the Electron Microscopy & Cryo Electron Microscopy facility in the Center for Cellular and Molecular Imaging (CCMI), Yale. The average foot process width was determined as described elsewhere86. In brief, for all animals, three to five toluidine blue semi thin sections were reviewed, and those containing more than five glomeruli were selected for ultrathin sectioning on grids. These grids were stained with uranyl acetate and lead citrate using the standard protocol employed by the central CCMI facility at Yale. Images of free glomerular capillary loops were captured at 1200× to 4800× (direct magnification), and micrographs from each group were analyzed to determine the mean foot process width (FPW) using ImageJ v2.1.0.

Development of P2I inhibitors
The crystal structure of SHROOM3-ROCK interaction is not well known, while the interaction sequences in the SBD–i.e., ROCK1 (AA 836- 913), ROCK2 (AA 856–936) with SHROOM3 ASD2 domain (AA 1563- 1986), are known36. The interaction is conserved cross-species41,42. The crystal structure of the SBD of human Rock1 (PDB ID: 4L2W) was first prepared, and possible binding cavities were identified for ligand interaction. Using a rational and fragment-based drug discovery (FBDD) approach, taking Pdb ID: 4L2W (Shroom-Binding domain of human Rock1) into consideration, we synthesized initial compounds and performed screening to identify the hit compound. We were informed by a prior Shroom3-Rock2 interaction inhibitor that had been screened in neuronal cells in vitro36. Virtual screening was carried out to dock each of the P2Is, selecting the lowest energy poses for the most favorable interactions between the ligands and the protein. Using this, we developed 7 P2Is for initial screening (D1–D7). Our analysis revealed that primary interactions between P2Is and the protein were hydrophobic and π–π interactions [Fig. 7b]. D1-to-D7 were screened using ROCK assay (below) and pMypt levels, to identify our hit compound BT584 (D4). D4 was active in the 50 micromolar range. SAR studies were then performed for D4 (as shown in Fig. S7c) to develop and synthesize seven more compounds, D4A-D4G. D4A-D4G were screened in two cell types of WT-Sh3 overexpression–IMCD (tubular cells) and 3T3 (fibroblasts) for toxicity and efficacy studies (shown in Figs. 7, S7). Each lot of drugs synthesized for in vitro and in vivo studies was confirmed using their nuclear magnetic resonance spectra. Using our lead compound BT1137 (D4G) as an example, a sample fluoro-NMR spectra from BT1137 (confirming 3 Hydrogen, 13 Carbon, and 3 Fluorine moieties), and representative drug synthesis steps are included in Supplementary dataset S2.

Rock, Rock2 knockdown studies
Small interfering RNAs (siRNA) (from Horizon Discovery Bioscience) were used to silence either Rock1 (#L-003536-00-0005) or Rock2 (#L-004610-00-0005) in WT-SH3-HEK293T cells, and the effect of the drug D4G on residual Rock activity was assayed. Transfections of 20 pmol of the siRNAs were done using Lipofectamine RNAiMax (Invitrogen, #13778150) following the manufacturer’s instructions and the knockdown efficiency was assessed by Western blot. Drug treatments were done 60h post-transfection, and cells were harvested at 72h for assays. Results were compared with cells transfected with ON-TARGETplus Non-targeting Pool (Horizon Discovery, # D-001810-10-05).

Rock activity assay
For the primary screening of the first set of drugs, HEK-293T cells were treated with HF and the compounds for 12h and lysed with the cell lysis buffer available with the ROCK activity assay kit (#CY-1160, MBL Life Science). For screening the derivatives of the drug D4, IMCD cells overexpressing WT-Sh3 were treated as above and lysed after 12h treatments. For evaluating the efficacy of the lead compound D4G on Rock1/2 separately, Rock1-knockdown and Rock2-knockdown WT-Sh3-HEK293T cells were treated with D4G during the last 12h of transfection and were harvested for cell lysate preparation. The assay was performed with the cell lysates (prepared using 2x Cell Lysis Buffer, #RABLYSIS1; MilliporeSigma) as per kit instructions.

In vitro drug screening
IMCD cells were treated with D1–D7 at 50μM and 100μM for 6h and assayed for pMypt levels for initial screening. After developing the derivatives of D4, WT-Sh3 overexpressing IMCD and 3T3 cells were treated with 50μM of D4A-D4G for protein analysis-IMCD (6h), qPCR analysis (12h) and toxicity and proliferation assays (24h). On identifying the potential drug candidates, D4E-D4G were tested at 1–10μM concentrations for protein analysis, qPCR analysis and toxicity/proliferation assays as described above.

In vivo drug testing (D4G)
Adult Pax8-rtTA/WT-Sh3 mice were DOX fed to induce tubular-specific transgene induction. After 4 weeks, the right UUO was performed (as above). Starting at day 1 after UUO on day-0, either D4G (50mg/kg/dose) or DMSO (5%) (#D2653; MilliporeSigma) suspended in corn oil (#C8267; MilliporeSigma) was injected intraperitoneally every 48h till sacrifice on day-7. Serial weights were monitored. Urine and blood were collected at sacrifice, and kidney tissue was harvested, processed and stored for RNA, protein, and histology evaluation.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 10 (Dotmatics, CA). Data are presented as the mean±SEM. Two-tailed Unpaired t-tests or Mann–Whitney tests (where data were non-normally distributed) were performed for univariate comparisons between two groups. The EnrichR analysis for the DEGs used Fisher’s exact test, while the DESeq2 analysis of the DEGs involved a negative binomial generalized linear model. One-way ANOVA was used while comparing more than 2 groups, with Tukey’s/Dunnett’s or Holm’s-Sidak/Bonferroni-Holm’s tests for subgroup comparisons. Statistical significance was considered with a two-tailed p<0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Dr. Menon and team investigate the role of the Shroom3-ROCK interaction in fibrosis and explores the potential of Shroom3-ROCK interaction inhibitors (P2Is) as a therapeutic strategy for CKD. The work is comprehensive, employing in vitro, in vivo, and computational approaches. The use of different transgenic mouse models strengthens the study, and the development of D4G as a potential therapeutic is a notable advancement. However, several areas require clarification and improvement. Key mechanistic aspects remain unclear, particularly regarding ROCK1 vs. ROCK2 specificity, the dominant-negative effect of ASD2Δ-Sh3, and the role of fibroblast-derived Shroom3 in fibrosis. Additional protein-level validation would support RNA-seq findings if possible.\nBelow are my specific comments.\nFigure 1C, D, and Figure S1H: The study primarily focuses on ROCK1, but it remains unclear whether ASD2Δ-Sh3 also affects ROCK2. Given that ROCK1 and ROCK2 have overlapping yet distinct functions, including Western blot data for ROCK2 in Figure 1E would clarify whether ASD2Δ-Sh3 selectively impacts ROCK1 or both isoforms. If ROCK2 was not assessed, a brief explanation of the rationale would be helpful\nFigure 1E: The pMYPT1 blot is normalized to β-Actin rather than total MYPT1, which may not accurately reflect phosphorylation changes. Normalization to total MYPT1 would provide a more precise assessment.\nFigure S1H, S7B, and S7F (lower panel): pMYPT1 is shown without a corresponding total MYPT1 blot, which is provided in Figure S3E. Including a total MYPT1 blot here would improve data interpretation and completeness.\nFigure 1F: The wound healing assay is an appropriate functional test, but the inclusion of ROCK inhibitors in WT-Sh3 and ASD2Δ-Sh3 cells would confirm that ASD2 deletion directly affects migration through ROCK inhibition.\nFigure 2B: Prior scRNA-seq data suggest that ASD2Δ-Sh3 expression is minimal in podocytes. Could the authors clarify whether ASD2Δ-Sh3 primarily affects tubular cells and does not influence podocyte function? Explicitly confirming this would strengthen the interpretation of ASD2Δ-Sh3’s tissue-specific effects.\nFigure 2I-J: Electron microscopy results show that ASD2Δ-Sh3 does not cause podocyte foot process effacement, whereas FBDM-Sh3 induces podocyte structural abnormalities and albuminuria (Figure S2F). Given the essential role of ROCK in podocyte cytoskeletal regulation, it would be helpful if the authors could elaborate on why ASD2Δ-Sh3 does not disrupt podocyte structure.\nIs this due to a cell-type-specific effect, or could differences in ROCK activation between podocytes and tubules explain this phenomenon? A brief discussion on this point would provide additional mechanistic insight.\nFigures 3C-E and 4C-F: While the study demonstrates that ASD2Δ-Sh3 reduces fibrosis in multiple models, it remains unclear whether its protective effects are solely fibrosis-related or if they also extend to direct tubular protection. Since Figure S3A suggests potential tubular preservation in ASD2Δ-Sh3 mice, including tubular injury markers such as Kim-1, NGAL, and Lcn2 would help distinguish whether ASD2Δ-Sh3 protects tubules beyond its effects on fibrosis. Providing this information would strengthen the interpretation of ASD2Δ-Sh3’s role in kidney injury.\nFigures 3I-K and 4G: RNA-seq analysis identifies fibrosis-related gene changes, but key fibrosis markers (TGF-β, COL1A1, α-SMA) were not validated at the protein level. Western blot or IHC confirmation of these markers would strengthen the mechanistic conclusions.\nFigure 5: ASD2Δ-Sh3 overexpression in Pax8-rtTA mice reduces fibrosis, but its direct effects on tubular injury remain unclear. Similar to Figures 3 and 4, including tubular injury markers (Kim-1, NGAL, Lcn2) would clarify whether ASD2Δ-Sh3 protects tubules independently of its fibrosis-reducing effects.\nIn addition, analyzing the correlation between fibrosis severity and ACR would provide further insights into ASD2Δ-Sh3’s functional impact on kidney health.\nFigure 6B, D: Most of the figures in this manuscript, Collagen I was primarily used to assess fibrosis, while Figure 6 focuses on Collagen III staining. Could the authors share the rationale behind this choice? Was the collagen I also analyzed in this fibroblast-specific ASD2Δ-Sh3 overexpressing mice compared to WT mice ?\nFigure 7D-F: D4G effectively inhibits ROCK activity, but its selectivity for ROCK1 vs. ROCK2 remains unclear. Conducting ROCK1 vs. ROCK2 knockdown experiments would clarify whether D4G preferentially inhibits one isoform.\nFigure 8: D4G reduces fibrosis and ROCK activity in UUO-induced injury, but further validation is needed. Western blot analysis for fibrosis markers such as α-SMA, COL1A1, and TGF-β1 would provide further mechanistic confirmation of D4G’s anti-fibrotic effects.\nAdditional Minor comments:\nFigure S2A: The expression of the Shroom3 mutant in Figure S2A is not clearly distinguishable. It would be helpful if the authors could indicate the expression regions with arrows or other markers to improve clarity. This would make it easier for readers to interpret the data.\nFigure Organization & Consistency : For better readability and consistency, it would be beneficial if all figures followed a uniform layout and orientation. Aligning the figure panels in a more structured and consistent manner would improve the overall presentation of the data.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This manuscript describes the identification of a Shroom3 and ROCK interaction site and its potential applications for developing drugs to treat CKD. The manuscript is well written and organized, and the MOA studies are clear and well demonstrated. The identification of hit/probe molecules for this PP interaction by AI drug discovery were also well described and the compound optimization (limited) is also rational. The identifications of both the PP interaction and the probe compounds are novel. Therefore, the manuscript could be accepted for publication in Nat Comm. From the view point of drug discovery and medicinal chemistry, for both the authors and the editor to consider, the following comments might be taken into consideration (optional):\n1, The potassium trifluoroborate moiety (in cmpd 4G4 or BT1137) has never before be used in a drug-like compound, what is the rational the researchers advanced this compound into animal studies? any anticipated tox and side effects?\n2, In animal studies, Cmpd 4G4 was dosed IP at 50 mg/kg. What is the rational for this dosage and the rational for IP dosing (not po dosing). Any in vitro and in vivo PK data obtained for cmpd 4G4? What is or expected to be the plasma exposure level of cmpd 4G4 at this IP dosage? Is this exposure level much higher than the cell-based EC50/IC50 (also need to take into calculation for the plasma-protein binding of cmpd 4G4 in mice plasma). If the 50 mg/kg dosage was chosen to present just because it showed good enough efficacy? If so, maybe the dose-response graph should be shown.\n3, what is the formulation for the IP dosing? Does this formulation help the complete dissolution of 4G4 after IP dosing? A common problem is that, for some compounds which are not so soluble, IP dosing might over-estimate the efficacy due to the solubility issues. If 4G4 precipitated after IP dosing, 4G4 will be slowly released, and thus IP dosing will functionally enhance the psudo in vivo stability of 4G4 and hence increase the in vivo half-life.\n4, I noticed that cell assays in HEK293 cells were used to screen compounds and to evaluate the cell-based efficacy of the compounds. I have no objections for this and it actually served very well the purpose of MOA studies. However, I also noticed that the IC50/EC50 in Hek293 cells are all expected to be higher than 20 uM based on the data shown. My question is why not to use a more relevant cell-line to do these studies (more related to CKD)? My personal observation is that, many compounds have lower solubility and/or lower cell-permeation in the HEK293 cell media, thus resulting in much lower activity (higher EC50/IC50 values) in HEK293 cells than in those more disease-related cells. For drug discovery, this might give misleading information. For example, the IC50 value in HEK293 cells is 25 uM, but in CDK related cells, it might be ~ 1 uM. In vivo PK of 4G4 in mice might show that 50 mg/kg IP dosing is not enough if based on the cell efficacy data in HEK293 cell (EC50: 25 uM), but it will be enough if based on the cell efficacy data in a CDK related cell line (EC50: ~ 1 uM).", "reviewer_4": "The authors elucidated the interaction with Rock and ASD2 domain in Shroom3, using mice with ablation of ASD2 domain. They also created the compound that interrupts the interaction with Rock and ASD2, which might be a promising drug for kidney disease. However, there are some points to be addressed.\n[Major]\n1. The dataset used by the authors in Figure 1A includes the IRI and UUO model at different time points. Therefore, it is necessary to clarify which model and timepoint the authors analyzed. In addition, according to the result from the website (https://humphreyslab.com/SingleCell/), the expression patterns of Shroom3 and Rock are different, suggesting that Rock expression is likely regulated by other molecules.\n2. Figure 1A and supplemental Figure 1a show injured PT and fibroblast expressed Shroom3, Rock1 and Rock2 higher than other cells, however, the expression patterns were quite different.\nIn Figure 1A, Fibroblast expressed these three genes higher, and injured PT had less expression of shroom3. On the other hand, in supplemental Figure 1A, fibroblast expressed Rock1 and Rock2, but did not express Shroom3 while injured PT expressed Shroom3 and Rock2.\n3. You adopted UUO model in the experiment of Figure 4 and 6 while you used AAN model in Figure 5. In UUO model, kidney has less expression of Shroom3 until day 7 according to Humphreys website (see attached file).\nTherefore, you could not evaluate how ASD2delta-Sh3 in fibroblast affected kidney in UUO model. Please explain the reason that you adopt UUO model, not AAN model in Figure 6.\n4. Regarding Figure 1G and 1I, the authors concluded that tubular cells from ASD2delta-Sh3 mice had less ability to migrate and proliferate. And ASDdelta-Sh3 was protective against kidney injury. However, in previous reports (J Clin Invest. 2019;129(12):5501–5517, Nat Rev Nephrol 16, 269–288 (2020)), recovery from injury requires proliferative ability in tubular cells, which is not consistent with your result.\n5. In Figure 8d, please add the data on Vimentin, Col1a1, Fsp1, Mmp2 as shown in Figure 4.\n6. To confirm that D4G is effective for fibrosis, the authors should perform an additional experiment with AAN model.\n7. Please clarify in which area of kidney and who evaluated fibrotic score.\n[Minor]\n1. Another research team has reported that a different SNP (rs142647267 ) was significantly associated with a decline in eGFR in a study of the Shroom3 locus. Please discuss these other studies in detail.\n2. There are many abbreviations in the text without initial spell-out, making it difficult to read, and this needs to be resolved."}, "author_response": "To our Editor and Reviewers,\nWe would like to thank you for the thorough evaluation of our manuscript and providing us with constructive\nfeedback. We greatly appreciate the opportunity to revise and resubmit our work.\nWe have carefully considered all the comments and concerns raised by the reviewers and the editorial team. In\nresponse, we have done substantial revisions and additional experiments, which we believe have significantly\nstrengthened the rigor and impact of the study.\nAll reviewer comments have been addressed in detail in the accompanying point-by-point response document.\nRevisions to the manuscript have been clearly highlighted as per journal guidelines.\nWe have ensured full compliance with all editorial policies and requirements, including:\n Completion of the Editorial Policy Checklist and attached to the Reporting Summary.\n Updated Data Availability section with deposition of datasets in appropriate public repositories.\n Providing a comprehensive Source Data files for all main figures as ppt and tables in Excel format.\n Included more data into the panels from new experiments suggested by all the reviewers.\n Performed adjustments of all figures and bar graphs to appropriate formats for displaying data\ndistribution.\n Consideration and appropriate reporting of sex and gender in line with SAGER guidelines\nWe enclose in the revised submission (i) a marked copy (yellow highlights for changes in revision), (ii) a clean\ncopy of the manuscript file, (iii) Supplementary information (Supplementary figures/legends and Reporting\nsummary as separate documents) and (iv) Supplementary datasets 1-4.\nWe confirm that there have been no changes to the author's list. In order to give due credit to Dr Bhaskar Das’s\ncontributions in drug discovery/drug development/synthesis part of this manuscript (including the revision) we\nhave included Dr Das as a co-corresponding author for this manuscript.\nWe sincerely hope that the revised version addresses all the concerns raised and meets the expectations of the\nreviewers and editorial board.\nPoint by point response to all reviewers’ comments are listed below:\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nDr. Menon and team investigate the role of the Shroom3-ROCK interaction in fibrosis and explores the potential\nof Shroom3-ROCK interaction inhibitors (P2Is) as a therapeutic strategy for CKD. The work is comprehensive,\nemploying in vitro, in vivo, and computational approaches. The use of different transgenic mouse models\nstrengthens the study, and the development of D4G as a potential therapeutic is a notable advancement.\nHowever, several areas require clarification and improvement. Key mechanistic aspects remain unclear,\nparticularly regarding ROCK1 vs. ROCK2 specificity, the dominant-negative effect of ASD2Δ-Sh3, and the role\nof fibroblast-derived Shroom3 in fibrosis. Additional protein-level validation would support RNA-seq findings if\npossible.\nWe appreciate the reviewer’s positive assessment of our study and their thoughtful suggestions, which have\nhelped us to strengthen and improve this work manuscript.\nQR1.1. Figure 1C, D, and Figure S1H: The study primarily focuses on ROCK1, but it remains unclear whether\nASD2Δ-Sh3 also affects ROCK2. Given that ROCK1 and ROCK2 have overlapping yet distinct functions,\nincluding Western blot data for ROCK2 in Figure 1E would clarify whether ASD2Δ-Sh3 selectively impacts\nROCK1 or both isoforms. If ROCK2 was not assessed, a brief explanation of the rationale would be helpful\nReply: We thank the reviewer for raising this important question regarding the potential role of ROCK2 in the\ncontext of SHROOM3-ASD2 deletion. As ROCK1 and ROCK2 contain conserved Shroom3-binding domains\n(SBD), the ASD2 deletion is expected to disrupt interactions with both paralogues.\nIn the revision to address this question by the reviewer, we have included both in vitro and in vivo data that\nassesses changes in ROCK2. (Supplementary Fig 1)\n1. First, we show data confirming Rock2 binding is inhibited by ASD2 deletion (similar to ROCK1 already\nshown in Figure 1c). Here in figure R1, lysates from HEK cells transfected with GFP, WT-Sh3, and\nASD2Δ-Sh3 plasmids were immunoprecipitated using V5-conjugated agarose beads. The resulting\ndata are included in the revised manuscript as Fig S1c.\nR1\n1. We repeated the experiment shown in Fig 1D-E comparing IMCD cells expressing either WT-\nSh3 or ASD2Δ-Sh3, this time probing for ROCK2. Consistent with the effect observed for\nROCK1 (shown in Fig 1 D-E), we detected a reduction in ROCK2 expression in the ASD2Δ-\nSh3 group. These data are presented here as Fig R2 for the reviewer/editor reference but are not\ncurrently included in the revised manuscript to avoid redundancy, as ROCK2 down regulation is\nreferenced in multiple sections of the text.\nR2\n2. Similarly, in vitro experiments using 3T3 cells expressing ASD2Δ-Sh3 also showed reduced levels of\nboth ROCK2 and ROCK1. These immunoblot data are shown below in response to reviewer comment\nQR1.3 and are included in the revised manuscript in Fig S1i.\nR3\n3. Our P2I compounds, which inhibit ASD2-Rock SBD interaction, led to a reduction in both ROCK1 and\nROCK2 levels. These data, included in revised manuscript as Figure 8/S8, further support the\ninvolvement of both ROCK paralogues in SHROOM3 ASD2-domain.\n2. Analogous with our data on the effect of Shroom3 ADS2 domain on Rock2 activation, an independent\nstudy used in vitro screening to identify a relatively specific Shroom3-Rock2 interaction inhibitor focused\non neuronogenesis after stroke. We have cited this paper 1 in Result #7 and in Methods under the heading\n‘Development of P2I inhibitors’. To clarify this question in the revised manuscript, we also included the\nfollowing in the discussion to highlight inhibition of both ROCK paralogues by the Shroom3-ASD2 directed\ninterventions.\n“Critically, the protein-protein interaction-based, pro-injury signaling mechanism that we describe allowed\nfor the development of P2Is that inhibited Shroom3-Rock interaction and mitigated TIF1.”\nQR1.2. Figure 1E: The pMypt1 blot is normalized to β-Actin rather than total Mypt1, which may not accurately\nreflect phosphorylation changes. Normalization to total Mypt1 would provide a more precise assessment.\nReply: Reply: We thank the reviewer for this important comment. . In response, we quantified phosphor-MYPT1\n(pMYPT1) normalized to total MYPT1 (tMYPT1) rather than β-Actin. As shown in Fig. R4 densitometric analysis\nof pMYPT1 normalized to total MYPT1 confirms the results presented in Fig. 1E, supporting the conclusion that\nphosphorylation changes are independent of total protein abundance\nR4\nQR1.3. Figure S1H, S7B, and S7F (lower panel): pMypt1 is shown without a corresponding total Mypt1 blot,\nwhich is provided in Figure S3E. Including a total Mypt1 blot here would improve data interpretation and\ncompleteness.\nReply: We apologize to the reviewer for this oversight. We have now corrected Figs S1h, S7b and S7g (earlier\nS7F lower panel) to include Mypt1(blots shown below).\nFigure S1h has been added with blots of Rock2 and total Mypt1 from 3T3 lysates- WT-Sh3 and ASD2-Sh3 as\nshown above in Figure R3.\nFigure S7b, Drug D4-D7 total-Mypt1\nR5\nFigure S7g has been added with a blot of total Mypt1 from 3T3 lysates- Drug E, F, G 1-10M\nR6\nQR1.4. Figure 1F: The wound healing assay is an appropriate functional test, but the inclusion of ROCK\ninhibitors in WT-Sh3 and ASD2Δ-Sh3 cells would confirm that ASD2 deletion directly affects migration through\nROCK inhibition.\nReply: We thank the reviewer for this valuable suggestion. We repeated the wound healing assay in IMCD cells,\nadding HF at 10 μM. We used lower dose of HF to avoid any confounding by cell death observed at 30 μM.\nThe percentage of migrated area was then calculated at 16-hrs (as before) and normalized to the migration by\nthe WT-Sh3-IMCD in the same experiment. We observed that when ROCK activity was reduced by HF-10μM,\nthe difference in migration between ASD2Δ-Sh3 cells vs. WT-Sh3 significantly narrowed but was not abolished.\nThis lack of complete abolition of differences in migration could be because of two reasons. (1) As shown in Fig\n1d-e, MYPT phosphorylation is still detectable in IMCD lysates even at 30 μM HF, suggesting residual Rock\nactivity persists and is not completely abolished by HF. (2) The significantly reduced total endogenous Rock1,\nRock2 levels observed with ASD2Δ-Sh3 also represent a difference that would persist regardless of the presence\nof HF. Hence these data support the role of Shroom3-Rock interaction in mediating differences in migration\nbetween ASD2Δ-Sh3 and WT-Sh3.\nWe have included this data in updated Figure 1f and Supplementary Figure S1h-i and shown here as R7.\nR7\nQR1.5-6: Questions pertaining to the role of ASD2-domain of Shroom3 in Podocytes vs Tubular cells.\nQR1.5. Figure 2B: Prior scRNA-seq data suggest that ASD2Δ-Sh3 expression is minimal in podocytes.\nCould the authors clarify whether ASD2Δ-Sh3 primarily affects tubular cells and does not influence\npodocyte function? Explicitly confirming this would strengthen the interpretation of ASD2Δ-Sh3’s tissue-\nspecific effects.\nQR1.6. Figure 2I-J: Electron microscopy results show that ASD2Δ-Sh3 does not cause podocyte foot process\neffacement, whereas FBDM-Sh3 induces podocyte structural abnormalities and albuminuria (Figure S2F). Given\nthe essential role of ROCK in podocyte cytoskeletal regulation, it would be helpful if the authors could elaborate\non why ASD2Δ-Sh3 does not disrupt podocyte structure. Is this due to a cell-type-specific effect, or could\ndifferences in ROCK activation between podocytes and tubules explain this phenomenon? A brief discussion on\nthis point would provide additional mechanistic insight.\nReply: We thank the reviewer for highlighting this excellent point and allowing us to discuss in detail why ASD2-\ndeletion mediated lack of ROCK facilitation did not have significant in vivo consequences in podocytes. We\nbelieve the reviewer is pointing to 2B which is the immunofluorescence image (and not the scRNA data). The\nlack of podocyte phenotype in our model of ASD2Δ-Sh3 overexpression (therefore Rock inhibition) could be due\nto the following reasons:\n1. Regulation of Rho GTPase balance in podocytes: We acknowledge that simultaneous Rock1/Rock2\nknockout in podocyte-specific models, have not yet been performed, and is a limitation in the field. However,\nRho kinases (which Shroom3 facilitates) activate podocyte RhoA, are extensively studied with conflicting\nresults. This inconsistency may be due to the finely tuned balance of Rho-GTPases that ultimately regulate\nsusceptibility to injury (reviewed by Reiser et al 20122). For instance, dysfunctional RhoA over-expression in\npodocytes induced injury3; however, over-expression of active RhoA also induced injury. Podocyte specific\nRac1 knockout (i.e. with unopposed RhoA) offered protection in injury models4. In the specific context of\nShroom3, reports show that Shroom3 5 regulates Synaptopodin levels via 14-3-3 binding motif 6, which in\nturn can facilitate RhoA levels/signaling7. Hence, the overall podocyte phenotype involves a combination of\nfactors that regulate Rho-GTPAses in each transgenic model including ours.\n2. Potential beneficial effects of RhoA/Rock deficiency in podocytes: Next, Podocyte-specific RhoA-deficient\nmice themselves were normal without proteinuria 8. Podocyte-specific Rock2-deficient were also normal 9,\nand in the setting of injury, Rock2-Knockout mice exhibited protection from proteinuria in models of FSGS 9,\n10. Similarly, Rock1 deficiency also led to less albuminuria 9, 11. Analogously, data show that RhoA/Rho-kinase\npathway is upregulated in glomerular injury accompanying diabetes 12. In this milieu, Rho-kinase inhibitors\nreduced glomerular damage from diabetes 13, 14, 15, 16. The Rho-kinase inhibitor (Fasudil) also had beneficial\neffects on podocyte foot process effacement 12. Together these data point to a degree of dispensability of the\nRock-RhoA axis downstream of Shroom3 ASD2-domain in podocytes and support the lack of podocyte\nphenotype in our ASD2Δ-Sh3 mice.\n3. High endogenous Shroom3 expression in Podocytes: We also note that in single cell datasets (including\nthose shown in Figure 1a and S1a-b), constitutive Shroom3 expression in podocytes is indeed high both in\nhomeostasis and during injury. As shown in Figure 2b endogenous Shroom3 protein was higher (by\nimmunofluorescence) in glomeruli of uninjured control mice vs the tubulo-interstitial compartment. After DOX\nfeeding, we also observed overexpression to a similar extent in glomeruli of ASD2Δ-Sh3 and WT-Sh3 mice\nby immunofluorescence. Conjecturally, in the presence of high constitutive Shroom3 levels, the\noverexpressed ASD2-deletion mutant could have less dominant negative effect (especially if the effect is\nmild) over the endogenous protein’s activity. However, the use of D4G (Figure S8d) also did not induce\nproteinuria.\n4. We accept that proteinuric phenotype could also result from defects in reabsorption of albumin as a tubular\neffect of ASD2-deleted Shroom3 which we did not specifically test.\nHence in summary, the protective role of RhoA-Rock axis in podocytes in homeostasis is controversial, and\ndependent on Rho-GTPAse balance in each model, and inhibition of Shroom3-Rock interaction alone in our\nmodel likely does not induce significant podocyte dysfunction. This is consistent with the absent podocyte foot\nprocess effacement and/or proteinuria seen in vivo in global ASD2Δ-Sh3 excess.\nOn the other hand, FBDM-Sh3 mice exhibit podocyte FPE due to alteration of a different motif in Shroom3. The\nmutation in the Fyn-binding motif (i.e. -PxxP- to -AxxA-) leads to reduced interaction with Fyn which is crucial for\nnephrin cytoplasmic tail phosphorylation in podocytes and maintenance of podocyte cytoskeleton by binding Nck\nadapter proteins 17. The role of Fyn inactivation or deficiency in podocytes impacting Nphs1 phosphorylation,\nactin cytoskeleton has been independently reported in animal models18, 19 and proteinuric human diseases20.\nAs suggested, and to clarify the reviewer’s point regarding podocyte function, we have included the following in\nthe revision:\n” Indeed, in the setting of diabetic kidney injury, global Rock1 deficiency or podocyte-specific Rock2 deficiency\nled to less albuminuria and a protective phenotype 9, 11. Furthermore, via a distinct 14-3-3 binding motif6 Shroom3\ncould regulate Synaptopodin levels in podocytes 17 and facilitate RhoA signaling7, providing a potential\nexplanation for the minimal impact of ASD2-motif antagonism on podocyte phenotypes”\n“…high endogenous expression of Shroom3 may attenuate the dominant negative effect of ASD2Δ-Sh3\nin cell types such as fibroblasts and podocytes with high constitutive Shroom3 levels.”\nQR1.7. Figures 3C-E and 4C-F: While the study demonstrates that ASD2Δ-Sh3 reduces fibrosis in multiple\nmodels, it remains unclear whether its protective effects are solely fibrosis-related or if they also extend to direct\ntubular protection. Since Figure S3A suggests potential tubular preservation in ASD2Δ-Sh3 mice, Including\ntubular injury markers such as Kim-1, NGAL, and Lcn2 would help distinguish whether ASD2Δ-Sh3 protects\ntubules beyond its effects on fibrosis. Providing this information would strengthen the interpretation of ASD2Δ-\nSh3’s role in kidney injury.\nReply: We thank the reviewer for this most helpful suggestion. We have now systematically evaluated profibrotic,\ntubular injury/inflammation and tubular homeostasis markers in the mRNA expression panel in figure 3k and\nsimilarly under each relevant invivo experiment. The injury markers Kim1, Lcn2, Hmgb1, and Il18 tended to be\nreduced in the ASD2Δ-Sh3 in comparison with the WT-Sh3 kidneys, whereas the tubular homeostasis markers\nLrp2 and Cdh16 were upregulated in the ASD2Δ-Sh3 mice.\nR8\nTo evaluate the protein level expression of markers of tubular health in kidney sections, we stained paraffin-\nembedded sections from AAN kidneys for KSP-cadherin (CDH16) a pan-tubular homeostatic marker using IF.\nKSP-cadherin was significantly higher in AAN kidneys from ASD2Δ-Sh3 as compared to WT-Sh3 kidneys,\nsuggesting better preservation of homeostatic gene expression in tubules of ASD2Δ-Sh3 mice with reduced TIF.\nThese data have been added in manuscript (Figure S3i-j).\nR9\nWe also evaluated Megalin (LRP2) which showed similar trend of increased expression in ASD2Δ-\nSh3, however, this data is only shown here for reviewers’ and not included in the manuscript to\nminimize redundancy and for space concerns.\nR10\nQR1.8. Figures 3I-K and 4G: RNA-seq analysis identifies fibrosis-related gene changes, but key fibrosis markers\n(TGF-β, COL1A1, α-SMA) were not validated at the protein level. Western blot or IHC confirmation of these\nmarkers would strengthen the mechanistic conclusions.\nReply: We thank the reviewer for suggesting additional validation of RNA-seq data using protein estimation for\nkey fibrosis markers. We have already shown protein levels for COL-I and COL-III for AAN by immunostaining.\nWe now included FIBRONECTIN1 immunostaining on paraffin sections of AAN and UUO kidneys (WT-Sh3 vs.\nASD2Δ-Sh3) to show tissue localization. Fibronectin expression was quantified and found consistent with qPCR\ndata, i.e., significantly higher in WT-Sh3 as compared to ASD2Δ-Sh3 in both AAN (Figure-S3c-d) and UUO\n(Figure-4f-g) models (Figure-R11A, B respectively). We did not include α-SMA in the fibrosis assessment as our\nstaining proved inconsistent for this marker, and α-SMA has been reported to be an unreliable marker for\nfibrogenic cells in murine experimental models of organ fibrosis 21, 22.\nTo test intrarenal canonical Tgfb1 signaling, we probed for phosphorylated-Smad3/Smad3 in kidney lysates from\nour AAN TIF model. We identified reduced P-Smad3 in ASD2Δ-Sh3 vs WT-SH3- AAN lysates. These data are\nshown in figure 3l-m, below (R11C) and are consistent with similarly reduced PSmad3 which was seen in ASD2Δ-\nSh3 UUO kidneys. We chose P-Smad3 levels, as Total TGFB1 levels are an unreliable assay since TGFB1 is\nactivated extracellularly before receptor binding by cleavage of Latent peptide 23, 24, 25, 26\nR11A\nR11B\nR11C\nQR1.9. Figure 5: ASD2Δ-Sh3 overexpression in Pax8-rtTA mice reduces fibrosis, but its direct effects on tubular\ninjury remain unclear. Similar to Figures 3 and 4, including tubular injury markers (Kim-1, NGAL, Lcn2) would\nclarify whether ASD2Δ-Sh3 protects tubules independently of its fibrosis-reducing effects.\nReply: We thank the reviewer for this important suggestion. We have now analyzed tubular injury and\nhomeostasis markers in AAN-kidneys of Pax8-rtTA mice as described below and confirmed that tubules are more\npreserved in the ASD2Δ-Sh3 kidneys. The tubular injury/inflammation markers Kim1, Lcn2 and Il18, and Ccl2\nwere significantly lower in tubular-specific ASD2Δ-Sh3 overexpressing kidneys vs. WT-SH3 (R12).\nR12\nOn the other hand, the tubular homeostasis markers Lrp2 and Cdh16 were more preserved in ASD2Δ-Sh3\n(R12 and Figure 5i). Similar findings were noted at the protein level by IF, showing better-preservation of\ntubular expression of CDH16 in ASD2Δ-Sh3 kidneys compared to WT-Sh3 (Figure R13 and Figure 5c & f).\nR13\n50 ✱✱\n)%40\n(\na\ne\nrA\n+30\nn\nire\nh d20\na\nC\n-P n=4 n=5 n=6\nS10\nK\nA\nh3 SD2Δ-Sh3\nQR1.10. In addition, analyzing the correlation between fibrosis severity and ACR would provide further insights\ninto ASD2Δ-Sh3’s functional impact on kidney health.\nReply: We believe the reviewer is referring to 5H and S5D and appreciate the interesting observation. We\nperformed linear regression (person correlation) analyses comparing the Fibrosis scores (Masson’s staining)\nand the albuminuria (Albumin Creatinine Ratio). These plots are shown below for the reviewer Figure R14 (red\ntriangles represent ASD2Δ-Sh3 mice in each plot). We could not identify clear or significant correlations in these\nanalyses in either global or tubular specific Shroom3 excess (P>0.05), although the reduced fibrosis along with\ntendency to reduced ACR was suggested in the tubular specific ASD2Δ-Sh3 overexpression mice.\nR14\nQR1.11. Figure 6B, D: Most of the figures in this manuscript, Collagen I was primarily used to assess fibrosis,\nwhile Figure 6 focuses on Collagen III staining. Could the authors share the rationale behind this choice? Was\nthe collagen I also analyzed in this fibroblast-specific ASD2Δ-Sh3 overexpressing mice compared to WT mice?\nReply: As the reviewer requested, we now R15\nalso measured Collagen I in the UUO kidneys\nof the fibroblast-specific Shroom3 (ASD2Δ-\nSh3 vs. WT) by immunofluorescence. The\ndata shown below for the reviewer (R15) are\nincluded in the revised manuscript Figure 6d-\ne. Comparing either Collagen I and III,\nfibroblast-specific ASD2Δ-Sh3 vs WT-Sh3\nUUO kidneys showed no significant\ndifferences in fibrosis.\nQR1.12. Figure 7D-F: D4G effectively inhibits ROCK activity, but its selectivity for ROCK1 vs. ROCK2 remains\nunclear. Conducting ROCK1 vs. ROCK2 knockdown experiments would clarify whether D4G preferentially\ninhibits one isoform.\nReply: We are grateful for the reviewer’s important suggestion.\nWe did find a significant reduction in both Rock1 and Rock2 protein levels in vivo on D4G treatment (Revised\nFigure 8/S8). Our in vitro experiments also showed that in tubular and fibroblast cell lines overexpressing the\nShroom3 variants, Rock2 levels were lower in the AS D2Δ-Sh3 vs WT-Sh3. All these are consistent with the\nconcept that both Rock paralogs are expected to be inhibited by D4G.\nTo investigate whether D4G had preferential inhibitory effects on either ROCK1 or ROCK2 paralogs, we silenced\neither Rock1 or Rock2 using specific siRNA separately in WT-Sh3 overexpressing- HEK293T cells. We confirmed\ngene knockdown in each case by immunoblot (R16 or Fig 7n).\nR16\nWe then used the Rock assay (#MBL-CY-1160.) to test Rock activity inhibition by D4G in conditions of either\nRock1 or Rock2 knockdown in WT-Sh3 overexpressing HEK293 cells (representing activity of D4G on\nSHROOM3-ROCK2 or SHROOM3-ROCK1 interactions, respectively). Our lead P2I D4G inhibited ROCK2\nactivation in a dose-dependent fashion (by ROCK assay) when ROCK1 was selectively knocked down using\nsiRNA (Human-specific), and vice versa (Fig R17 or Fig 7o). The EC50 obtained from ROCK1 and ROCK2\nsilencing were similar (57 vs 50 μM).\nR17\nThese experiments confirmed similar activity of our lead P2I on both paralog interactions. These data have\nbeen included in the revised manuscript as Figure 7n-o.\n“To evaluate any paralog-specific effects of D4G, we knocked down either Rock1 or Rock2 in WT-Sh3 HEK-\n293 cells. After confirming ROCK1 and ROCK2 knockdown [Figure-7n], we performed ROCK activity assay\nusing D4G. In these experiments, D4G inhibited ROCK1 and ROCK2 nearly identically [7o], suggesting the\nlack of any preferential inhibition of either paralog by our lead P2I “\nQR1.13. Figure 8: D4G reduces fibrosis and ROCK activity in UUO-induced injury, but further validation is\nneeded. Western blot analysis for fibrosis markers such as α-SMA, COL1A1, and TGF-β1 would provide\nfurther mechanistic confirmation of D4G’s anti-fibrotic effects.\nReply: As the reviewer suggested, we have measured more differentially expressed genes, including tissue\nexpression of Fibronectin and Collagen-III. We also performed protein expression of Fn1 and Cdh16 by western\nblot. Collagen-III and Fibronectin IF images are now included in the revised Figure S8(j-m) and shown below as\nR18.\nR18\nO\nS\nM\nD\nG\nD4G treated Pax8-WT-Sh3 UUO kidneys also showed significantly preserved CDH16 levels by both IF and\nimmunoblotting in D4G vs Dmso treated Pax8-WT-Sh3 UUO kidneys. These data are shown below for the\nreviewer/editors, and included in revised supplementary figure as S8g-h. We did not include Cdh16 or Fn1\nimmunoblot lanes in the manuscript as we show significant differences in IF for these same proteins (to avoid\nredundancy).\nR19\n60\n****\nA\n)% \n( \na\ne\nrA40\n+ \nn\nire\nh \nd20\na\nC\n-P \nS\nK\nms\no\nD4\nG\nB\nThese new data show consistent changes at the protein and mRNA levels and validating the anti-TIF and tubular\npreserving effect of D4G on TIF outcomes, and mimicking signals seen with ASD2Δ-Sh3 overexpressing mice.\nAdditional Minor comments:\nQR1.14. Figure S2A: The expression of the Shroom3 mutant in Figure S2A is not clearly distinguishable. It would\nbe helpful if the authors could indicate the expression regions with arrows or other markers to improve clarity.\nThis would make it easier for readers to interpret the data.\nReply: We now added arrows to clearly mark the Shroom3 bands.\nQR1.15. Figure Organization & Consistency: For better readability and consistency, it would be beneficial if all\nfigures followed a uniform layout and orientation. Aligning the figure panels in a more structured and consistent\nmanner would improve the overall presentation of the data.\nReply: We apologize for this inconvenience and thank the reviewer for bringing this concern here. We have re-\nformatted all figure panels again to fit into A4 size as recommended by the journal. All figures are in a portrait\norientation except Fig 3, which needed to be in landscape orientation to accommodate the data panels without\nimpeding legibility.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition for\nEarly Career Researchers who co-review manuscripts.\nWe greatly appreciate the overall positive feedback from Reviewer 1 and 2 and believe the revised manuscript\nhas improved with the incorporation of these suggestions.\nReviewer #3 (Remarks to the Author):\nThis manuscript describes the identification of a Shroom3 and ROCK interaction site and its potential applications\nfor developing drugs to treat CKD. The manuscript is well written and organized, and the MOA studies are clear\nand well demonstrated. The identification of hit/probe molecules for this PP interaction by AI drug discovery were\nalso well described and the compound optimization (limited) is also rational. The identifications of both the PP\ninteraction and the probe compounds are novel. Therefore, the manuscript could be accepted for publication in\nNat Comm. From the viewpoint of drug discovery and medicinal chemistry, for both the authors and the editor to\nconsider, the following comments might be taken into consideration (optional):\nWe appreciate the thoughtful and very positive review by the reviewer, which has allowed us to provide details\non the drug discovery aspect of this manuscript and improve this work.\nQR3.1, The potassium trifluoroborate moiety (in cmpd 4G4 or BT1137) has never before been used in a drug-\nlike compound, what is the rational the researchers advanced this compound into animal studies? any\nanticipated tox and side effects?\nReply: We appreciate this interesting question from the reviewer. We used a boron-based compound since when\nBoron atoms are introduced into biologically active molecular frameworks, they interact with target proteins\nproducing potent biological activity. Boron-containing pharmacophore groups interact with a target protein not\nonly through hydrogen bonds but also through reversible covalent bonds to produce potent biological activities\n(i.e. antifungal, antiparasitic, protease inhibitors, and other drugs). This concept is well supported in the literature\n27, 28. Proof of this concept is further provided by boron-containing compounds that are FDA approved such as\nthe proteasome inhibitor Velcade (bortezomib) for multiple myeloma, antifungal Keydin (tavaborole, approved in\n2014), Crisaborole (eucrisa, approved in 2016) for atopic dermatitis, and Vabomere (vaborbactam, approved\n2017), an antibacterial β-lactamase inhibitor for complicated urinary tract infections and pyelonephritis. Notably,\nas formulated, tavaborole is only FDA approved for topical use for as a 5% solution to treat onychomycosis (nail\nfungus). A boron-based small molecule was used as a TYRO3 agonist and reported as a therapy for glomerular\ndisease in a JCI-Insight article 29 by our co-authors.\nFor the lead compound we have performed toxicity studies in vitro (WT-Sh3 overexpressing IMCD) and is\nillustrated in Figure 7. In the in vivo setting, no overt adverse effects, ie no change in bodyweight and albuminuria\n(Figure S8c-d) were observed in D4G treated mice compared to DMSO treated mice. We also evaluated kidney\ninjury in non-obstructed left kidneys from D4G-mice, which did not demonstrate increases in mRNA expression\nof injury markers, vs vehicle [shown as Figure-S8i] nor did they exhibit overt injury in PAS-stained sections\n[shown as Figure S8j]. We also performed targeted kinase profiling (in comparison with HF – a ROCK inhibitor)\nwhere D4G was incubated with 8 kinases (that have been reported variably to be inhibited by HF) at an ATP\nconcentration of 10mM (Figure S7). In this kinase assay, D4G showed no off target inhibition of these kinases.\nIn this work, we identified our Target-to-Hit D4G compound, and structure activity relationship (SAR) studies are\nongoing to develop further Hit-to-Lead compounds. As noted by the reviewer, while reflecting proof-of-concept\ninhibition and in vivo anti-TIF effect, the EC50 for D4G does not show high potency (in micromolar range; see\nresponse to QR3.2 below). Therefore, to design potent Hit-to-lead compounds in future work, we will be guided\nby the Lipinski rule of 5. New P2Is will be designed with considerations of the propensity for (1) biological activity,\ni.e., more sp3 carbon atoms; (2) ease of syntheses; and (3) moderate compound complexity, to minimize toxicity\n& off-target effects30, 31. We will use ACDLabs ADMET suites (https://www.acdlabs.com /products/percepta-\nplatform/) and Toxicity suites to predict drug-like properties of our designed compounds, then use Go/No-Go\ncriteria to develop new compounds. These points will guide our molecular design choices and minimize the risk\nof potential toxic effects as we develop more potent P2Is in future studies.\nQR3.2, In animal studies, Cmpd D4G was dosed IP at 50 mg/kg. What is the rational for this dosage and the\nrational for IP dosing (not po dosing). Any in vitro and in vivo PK data obtained for cmpd 4G4? What is or expected\nto be the plasma exposure level of cmpd 4G4 at this IP dosage? Is this exposure level much higher than the cell-\nbased EC50/IC50 (also need to take into calculation for the plasma-protein binding of cmpd 4G4 in mice plasma).\nIf the 50 mg/kg dosage was chosen to present just because it showed good enough efficacy? If so, maybe the\ndose-response graph should be shown.\nReply: We greatly appreciate these questions regarding the dosage and route we chose for our in vivo data.\nIn our pilot in vivo studies, we used the IP route to avoid any incidental reduction in bioavailability (and therefore\nefficacy) of the compound D4G due to the digestive enzymes, gastric acidity, or first pass liver metabolism. We\nacknowledge that formal in vivo PK/PD studies in small animals have not been done yet, which we have\nacknowledged as a limitation of the manuscript in its original submission. However, we did apriori insilico ADMET\nstudies 2.0 and identified good-to-excellent predicted T1/2 and a predicted clearance of 1.39 ml/min/kg. The\npredicted plasma protein binding was 98.5% for D4G.\nWe performed in vitro PK studies using ROCK assay (#MBL-CY-1160) on WT-Sh3-IMCD cells. The EC50 of\nD4G is calculated to be 57.92μM from the equation derived from the percentage Inhibition plot (R20A). EC50\nobtained in HEK-293T cells overexpressing WT-SH3 were also similar after either ROCK1 or ROCK2 knockdown\n(Fig R20B; 58.9 and 50.2 M respectively). These data are included now as S8a-b.\nR20A R20B\nEC50= 57.92μM\nOn the other hand, the in vitro IC50 calculated from the Cell-glo toxicity assay on WT-SH3-IMCD cells was 613.2\nμM (shown in Figure7 and the non-linear regression shown in R21).\nR21 WT-SH3-IMCD\n150\nlo\nr\ntn y=0.2437x2 - 48.761x\no + 2442\nC\nr\nR² = 0.9938\ne\nv o 100\ny\ntilib\na\niV IC50=613.2\n%\n50\n0 50 100 150\nD4G concentration\nHence, we surmised that the initial efficacious dose, without risking significant toxicity was between EC50 and\nIC50 (57 to 613.2 μM).\nAs we observed EC50 of ~57μM, we first calculated an in vivo dose of 20mg/kg to approximate the EC50\n(assuming 100% bioavailability of the IP route). In these studies using WT-Sh3 mice we did not observe\nsignificant reduction in TIF in a UUO model. These data are now shown below for the reviewer (R22).\nR22\ne\ng\nh\nd\nnp\naa\nhG2\nc\nd lo o t d\nFe\ne v ita\nle\nz ila m\nro\n0\n600μm\n600μm\nSince we did not observe protection at 20 mg/kg, we empirically escalated the dose by ~2.5 times the EC50 (i.e.\n~142.5μM or 50 mg/kg), which is still well below the IC50 and repeated the efficacy study. This dose i.e. 50\nmg/kg – based on which in vivo data included in Fig 8 is based in the manuscript – showed efficacy without\nsignificant toxicity (weight loss, proteinuria or histologic injury in control kidneys).\nOur goal in ongoing work is to develop further potent Hit-to-lead compounds and perform formal PK/PD\nstudies, which will include Plasma stability, protein binding, T1/2 and maximum tolerated dose studies on\nnewer compounds.\nQR3.3, what is the formulation for the IP dosing? Does this formulation help the complete dissolution of 4G4\nafter IP dosing? A common problem is that, for some compounds which are not so soluble, IP dosing might over-\nestimate the efficacy due to the solubility issues. If 4G4 precipitated after IP dosing, 4G4 will be slowly released,\nand thus IP dosing will functionally enhance the pseudo in vivo stability of 4G4 and hence increase the in vivo\nhalf-life.\nReply: The compound was first dissolved in DMSO and then diluted to a final working concentration with corn oil\nto keep it in a non-aqueous solvent mixture. When we solubilized in DMSO, our compound was fully soluble\nwithout visible precipitation.\nAs we continue our studies with D4G, and develop refined formulations using SAR studies modifying the D4G\npharmacophore, we anticipate formulating the drugs using cyclodextrin and Captisol, which will further increase\nthe solubility.\nWe acknowledge the limitation that precipitation of the drug is a possibility; however, since the drug-treated mice\nshowed a response, we conclude that there are significant amount of active molecules in circulation using our\nIP dosing schedule.\nQR3.4, I noticed that cell assays in HEK293 cells were used to screen compounds and to evaluate the cell-\nbased efficacy of the compounds. I have no objections for this and it actually served very well the purpose of\nMOA studies. However, I also noticed that the IC50/EC50 in Hek293 cells are all expected to be higher than 20\nuM based on the data shown. My question is why not to use a more relevant cell-line to do these studies (more\nrelated to CKD)? My personal observation is that, many compounds have lower solubility and/or lower cell-\npermeation in the HEK293 cell media, thus resulting in much lower activity (higher EC50/IC50 values) in HEK293\ncells than in those more disease-related cells. For drug discovery, this might give misleading information. For\nexample, the IC50 value in HEK293 cells is 25 uM, but in CKD related cells, it might be ~ 1 uM. In vivo PK of\n4G4 in mice might show that 50 mg/kg IP dosing is not enough if based on the cell efficacy data in HEK293 cell\n(EC50: 25 uM), but it will be enough if based on the cell efficacy data in a CKD related cell line (EC50: ~ 1 uM).\nReply: We appreciate the reviewers point about the varying efficacy (EC50) that could be observed due to\ndifferent cell lines, specifically HEK-293 cells. We would like to clarify that we used 3 types of Shroom3-\noverexpressing cell lines in our efficacy and toxicity studies in experiments in Fig 7/S7/S8. For the majority of\nexperiments, we used a WT-SH3-IMCD cell line to simulate the Shroom3 excess rendered by the CKD-\nassociated SNPs, to assay the EC50 and toxicity in vitro. We also used 3T3 cell lines (human fibroblast) to\nperform additional studies in fibroblasts. Finally, we also used HEK-293 cells in ROCK assay screening for\ncompounds D1-D7, and in ROCK1, ROCK2 knockdown experiments with D4G (shown in response to QR1.12\nwhere EC50 was calculated). The EC 50 reported in Fig S8 in the revision are from WT-SH3 IMCD (57 mcM).\nThis was the basis of our initial low dose in vivo testing using 20 mg/Kg (57mcM) (shown in response to reviewer\nQR2.2). This initial dose lacked in vivo efficacy against TIF in UUO as shown, and we thus used a higher dose\n50 mg/Kg (as described above and shown in Fig 8/S8). We accept that small animal models\npharmacokinetic/pharmacodynamic studies have not been done yet, but we plan to do these steps after this\ninitial proof-of-principle work. SAR studies are also ongoing with the goal to improve potency, improve solubility\nand minimize any toxicity, before subsequent PK/PD studies.\nReviewer #4 (Remarks to the Author):\nThe authors elucidated the interaction with Rock and ASD2 domain in Shroom3, using mice with ablation of\nASD2 domain. They also created the compound that interrupts the interaction with Rock and ASD2, which might\nbe a promising drug for kidney disease. However, there are some points to be addressed.\n[Major]\nQ.R4.1. The dataset used by the authors in Figure 1A includes the IRI and UUO model at different time points.\nTherefore, it is necessary to clarify which model and timepoint the authors analyzed.\nReply: We appreciate the reviewer’s suggestion and apologize for the lack of clarity in the figure 1A. The dot plot\nis a depiction of gene expression from cells of healthy (baseline), unilateral-IRI and UUO kidneys including all\ntimepoints used in the cited study. We have now added this information to the revised manuscript and Figure\nlegend.\nQ.R4.2 In addition, according to the result from the website (https://humphreyslab.com/SingleCell/), the\nexpression patterns of Shroom3 and Rock are different, suggesting that Rock expression is likely regulated by\nother molecules.\nReply: We thank the reviewer for this insightful observation. In Figure 1A, our primary goal was to illustrate that\nShroom3-Rock protein-protein interaction is possible in the highlighted cell types since both Shroom3 and Rock\nmRNAs are expressed in those specific cell types (including in injured proximal tubular cells and Fibroblasts).\nHowever, we agree with the reviewer that Shroom3 and ROCK are likely regulated by distinct upstream signals,\nand their expression may diverge across other cell populations or states. For instance, both Rock isoforms have\nmultiple promoter binding sites for KLF family transcription factors, and experimental data show that SP6 and\nSP1 (KLF family members) can increase Rock1 levels32, 33. During cell division/mitosis, proteins required for\nmitosis (including Rock1, Rock2 needed for cytokinesis) are upregulated by multiple transcription factors\nincluding E2F/SP1, FOXM1, and NF-Y34, 35 Similarly TFAP2C (AP-2 family) increases expression of both Rock\nparalogs36. To date, the roles of these TFs in Shroom3 regulation have not yet been reported.\nOn the other hand, multiple groups including ours, have reported Shroom3 to be upregulated by pro-fibrogenic\nsignaling pathways including Wnt/Ctnnb1 and Tgfβ137, 38, 39, 40. Notably, both Rock1 and Rock2 promoters also\nhave SMAD4 binding sequences, and experimental data from multiple cell lines have shown increased Rock\nexpression with TGFB1 stimulation41, 42. Rock1 is also regulated by Tgfβ in a Smad-independent manner43, 44.\nHence, TGFB1 is a potential signal, which in the setting of TIF could simultaneously upregulate both Shroom3\nand Rock1/Rock2. To reflect the reviewer’s important point, we have included the following in the revised\nmanuscript.\n“Rock1 and Rock2 are regulated by distinct set of transcription factors during cell proliferation50, 51, but during\nTIF states may be regulated by TGFB1-signaling52, 53, which also upregulates Shroom3 54, 55, 56.”\nQ.R4.3. Figure 1A and supplemental Figure 1a show injured PT and fibroblast expressed Shroom3, Rock1 and\nRock2 higher than other cells, however, the expression patterns were quite different. In Figure 1A, Fibroblast\nexpressed these three genes higher, and injured PT had less expression of shroom3. On the other hand, in\nsupplemental Figure 1A, fibroblast expressed Rock1 and Rock2, but did not express Shroom3 while injured PT\nexpressed Shroom3 and Rock2.\nReply: As the reviewer correctly observed, Figure 1A is a representation of cell-type specific gene expression of\nShroom3, Rock1, Rock2 in UUO/IRI kidneys. Hence, it represents the average expression of genes in each cell\ntype regardless of condition, or time points of the injuries.\nFigure S1a data was based on cell-specific gene expressions at different time points (Days 7, 30) of Unilateral\nIRI kidney compared to the sham control kidney performed by our collaborator/co-authors45. Notably, this is a\nsingle cell RNA-seq (scRNA-seq) dataset. We agree with the reviewer that in this dataset shown in Fig S1A,\nPdgfrb+ myofibroblast cells exhibit low expression level of Shroom3 at the IRI timepoints. In this dataset\ngenerated by our collaborator using a fibrotic mouse model, fibroblasts and myofibroblasts could not be\nseparated, likely accounting for the lower overall expression level of Shroom3 due to the inclusion of\nmyofibroblasts along with fibroblasts 45. This distinction was apparent when examining data from Li et al 2022 (a\nsingle nuclear RNA-seq i.e. snRNA-seq), fibroblast and myofibroblast were separately annotated (shown below\nfor the reviewer Fig R23). As shown, Fibroblasts in UUO kidneys express Shroom3 from Day2-Day14 (now\nincluded in Revised Figure S1a), while myofibroblasts display markedly lower expression level of Shroom3.\nExpression of Shroom3 and Rock1/2 in fibroblasts of UUO kidneys from Li et al., 2022\nR23\nWe agree with the reviewer’s observation that Rock1 and Rock2 are differentially expressed spatially and\ntemporally in each model of TIF. Either/both paralogs could be involved in the profibrotic signaling in the context\nof excess Shroom3. To specifically investigate the individual roles of the Rock paralogs in the pro-fibrotic\nsignaling in vivo, we obtained Rock1f/f 46 and Rock2f/f 47, 48 mice from Dr Anne Eichmann (collaborator, coauthor)\nto be crossed with inducible Shroom3 overexpression mice. These studies will dissect paralog-specific\ncontributions to Shroom3-mediated fibrotic signaling and are planned future directions.\nWe also want to point out that the lack of expression of Rock1 in injured tubular cells in later phases of IRI\n(shown in Fig S1b), was not observed in Li et al’s dataset. As shown below [Fig R24], from Li et al’s dataset\nRock1 expression was also present in iPTs at day 28 (in addition to Rock2). This contrasts with Xu et al.’s\nscRNA-seq dataset and may reflect methodological differences (snRNA-seq vs. scRNA-seq). We therefore\nhave used two datasets and which include two injury models, and two modalities of single cell transcriptome\nassessment to identify putative cells of origin for Shroom3-Rock interaction for this manuscript.\nRock1 Rock2\nR24\nRepairing\nInjured\nFailed repair\nAc Injured\nDays 0.5 2 7 14 28 0.5 2 7 14 28\nQ. R4.4. You adopted UUO model in the experiment of Figure 4 and 6 while you used AAN model in Figure 5.\nReply: We thank the reviewer for this important point. We have adopted both AAN (Figure 3) and UUO (Figure\n4) models in global WT-Sh3 and ASD2Δ-Sh3 mice.\nIn response to the question raised by the reviewer, we now performed UUO model with tubular specific, Pax8-\nWTSh3/ASD2Sh3 mice. As shown in Figure 5 and S5 of the revision, Pax8-rtTA/ ASD2Sh3 mice showed\nbenefit in TIF outcomes vs Pax8-rtTA/ WT-Sh3 (n=4 vs 6). These data are summarized below as R25.\nR25\nHence, we now conclude that both in the global- or tubular-specific overexpression mice, impairing Shroom3-\nRock interaction by overexpressing the ASD2 deletion provided benefit in both AAN- & UUO- models of TIF.\nThese data support the model-independent beneficial effect of impairing Shroom3-Rock interaction, and\nsupport the strategy of using either model to test the efficacy of our P2Is.\nIn UUO model, kidney has less expression of Shroom3 until day 7 according to Humphrey’s website (see\nattached file).\nTherefore, you could not evaluate how ASD2delta-Sh3 in fibroblast affected kidney in UUO model.\nReply: We appreciate this question raised by the reviewer. We want to first note that in our model, Shroom3\nexpression itself is driven by DOX induction, and not dependent on regulation by endogenous stimuli. However,\nwe admit Rock1 and Rock2 expression would be temporally and spatially regulated by endogenous transcription\nfactors and signals.\nIn response to this important question raised by the reviewer, we show here data from the Li et al, 2022 [Fig\nR26], SnRNA dataset that Shroom3, Rock1 and Rock2 are all expressed as early as day 2 post-UUO and remain\ndetectable through day 6 after UUO. Specifically, injured and repairing tubular cells express these genes as\nobserved in Fig R26 (lower panel) and in S1a. Furthermore, as shown in response to QR4.3, fibroblasts also\nexpress Shroom3 and Rock1, Rock2 in early stages of UUO. Hence, for these reasons we used the UUO model\nas a TIF model in this work.\nR26\nShroom3 Rock1 Rock2\nRepairing\nInjured\nFailed repair\nAc Injured\nDays 2 4 6 10 14\nQR4.5. Please explain the reason that you adopt UUO model, not AAN model in Figure 6.\nReply: We appreciate this concern raised by the reviewer. We now performed UUO model (in addition to the\nprevious AAN model) with Pax8-WTSh3/ASD2Sh3 mice and found mitigation of fibrosis in the ASD2Sh3 mice\nsimilar to what was observed in the AAN model (data shown above in response to QR4.4). This indicates that\nreadouts from either of the TIF models could test the antifibrotic effect of ASD2 domain deletion, if present.\nFor Figure 6, in fibroblast-specific overexpression mice, UUO model did not reveal any difference in fibrosis\nprogression WTSh3 vs. ASD2Sh3. Hence, we did not proceed with a second model of fibrosis-AAN with the\nfibroblast-specific overexpression mice.\nQR4.6. Regarding Figure 1G and 1I, the authors concluded that tubular cells from ASD2delta-Sh3 mice had\nless ability to migrate and proliferate. And ASDdelta-Sh3 was protective against kidney injury. However, in\nprevious reports (J Clin Invest. 2019;129(12):5501–5517, Nat Rev Nephrol 16, 269–288 (2020)), recovery from\ninjury requires proliferative ability in tubular cells, which is not consistent with your result.\nReply: We thank the reviewer for this important and interesting point and agree that we observed that ASD-Sh3\noverexpression inhibited tubular cell proliferation. We believe this is likely due to the role of Rho-kinases in\nspindle formation and cytokinesis during cell division, and inhibiting activation of ROCKs via ASD2 may underlie\nthis observation.\nWe agree that this could potentially worsen recovery from acute kidney injury, and admit we did not evaluate\nacute injury or test recovery from AKI here. Our focus in this work was on the later TIF outcome after remodeling/\nprogressive injury. To test the role of ASD-Sh3 overexpression in acute injury, would require sequential time\ncourse experiments which we have not performed here.\nHowever, we do want to note that we continuously induced ASD2 mutant expression in UUO and AAN models,\nthroughout the acute and post-injury phases and still observed a benefit in TIF phenotype, suggesting that the\nacute benefit from Rock activation during recovery may ultimately not be of greater importance in TIF outcome.\nWe have discussed this in the last paragraph of Discussion listing the limitations of the study.\n“First, in contra-distinction to multiple reported adverse associations of Shroom3-SNPs with CKD, a potential\nprotective role for a linked intronic Shroom3-SNP in AKI in humans was reported recently49, and increased AKI\nalso occurred in heterozygous global Shroom3-knockout mice50. Hence, delineating the role of increased\nShroom3-Rock interaction during acute injury, recovery from AKI vs TIF/CKD will avoid any potential harm during\ntherapeutic use. Although we did not conduct such time course experiments here, we note that we continuously\ninduced ASD2Δ-Sh3 throughout the injury phase and still observed benefit in TIF.”\nQR4.7. In Figure 8d, please add the data on Vimentin, Col1a1, Fsp1, Mmp2 as shown in Figure 4.\nReply: We appreciate the reviewer’s suggestion and have included more genes (including Vim, Col1a1, Fsp1\nand Mmp2) to the mRNA expression profile (Revised Figure 8n) and improved the data with more animals (n=6\neach condition). This data is shown in Fig R27. This data have improved the rigor of our conclusions regarding\nthe benefit of D4G in TIF in vivo.\nR27\nQR4.8. To confirm that D4G is effective for fibrosis, the authors should perform an additional experiment with\nAAN model.\nReply: We thank the reviewer for the suggestion. While our invitro data shows clear efficacy with minimal toxicity\nof P2Is, we do acknowledge that a second in vivo model (AAN) would provide greater reassurance of the effect\nof P2Is on TIF in vivo.\nIn data included here, we now show that interfering with Shroom3-Rock interaction in states of global or tubular\nspecific Shroom3 excess using ASD2Δ-Sh3 mice was similarly protective for TIF from either AAN or UUO\nmodels. Our data shows clear impact on Rock signaling mechanisms, profibrotic TGFB1 or Wnt signals,\nproinflammatory genes, as well as preservation of genes of tubular homeostasis in ASD2Δ-Sh3 mice. Further,\nin the in vivo experiments with D4G, we have now expanded the sample size in our UUO experiment in the\nrevision, and included robust protein level data from this model which shows similar alterations in Rock signaling\npathway, profibrotic and proinflammatory genes, as well as preservation of tubular homeostasis mechanisms\nwith D4G (analogous to observations with ASD2Δ-Sh3) [Revised Figures 8/S8; and response to QR1.13]. These\ndata in the revision provide proof-of-concept for feasibility and efficacy of our novel Shroom3-Rock P2Is as\nantifibrotic therapy in conditions of Shroom3 excess.\nDespite the requirement of additional time and resources for a potential AAN experiment (beyond the 3-month\ntimeline provided for the revision), we can provide this if felt to be critical by the reviewer/editors for publication.\nWe have also acknowledged the lack of a second model as a limitation in the revision as follows.\n“Finally, although the beneficial role of inhibiting Shroom3-Rock interaction on TIF was not model-specific,\nand we demonstrated that D4G has in vivo efficacy as a P2I in a UUO model of TIF, evaluation of P2Is in other\nin vivo models including pharmacokinetics/pharmacodynamic profiles, human organoid testing and, longer term\ntoxicity studies, are all essential. All these studies are critical for translational potential and the ultimate\ndevelopment of refined Shroom3-Rock P2Is to treat CKD related to Shroom3-excess & Shroom3 SNPs.”\nQR4.9. Please clarify in which area of kidney and who evaluated fibrotic score.\nReply: We thank the reviewer for the query. We quantified fibrosis scores from the cortical area of the kidney.\nFor evaluating cortical fibrosis, slides were reviewed by Pathologist from Yale renal Pathology (S.P.) and digital\nimages were evaluated by G. B. using ImageJ (published Macro). The fibrosis scores estimated using ImageJ\nwere similar to the Pathologist’s scoring (both blinded).\n[Minor]\nQR4.10 Another research team has reported that a different SNP (rs142647267) was significantly associated\nwith a decline in eGFR in a study of the Shroom3 locus. Please discuss these other studies in detail.\nReply: We appreciate this observation by the reviewer. Indeed, multiple studies have identified intronic SNPS in\nShroom3 as associated with eGFR traits (eGFR-creatinine; eGFR-Cystatin C; prevalent CKD and recently AKI).\nThe SNP mentioned by the reviewer has been identified in a Japanese population study. We have now included\nsentences in the discussion to allude to these many studies and call out these SNP variants in the text.\n“For instance, intronic variants in the Shroom3 gene have been repeatedly identified as associated with CKD 51,\n52, 53.”\n“Independent work has also supported a regulatory role for intronic Shroom3 variants (rs17219731 ,\nrs142647267, rs4859682) in renal epithelial cells5, 54, and more specifically in proximal tubular cells52.”\n“..a potential protective role for a linked intronic Shroom3-SNP in AKI in humans was reported recently49,…”\nReply: We believe these multiple studies repeatedly support the relevance of Shroom3 protein in CKD and the\nrationale of our experimental work in this manuscript.\nQR4.11. There are many abbreviations in the text without initial spell-out, making it difficult to read, and this\nneeds to be resolved.\nReply: As per the reviewer’s suggestion, we revised the manuscript for uniformity in abbreviations in the\nrunning text.\nWe greatly appreciate the expert reviews and feedback by the reviewers and editors which have improved our\nmanuscript considerably.\nReviewer References:\n1. Dickson HM, Wilbur A, Reinke AA, Young MA, Vojtek AB. Targeted inhibition of the Shroom3-\nRho kinase protein-protein interaction circumvents Nogo66 to promote axon outgrowth. BMC\nNeurosci 16, 34 (2015).\n2. Kistler AD, Altintas MM, Reiser J. Podocyte GTPases regulate kidney filter dynamics. Kidney\nInt 81, 1053-1055 (2012).\n3. Wang L, et al. Mechanisms of the proteinuria induced by Rho GTPases. Kidney Int 81, 1075-\n1085 (2012).\n4. Blattner SM, et al. Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte\ninjury. Kidney Int 84, 920-930 (2013).\n5. Prokop JW, et al. Characterization of Coding/Noncoding Variants for SHROOM3 in Patients\nwith CKD. J Am Soc Nephrol, (2018).\n6. Faul C, et al. The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric\neffect of cyclosporine A. Nature medicine 14, 931-938 (2008).\n7. Asanuma K, Yanagida-Asanuma E, Faul C, Tomino Y, Kim K, Mundel P. Synaptopodin\norchestrates actin organization and cell motility via regulation of RhoA signalling. Nature cell\nbiology 8, 485-491 (2006).\n8. Scott RP, et al. Podocyte-specific loss of Cdc42 leads to congenital nephropathy. J Am Soc\nNephrol 23, 1149-1154 (2012).\n9. Matoba K, et al. ROCK2-induced metabolic rewiring in diabetic podocytopathy. Commun Biol\n5, 341 (2022).\n10. Matoba K, et al. Deletion of podocyte Rho-associated, coiled-coil-containing protein kinase 2\nprotects mice from focal segmental glomerulosclerosis. Commun Biol 7, 402 (2024).\n11. Zhou L, et al. Amelioration of albuminuria in ROCK1 knockout mice with streptozotocin-\ninduced diabetic kidney disease. American journal of nephrology 34, 468-475 (2011).\n12. Peng F, et al. RhoA/Rho-kinase contribute to the pathogenesis of diabetic renal disease.\nDiabetes 57, 1683-1692 (2008).\n13. Komers R. Rho kinase inhibition in diabetic kidney disease. Br J Clin Pharmacol 76, 551-559\n(2013).\n14. Gojo A, et al. The Rho-kinase inhibitor, fasudil, attenuates diabetic nephropathy in\nstreptozotocin-induced diabetic rats. Eur J Pharmacol 568, 242-247 (2007).\n15. Komers R, et al. Rho kinase inhibition protects kidneys from diabetic nephropathy without\nreducing blood pressure. Kidney Int 79, 432-442 (2011).\n16. Kolavennu V, Zeng L, Peng H, Wang Y, Danesh FR. Targeting of RhoA/ROCK signaling\nameliorates progression of diabetic nephropathy independent of glucose control. Diabetes 57,\n714-723 (2008).\n17. Wei C, et al. SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects\nAlbuminuria in Allografts. J Am Soc Nephrol 29, 2641-2657 (2018).\n18. Verma R, et al. Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin.\nThe Journal of biological chemistry 278, 20716-20723 (2003).\n19. Verma R, Kovari I, Soofi A, Nihalani D, Patrie K, Holzman LB. Nephrin ectodomain\nengagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and\nactin polymerization. The Journal of clinical investigation 116, 1346-1359 (2006).\n20. Audard V, et al. Occurrence of minimal change nephrotic syndrome in classical Hodgkin\nlymphoma is closely related to the induction of c-mip in Hodgkin-Reed Sternberg cells and\npodocytes. Blood 115, 3756-3762 (2010).\n21. Sun KH, Chang Y, Reed NI, Sheppard D. alpha-Smooth muscle actin is an inconsistent marker\nof fibroblasts responsible for force-dependent TGFbeta activation or collagen production\nacross multiple models of organ fibrosis. Am J Physiol Lung Cell Mol Physiol 310, L824-836\n(2016).\n22. Zhao W, Wang X, Sun KH, Zhou L. alpha-smooth muscle actin is not a marker of fibrogenic\ncell activity in skeletal muscle fibrosis. PLoS One 13, e0191031 (2018).\n23. Miyazono K, Heldin CH. Latent forms of TGF-beta: molecular structure and mechanisms of\nactivation. Ciba Found Symp 157, 81-89; discussion 89-92 (1991).\n24. Miyazono K, Olofsson A, Colosetti P, Heldin CH. A role of the latent TGF-beta 1-binding protein\nin the assembly and secretion of TGF-beta 1. EMBO J 10, 1091-1101 (1991).\n25. Sun Z, et al. Multiscale genetic architecture of donor-recipient differences reveals intronic\nLIMS1 mismatches associated with kidney transplant survival. The Journal of clinical\ninvestigation 133, (2023).\n26. Wong MG, Panchapakesan U, Qi W, Silva DG, Chen XM, Pollock CA. Cation-independent\nmannose 6-phosphate receptor inhibitor (PXS25) inhibits fibrosis in human proximal tubular\ncells by inhibiting conversion of latent to active TGF-beta1. Am J Physiol Renal Physiol 301,\nF84-93 (2011).\n27. Das BC, et al. Boron chemicals in diagnosis and therapeutics. Future Med Chem 5, 653-676\n(2013).\n28. Fernandes GFS, Denny WA, Dos Santos JL. Boron in drug design: Recent advances in the\ndevelopment of new therapeutic agents. Eur J Med Chem 179, 791-804 (2019).\n29. Zhong F, et al. Tyro3 is a podocyte protective factor in glomerular disease. JCI Insight 3,\n(2018).\n30. Lovering F, Bikker J, Humblet C. Escape from flatland: increasing saturation as an approach to\nimproving clinical success. J Med Chem 52, 6752-6756 (2009).\n31. Maurer TS, Edwards M, Hepworth D, Verhoest P, Allerton CMN. Designing small molecules for\ntherapeutic success: A contemporary perspective. Drug Discov Today 27, 538-546 (2022).\n32. Yanuaryska RD, et al. Sp6 regulation of Rock1 promoter activity in dental epithelial cells. J\nMed Invest 61, 306-317 (2014).\n33. Elliott EG, Ettinger AS, Leaderer BP, Bracken MB, Deziel NC. A systematic evaluation of\nchemicals in hydraulic-fracturing fluids and wastewater for reproductive and developmental\ntoxicity. J Expo Sci Environ Epidemiol 27, 90-99 (2017).\n34. Zanotto E, Shah ZH, Jacobs HT. The bidirectional promoter of two genes for the mitochondrial\ntranslational apparatus in mouse is regulated by an array of CCAAT boxes interacting with the\ntranscription factor NF-Y. Nucleic Acids Res 35, 664-677 (2007).\n35. Wang IC, et al. Forkhead box M1 regulates the transcriptional network of genes essential for\nmitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase. Mol Cell Biol\n25, 10875-10894 (2005).\n36. Farooqi AA, et al. Regulation of ROCK1/2 by long non-coding RNAs and circular RNAs in\ndifferent cancer types. Oncol Lett 23, 159 (2022).\n37. Menon MC, et al. Intronic locus determines SHROOM3 expression and potentiates renal\nallograft fibrosis. The Journal of clinical investigation, (2014).\n38. Durbin MD, O'Kane J, Lorentz S, Firulli AB, Ware SM. SHROOM3 is downstream of the planar\ncell polarity pathway and loss-of-function results in congenital heart defects. Dev Biol 464, 124-\n136 (2020).\n39. Yoon J, Sun J, Lee M, Hwang YS, Daar IO. Wnt4 and ephrinB2 instruct apical constriction via\nDishevelled and non-canonical signaling. Nat Commun 14, 337 (2023).\n40. Li A, et al. Shroom3, a Gene Associated with CKD, Modulates Epithelial Recovery after AKI.\nKidney360 3, 51-62 (2022).\n41. Ji H, et al. Rho/Rock cross-talks with transforming growth factor-beta/Smad pathway\nparticipates in lung fibroblast-myofibroblast differentiation. Biomed Rep 2, 787-792 (2014).\n42. Igarashi N, Honjo M, Aihara M. mTOR inhibitors potentially reduce TGF-beta2-induced\nfibrogenic changes in trabecular meshwork cells. Sci Rep 11, 14111 (2021).\n43. Papadimitriou E, Kardassis D, Moustakas A, Stournaras C. TGFbeta-induced early activation\nof the small GTPase RhoA is Smad2/3-independent and involves Src and the guanine\nnucleotide exchange factor Vav2. Cell Physiol Biochem 28, 229-238 (2011).\n44. Wei YH, Liao SL, Wang SH, Wang CC, Yang CH. Simvastatin and ROCK Inhibitor Y-27632\nInhibit Myofibroblast Differentiation of Graves' Ophthalmopathy-Derived Orbital Fibroblasts via\nRhoA-Mediated ERK and p38 Signaling Pathways. Front Endocrinol (Lausanne) 11, 607968\n(2020).\n45. Xu L, Guo J, Moledina DG, Cantley LG. Immune-mediated tubule atrophy promotes acute\nkidney injury to chronic kidney disease transition. Nat Commun 13, 4892 (2022).\n46. Huang H, et al. Rho-kinase regulates energy balance by targeting hypothalamic leptin receptor\nsignaling. Nat Neurosci 15, 1391-1398 (2012).\n47. Okamoto R, et al. FHL2 prevents cardiac hypertrophy in mice with cardiac-specific deletion of\nROCK2. FASEB J 27, 1439-1449 (2013).\n48. Zarkada G, et al. Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption.\nCirc Res 133, 333-349 (2023).\n49. Siew ED, et al. Genome-wide association study of hospitalized patients and acute kidney\ninjury. Kidney Int 106, 291-301 (2024).\n50. Lawlor A, et al. Minimal Kidney Disease Phenotype in Shroom3 Heterozygous Null Mice. Can J\nKidney Health Dis 10, 20543581231165716 (2023).\n51. Kottgen A, et al. New loci associated with kidney function and chronic kidney disease. Nature\ngenetics 42, 376-384 (2010).\n52. Loeb GB, et al. Variants in tubule epithelial regulatory elements mediate most heritable\ndifferences in human kidney function. Nature genetics, (2024).\n53. Ghasemi-Semeskandeh D, et al. Clinical and Metabolic Signatures of FAM47E-SHROOM3\nHaplotypes in a General Population Sample. Kidney Int Rep 10, 1495-1508 (2025).\n54. Matsuura R, et al. SHROOM3, the gene associated with chronic kidney disease, affects the\npodocyte structure. Sci Rep 10, 21103 (2020).\nTo our Editor and Reviewers,\nWe would like to thank you for the thorough evaluation of our manuscript and providing us with constructive\nfeedback. We greatly appreciate the opportunity to revise and resubmit our work.\nAll comments from the reviewer have been addressed in the accompanying point-by-point response document.\nRevisions to the manuscript have been clearly highlighted as per journal guidelines.\nWe have ensured full compliance with all editorial policies and requirements, including:\n• Submission of the Editorial Policy Checklist and attached to the Reporting Summary (Similar to first\nrevision).\n• Updated Data Availability section with deposition of datasets in appropriate public repositories (Similar to\nfirst revision).\n• Providing a comprehensive Source Data files for all main figures as ppt and tables in Excel format (Similar\nto first revision).\n• Edited data in the Figure panels and Supplementary information as suggested by the reviewer #4.\n• Consideration and appropriate reporting of sex and gender in line with SAGER guidelines (Similar to first\nrevision).\nWe enclose in the revised submission (i) a marked copy (yellow highlights for changes in revision), (ii) a clean\ncopy of the manuscript file, (iii) Supplementary information: Supplementary figures/legends (Marked and\nUnmarked copies); Reporting summary and Editorial Policy Checklist and (iv) Supplementary datasets 1-4.\nWe confirm that there have been no changes to the author's list.\nWe sincerely hope that the revised version addresses all the concerns raised and meets the expectations of the\nreviewer and editorial board.\nPoint by point response to all reviewers’ comments are listed below:\nReviewer #1 (Remarks to the Author):\nAll my comments have been addressed and the manuscript is suitable for publication\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early\nCareer Researchers who co-review manuscripts.\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #3 (Remarks to the Author):\nThe authors have answered all of my comments. It is Ok for me to accept it now.\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #4 (Remarks to the Author):\nThank you for your special efforts to revise this manuscript. I believe it has improved considerably. However, to\npreserve the robustness of the work in Nat Commun, the following points still require attention; I would appreciate\nyour continued consideration.\nWe appreciate the reviewer’s comments and detailed suggestions which have helped us improve our manuscript,\nand appreciate the favorable review of our work.\n1. Contradiction in expression patterns in Figure 1A/S1a\n→ From the dot plots, it appears that at most ~10% of cells within the proximal tubule and myofibroblast/fibroblast\npopulations express the genes in question, and likely at quite low levels. If violin plots are shown, it will probably\nbecome clear that there is essentially no expression. It is not reasonable to discuss genes that are virtually not\nexpressed in the single-cell data, and this likely contributes to the apparent discrepancy between Figure 1A and\nS1a. Even without the single-cell data the manuscript is sufficiently interesting, so it would be better to remove\nthese data.\n-We believe the reviewer is referring to Figures 1a, S1a and S1b (all single cell or single nuclear data related\nfigures), and appreciate the reviewer pointing this issue of Shroom3, Rock1 and Rock2 expression levels.\nOverall, we appreciate this valuable suggestion from the reviewer.\n-To respond to this question raised by the reviewer we generated violin plots from our co-author’s publication 1.\nAs shown below, iPTs during the fibrosis stage show increased expression of Shroom3, Rock1 and Rock2,\nsupporting mechanistic experiments in tubular cell lines. Myofibroblast cluster (which includes myofibroblasts\nand fibroblasts together) robustly express Rock1 and Rock2, but inconsistently express Shroom3 in this IRI\ndataset. As observed in the Li et al dataset, myofibroblasts do have lower expression of Shroom3.\n-We also evaluated recent independent data from a multispecies-integrated Single-Cell Kidney Atlas, by\nKloetzer, Susztak et al 2, which evaluated ScRNA data from healthy and diseased murine kidneys from multiple\npublications (total cells after QC =447499 (Healthy =64948 ,diseased =382551) [located at\nhttps://susztaklab.com/SISKA/mouse]. Here the proportion of iPT cells expressing Shroom3 ranged from 33 to\n45%, and in stromal cells (which includes fibroblasts/ myofibroblasts) Shroom3 expression ranged from 52 to\n53%. These corresponding data for Rock1 were 33-36% in iPTs and 31-32% in stromal cells, and for Rock2\nwere 41-50% in iPTS and 47-56% in stromal cells, respectively. These again reflect variability in cell-type specific\nexpression of these genes in single cell transcriptomic data across different publications, but support their\nexpression in iPTs and stromal cells. We have now included this recent reference in the revision.\nHence, these data and others suggest that there is sufficient expression of these genes in PTs and fibroblast\ncells providing rationale for our sequential mechanistic experiments, which then allowed us to infer the central\nrole of Shroom3-Rock interaction in iPTs (vs fibroblasts) during TIF. As suggested by the reviewer, since these\nderivative data from prior publications are not essential for the manuscript, and to avoid inconsistency between\nfigures, we have now removed these panels from the revised manuscript. We have redrafted the paragraph\nreferring to these data as follows.\n“We previously reported that tubular-specific Shroom3 knockdown alone could mitigate TIF in a UUO\nmodel 8. Recent functional genomics studies evaluating CKD-associated-SNPs using single cell-ATACseq also\nconcluded that single-cell, cis-eqtl effects of Shroom3 intronic variants are identifiable in proximal tubular cells\n(PTs) in human CKD24. These pointed to PTs as key players for TIF from excess Shroom3. We also considered\nthat fibroblasts which are key cells for matrix production during TIF, express Shroom3 and show high Rock1,\nRock2 expression in published data from murine TIF models25, 26, 27, 28. Podocytes show high expression of\nShroom3 at rest and injury but are not expected to play key roles in these TIF models after PT cell injury25, 26, 27.\nHence, we focused on tubular and fibroblast cell lines which were potential sites of Shroom3 interaction with\nRock1 and/or Rock2 during TIF for subsequent mechanistic experiments.”\n2. Insufficient explanation for the contradiction that ASD2Δ-Sh3 suppresses proliferation yet confers\nrenoprotection\n→ Prior reports (e.g., JCI 2019) indicate that tubular cell proliferation is required for renal recovery, but this\napparent contradiction has not yet been addressed.\nWe appreciate this point raised by the reviewer. We have now added a paragraph in the discussion to detail\npotential explanations behind the dichotomous association seen in epidemiologic studies of enhancer Shroom3\nSNPs (ie with more Shroom3-Rock interaction) with improved AKI but consistently increased CKD risk. We\nbelieve that Rock-activation mediated Ccl2-excess from iPTs sets up cross-talk to with immune cells (via Ccr2)\nwhich in turn activate resident stromal cells setting up “fibro-inflammation” and CKD progression. Ccl2 was\nconsistently observed to be inhibited significantly during genetic or pharmacologic inhibition of ASD2-Rock\ninteraction in vitro and in vivo in our data. This is also suggested from the multiple data showing consistent\nbenefit of Rock-inhibitors in AKI 3, 4. We have also accepted as limitation that we have not currently done time-\ncourse studies where we induced Shroom3-Rock interaction inhibition (genetically or pharmacologically) during\nAKI, during AKI recovery or during TIF stage alone.\nTo summarize this in the manuscript, we have added the following to the discussion.\n“It is also important to discuss implications of our work for acute kidney injury (AKI). We note that in contra-\ndistinction to multiple reported adverse associations of Shroom3-SNPs with CKD, a potential protective role for\na linked intronic Shroom3-SNP in AKI in humans was reported recently65, and increased AKI also occurred in\nheterozygous global Shroom3-knockout mice31. Facilitation of Rho-kinases by SHROOM3 could aid in spindle\nformation and cytokinesis during cell division, and inhibiting activation of ROCKs via ASD2 could reduce PT\nproliferation after injury 66, 67, consistent with our observations in vitro in IMCD lines. Since cell proliferation is\nrequired for tubular repair 68, delineating the role of increased Shroom3-Rock interaction during AKI, recovery\nfrom AKI vs TIF/CKD is needed to avoid any potential harm during therapeutic use of P2Is. We acknowledge\nthat we did not conduct such time course experiments. However, we note that ASD2Δ-Sh3 was continuously\nexpressed in our models throughout the AKI phase and we still observed benefit in TIF. Next, in spite of this\npotential adverse effect of inhibiting ROCKs during AKI, Rock-inhibitors have consistently shown benefit in\nmodels of AKI66, 69. A possible explanation is the observation that inhibition of Shroom3-Rock interaction in tubular\ncells alone markedly inhibited Ccl2, a chemokine signal known to be facilitated by rho-kinase activation 32, 70, 71.\nCcl2 mediates immune cell recruitment via its receptor Ccr2 and is a potent facilitator of tubular-inflammatory\ncell crosstalk after injury28. Hence, in spite of potential deleterious effects on tubular cell proliferation during AKI,\nexcess Shroom3-Rock interaction in iPTs overexpressing SHROOM3 would enhance crosstalk between iPTs\nand interstitial - inflammatory and/or stromal -cells, culminating in increased “fibro-inflammation” and progressive\nCKD.”\n3. Mismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ Although the rebuttal compared multiple datasets to explain this, the main text does not explicitly state it.\nPlease incorporate this discussion into the Discussion section.\nWe apologize for not including these lines from the rebuttal statement in the manuscript. We have now included\nthe following in the limitations section of the discussion as follows to address the Reviewers initial concern.\n“From the multiple single cell expression datasets 24, 25, 27 26, it is apparent to us that cell-type specific expression\npatterns of each Rock paralog and Shroom3 are variable and sometimes mismatched between datasets. This\nvariability may have resulted from technical variability between datasets (single cell vs single nuclear, etc). To\nminimize the impact of this in our work, we mined multiple datasets which included multiple injury models and\nmodalities of single cell transcriptome assessment to provide cell-type rationale for our mechanistic studies.”\n4. Other\n→ Because numerous new references were added in the revision, many citation numbers have changed.\nPlease carefully check that numbering and cross-references are consistent.\nWe apologize for this oversight. We cross-checked the references and ran the reference library for the Response\ndocument separate from the main manuscript document.\n1. Xu L, Guo J, Moledina DG, Cantley LG. Immune-mediated tubule atrophy promotes acute\nkidney injury to chronic kidney disease transition. Nat Commun 13, 4892 (2022).\n2. Klotzer KA, et al. Analysis of individual patient pathway coordination in a cross-species single-\ncell kidney atlas. Nature genetics 57, 1922-1934 (2025).\n3. Prakash J, et al. Inhibition of renal rho kinase attenuates ischemia/reperfusion-induced injury. J\nAm Soc Nephrol 19, 2086-2097 (2008).\n4. Liang H, et al. CXCL16/ROCK1 signaling pathway exacerbates acute kidney injury induced by\nischemia-reperfusion. Biomed Pharmacother 98, 347-356 (2018).\nREVIEWERS' COMMENTS\nWe appreciate the reviewers detailed and favorable responses.\nReviewer #4 (Remarks to the Author):\nWith regard to the reviewer’s concerns, the authors have addressed the issues as\nfollows. As a result, the manuscript has become logically coherent, more readily\ncomprehensible, and in my view has been further polished.\nContradiction in expression patterns in Figure 1A/S1a\n→ The authors re-examined the data using violin plots, removed non-essential data in\naccordance with the reviewer’s recommendation, and revised the relevant paragraph in\nthe text.\nContradiction that ASD2Δ-Sh3 suppresses proliferation yet confers renoprotection\n→ A new paragraph has been added to the Discussion, elaborating on the apparent\ndiscrepancy within the context of AKI versus CKD, with additional explanation involving\nCcl2/ROCK signaling.\nMismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ The variability across datasets has now been explicitly acknowledged and\nincorporated into the Limitations section.\nInconsistencies in reference numbering and cross-references\n→ The authors have stated that they have carefully reviewed and corrected these\ninconsistencies.\n-We appreciate the positive evaluation of our revisions by the reviewer.\nMinor points:\n• As noted, it would be appropriate to spell out iPTs (injured proximal tubules) at its first\nappearance in the main text. In fact, the rebuttal document already provides this\nclarification as “iPTs (injured proximal tubular cells).”\nReply: We thank the reviewer for the correction. We added the expansion for iPTs at the\nfirst occurrence.\n• FBDM-Sh3 (Fyn-binding domain mutant Shroom3): This abbreviation should be\ndefined at its first occurrence.\nReply: We thank the reviewer for pointing out this. We have now expanded the\nabbreviation at the first occurrence.\n• P2Is (Shroom3-Rock interaction inhibitors): Although the definition is provided in the\ntext, the introduction of this abbreviation is somewhat abrupt. It would be clearer and\nmore reader-friendly to introduce it more explicitly at first mention, for example: “we\ndeveloped novel small molecule inhibitors of Shroom3-Rock interaction (P2Is).”\nReply: We appreciate the reviewer’s suggestion and have incorporated it.\n• Several references appear to be incomplete. In particular, references 7, 18, 24,\n55, and 79 seem to be missing page numbers.\nWe appreciate these issues that were pointed out. We have corrected and highlighted in\nmarked copy.\nReference 7 & 11 – we have included page numbers for these references\nReference 18 and 11 were duplicated. Hence in the revision Ref 18 has been deleted,\nand all references below 18 have been pushed up by 1.\nReferences 24, 55, 79 have now become 23, 54 and 78, respectively. We have added in\npage numbers obtained from pubmed and the journal websites.\n• Additional inconsistencies may also exist, for example in references 25–27 (murine\nTIF models) and 65–67 (recently added AKI-related studies). These should be checked\ncarefully to ensure that full bibliographic details, including page ranges, are provided.\nWe checked these references carefully. The endnote program picked these PMIDs from\npubmed and cited these in Nature communications output format. The only potentially\nmissing detail to be added in to the manuscript are the issue numbers – which we have\nnow added (marked copy). These may not be automatically included in Nature\ncommunications but regardless we have now added these references."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All my comments have been addressed and the manuscript is suitable for publication", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have answered all of my comments. It is Ok for me to accept it now.", "reviewer_4": "Thank you for your special efforts to revise this manuscript. I believe it has improved considerably. However, to preserve the robustness of the work in Nat Commun, the following points still require attention; I would appreciate your continued consideration.\n1. Contradiction in expression patterns in Figure 1A/S1a\n→ From the dot plots, it appears that at most ~10% of cells within the proximal tubule and myofibroblast/fibroblast populations express the genes in question, and likely at quite low levels. If violin plots are shown, it will probably become clear that there is essentially no expression. It is not reasonable to discuss genes that are virtually not expressed in the single-cell data, and this likely contributes to the apparent discrepancy between Figure 1A and S1a. Even without the single-cell data the manuscript is sufficiently interesting, so it would be better to remove these data.\n2. Insufficient explanation for the contradiction that ASD2Δ-Sh3 suppresses proliferation yet confers renoprotection\n→ Prior reports (e.g., JCI 2019) indicate that tubular cell proliferation is required for renal recovery, but this apparent contradiction has not yet been addressed.\n3. Mismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ Although the rebuttal compared multiple datasets to explain this, the main text does not explicitly state it. Please incorporate this discussion into the Discussion section.\n4. Other\n→ Because numerous new references were added in the revision, many citation numbers have changed. Please carefully check that numbering and cross-references are consistent."}, "author_response": "To our Editor and Reviewers,\nWe would like to thank you for the thorough evaluation of our manuscript and providing us with constructive\nfeedback. We greatly appreciate the opportunity to revise and resubmit our work.\nAll comments from the reviewer have been addressed in the accompanying point-by-point response document.\nRevisions to the manuscript have been clearly highlighted as per journal guidelines.\nWe have ensured full compliance with all editorial policies and requirements, including:\n• Submission of the Editorial Policy Checklist and attached to the Reporting Summary (Similar to first\nrevision).\n• Updated Data Availability section with deposition of datasets in appropriate public repositories (Similar to\nfirst revision).\n• Providing a comprehensive Source Data files for all main figures as ppt and tables in Excel format (Similar\nto first revision).\n• Edited data in the Figure panels and Supplementary information as suggested by the reviewer #4.\n• Consideration and appropriate reporting of sex and gender in line with SAGER guidelines (Similar to first\nrevision).\nWe enclose in the revised submission (i) a marked copy (yellow highlights for changes in revision), (ii) a clean\ncopy of the manuscript file, (iii) Supplementary information: Supplementary figures/legends (Marked and\nUnmarked copies); Reporting summary and Editorial Policy Checklist and (iv) Supplementary datasets 1-4.\nWe confirm that there have been no changes to the author's list.\nWe sincerely hope that the revised version addresses all the concerns raised and meets the expectations of the\nreviewer and editorial board.\nPoint by point response to all reviewers’ comments are listed below:\nReviewer #1 (Remarks to the Author):\nAll my comments have been addressed and the manuscript is suitable for publication\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early\nCareer Researchers who co-review manuscripts.\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #3 (Remarks to the Author):\nThe authors have answered all of my comments. It is Ok for me to accept it now.\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #4 (Remarks to the Author):\nThank you for your special efforts to revise this manuscript. I believe it has improved considerably. However, to\npreserve the robustness of the work in Nat Commun, the following points still require attention; I would appreciate\nyour continued consideration.\nWe appreciate the reviewer’s comments and detailed suggestions which have helped us improve our manuscript,\nand appreciate the favorable review of our work.\n1. Contradiction in expression patterns in Figure 1A/S1a\n→ From the dot plots, it appears that at most ~10% of cells within the proximal tubule and myofibroblast/fibroblast\npopulations express the genes in question, and likely at quite low levels. If violin plots are shown, it will probably\nbecome clear that there is essentially no expression. It is not reasonable to discuss genes that are virtually not\nexpressed in the single-cell data, and this likely contributes to the apparent discrepancy between Figure 1A and\nS1a. Even without the single-cell data the manuscript is sufficiently interesting, so it would be better to remove\nthese data.\n-We believe the reviewer is referring to Figures 1a, S1a and S1b (all single cell or single nuclear data related\nfigures), and appreciate the reviewer pointing this issue of Shroom3, Rock1 and Rock2 expression levels.\nOverall, we appreciate this valuable suggestion from the reviewer.\n-To respond to this question raised by the reviewer we generated violin plots from our co-author’s publication 1.\nAs shown below, iPTs during the fibrosis stage show increased expression of Shroom3, Rock1 and Rock2,\nsupporting mechanistic experiments in tubular cell lines. Myofibroblast cluster (which includes myofibroblasts\nand fibroblasts together) robustly express Rock1 and Rock2, but inconsistently express Shroom3 in this IRI\ndataset. As observed in the Li et al dataset, myofibroblasts do have lower expression of Shroom3.\n-We also evaluated recent independent data from a multispecies-integrated Single-Cell Kidney Atlas, by\nKloetzer, Susztak et al 2, which evaluated ScRNA data from healthy and diseased murine kidneys from multiple\npublications (total cells after QC =447499 (Healthy =64948 ,diseased =382551) [located at\nhttps://susztaklab.com/SISKA/mouse]. Here the proportion of iPT cells expressing Shroom3 ranged from 33 to\n45%, and in stromal cells (which includes fibroblasts/ myofibroblasts) Shroom3 expression ranged from 52 to\n53%. These corresponding data for Rock1 were 33-36% in iPTs and 31-32% in stromal cells, and for Rock2\nwere 41-50% in iPTS and 47-56% in stromal cells, respectively. These again reflect variability in cell-type specific\nexpression of these genes in single cell transcriptomic data across different publications, but support their\nexpression in iPTs and stromal cells. We have now included this recent reference in the revision.\nHence, these data and others suggest that there is sufficient expression of these genes in PTs and fibroblast\ncells providing rationale for our sequential mechanistic experiments, which then allowed us to infer the central\nrole of Shroom3-Rock interaction in iPTs (vs fibroblasts) during TIF. As suggested by the reviewer, since these\nderivative data from prior publications are not essential for the manuscript, and to avoid inconsistency between\nfigures, we have now removed these panels from the revised manuscript. We have redrafted the paragraph\nreferring to these data as follows.\n“We previously reported that tubular-specific Shroom3 knockdown alone could mitigate TIF in a UUO\nmodel 8. Recent functional genomics studies evaluating CKD-associated-SNPs using single cell-ATACseq also\nconcluded that single-cell, cis-eqtl effects of Shroom3 intronic variants are identifiable in proximal tubular cells\n(PTs) in human CKD24. These pointed to PTs as key players for TIF from excess Shroom3. We also considered\nthat fibroblasts which are key cells for matrix production during TIF, express Shroom3 and show high Rock1,\nRock2 expression in published data from murine TIF models25, 26, 27, 28. Podocytes show high expression of\nShroom3 at rest and injury but are not expected to play key roles in these TIF models after PT cell injury25, 26, 27.\nHence, we focused on tubular and fibroblast cell lines which were potential sites of Shroom3 interaction with\nRock1 and/or Rock2 during TIF for subsequent mechanistic experiments.”\n2. Insufficient explanation for the contradiction that ASD2Δ-Sh3 suppresses proliferation yet confers\nrenoprotection\n→ Prior reports (e.g., JCI 2019) indicate that tubular cell proliferation is required for renal recovery, but this\napparent contradiction has not yet been addressed.\nWe appreciate this point raised by the reviewer. We have now added a paragraph in the discussion to detail\npotential explanations behind the dichotomous association seen in epidemiologic studies of enhancer Shroom3\nSNPs (ie with more Shroom3-Rock interaction) with improved AKI but consistently increased CKD risk. We\nbelieve that Rock-activation mediated Ccl2-excess from iPTs sets up cross-talk to with immune cells (via Ccr2)\nwhich in turn activate resident stromal cells setting up “fibro-inflammation” and CKD progression. Ccl2 was\nconsistently observed to be inhibited significantly during genetic or pharmacologic inhibition of ASD2-Rock\ninteraction in vitro and in vivo in our data. This is also suggested from the multiple data showing consistent\nbenefit of Rock-inhibitors in AKI 3, 4. We have also accepted as limitation that we have not currently done time-\ncourse studies where we induced Shroom3-Rock interaction inhibition (genetically or pharmacologically) during\nAKI, during AKI recovery or during TIF stage alone.\nTo summarize this in the manuscript, we have added the following to the discussion.\n“It is also important to discuss implications of our work for acute kidney injury (AKI). We note that in contra-\ndistinction to multiple reported adverse associations of Shroom3-SNPs with CKD, a potential protective role for\na linked intronic Shroom3-SNP in AKI in humans was reported recently65, and increased AKI also occurred in\nheterozygous global Shroom3-knockout mice31. Facilitation of Rho-kinases by SHROOM3 could aid in spindle\nformation and cytokinesis during cell division, and inhibiting activation of ROCKs via ASD2 could reduce PT\nproliferation after injury 66, 67, consistent with our observations in vitro in IMCD lines. Since cell proliferation is\nrequired for tubular repair 68, delineating the role of increased Shroom3-Rock interaction during AKI, recovery\nfrom AKI vs TIF/CKD is needed to avoid any potential harm during therapeutic use of P2Is. We acknowledge\nthat we did not conduct such time course experiments. However, we note that ASD2Δ-Sh3 was continuously\nexpressed in our models throughout the AKI phase and we still observed benefit in TIF. Next, in spite of this\npotential adverse effect of inhibiting ROCKs during AKI, Rock-inhibitors have consistently shown benefit in\nmodels of AKI66, 69. A possible explanation is the observation that inhibition of Shroom3-Rock interaction in tubular\ncells alone markedly inhibited Ccl2, a chemokine signal known to be facilitated by rho-kinase activation 32, 70, 71.\nCcl2 mediates immune cell recruitment via its receptor Ccr2 and is a potent facilitator of tubular-inflammatory\ncell crosstalk after injury28. Hence, in spite of potential deleterious effects on tubular cell proliferation during AKI,\nexcess Shroom3-Rock interaction in iPTs overexpressing SHROOM3 would enhance crosstalk between iPTs\nand interstitial - inflammatory and/or stromal -cells, culminating in increased “fibro-inflammation” and progressive\nCKD.”\n3. Mismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ Although the rebuttal compared multiple datasets to explain this, the main text does not explicitly state it.\nPlease incorporate this discussion into the Discussion section.\nWe apologize for not including these lines from the rebuttal statement in the manuscript. We have now included\nthe following in the limitations section of the discussion as follows to address the Reviewers initial concern.\n“From the multiple single cell expression datasets 24, 25, 27 26, it is apparent to us that cell-type specific expression\npatterns of each Rock paralog and Shroom3 are variable and sometimes mismatched between datasets. This\nvariability may have resulted from technical variability between datasets (single cell vs single nuclear, etc). To\nminimize the impact of this in our work, we mined multiple datasets which included multiple injury models and\nmodalities of single cell transcriptome assessment to provide cell-type rationale for our mechanistic studies.”\n4. Other\n→ Because numerous new references were added in the revision, many citation numbers have changed.\nPlease carefully check that numbering and cross-references are consistent.\nWe apologize for this oversight. We cross-checked the references and ran the reference library for the Response\ndocument separate from the main manuscript document.\n1. Xu L, Guo J, Moledina DG, Cantley LG. Immune-mediated tubule atrophy promotes acute\nkidney injury to chronic kidney disease transition. Nat Commun 13, 4892 (2022).\n2. Klotzer KA, et al. Analysis of individual patient pathway coordination in a cross-species single-\ncell kidney atlas. Nature genetics 57, 1922-1934 (2025).\n3. Prakash J, et al. Inhibition of renal rho kinase attenuates ischemia/reperfusion-induced injury. J\nAm Soc Nephrol 19, 2086-2097 (2008).\n4. Liang H, et al. CXCL16/ROCK1 signaling pathway exacerbates acute kidney injury induced by\nischemia-reperfusion. Biomed Pharmacother 98, 347-356 (2018).\nREVIEWERS' COMMENTS\nWe appreciate the reviewers detailed and favorable responses.\nReviewer #4 (Remarks to the Author):\nWith regard to the reviewer’s concerns, the authors have addressed the issues as\nfollows. As a result, the manuscript has become logically coherent, more readily\ncomprehensible, and in my view has been further polished.\nContradiction in expression patterns in Figure 1A/S1a\n→ The authors re-examined the data using violin plots, removed non-essential data in\naccordance with the reviewer’s recommendation, and revised the relevant paragraph in\nthe text.\nContradiction that ASD2Δ-Sh3 suppresses proliferation yet confers renoprotection\n→ A new paragraph has been added to the Discussion, elaborating on the apparent\ndiscrepancy within the context of AKI versus CKD, with additional explanation involving\nCcl2/ROCK signaling.\nMismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ The variability across datasets has now been explicitly acknowledged and\nincorporated into the Limitations section.\nInconsistencies in reference numbering and cross-references\n→ The authors have stated that they have carefully reviewed and corrected these\ninconsistencies.\n-We appreciate the positive evaluation of our revisions by the reviewer.\nMinor points:\n• As noted, it would be appropriate to spell out iPTs (injured proximal tubules) at its first\nappearance in the main text. In fact, the rebuttal document already provides this\nclarification as “iPTs (injured proximal tubular cells).”\nReply: We thank the reviewer for the correction. We added the expansion for iPTs at the\nfirst occurrence.\n• FBDM-Sh3 (Fyn-binding domain mutant Shroom3): This abbreviation should be\ndefined at its first occurrence.\nReply: We thank the reviewer for pointing out this. We have now expanded the\nabbreviation at the first occurrence.\n• P2Is (Shroom3-Rock interaction inhibitors): Although the definition is provided in the\ntext, the introduction of this abbreviation is somewhat abrupt. It would be clearer and\nmore reader-friendly to introduce it more explicitly at first mention, for example: “we\ndeveloped novel small molecule inhibitors of Shroom3-Rock interaction (P2Is).”\nReply: We appreciate the reviewer’s suggestion and have incorporated it.\n• Several references appear to be incomplete. In particular, references 7, 18, 24,\n55, and 79 seem to be missing page numbers.\nWe appreciate these issues that were pointed out. We have corrected and highlighted in\nmarked copy.\nReference 7 & 11 – we have included page numbers for these references\nReference 18 and 11 were duplicated. Hence in the revision Ref 18 has been deleted,\nand all references below 18 have been pushed up by 1.\nReferences 24, 55, 79 have now become 23, 54 and 78, respectively. We have added in\npage numbers obtained from pubmed and the journal websites.\n• Additional inconsistencies may also exist, for example in references 25–27 (murine\nTIF models) and 65–67 (recently added AKI-related studies). These should be checked\ncarefully to ensure that full bibliographic details, including page ranges, are provided.\nWe checked these references carefully. The endnote program picked these PMIDs from\npubmed and cited these in Nature communications output format. The only potentially\nmissing detail to be added in to the manuscript are the issue numbers – which we have\nnow added (marked copy). These may not be automatically included in Nature\ncommunications but regardless we have now added these references."}, {"round": 3, "reviewer_comments": {"reviewer_4": "With regard to the reviewer’s concerns, the authors have addressed the issues as follows. As a result, the manuscript has become logically coherent, more readily comprehensible, and in my view has been further polished.\nContradiction in expression patterns in Figure 1A/S1a\n→ The authors re-examined the data using violin plots, removed non-essential data in accordance with the reviewer’s recommendation, and revised the relevant paragraph in the text.\nContradiction that ASD2Δ-Sh3 suppresses proliferation yet confers renoprotection\n→ A new paragraph has been added to the Discussion, elaborating on the apparent discrepancy within the context of AKI versus CKD, with additional explanation involving Ccl2/ROCK signaling.\nMismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ The variability across datasets has now been explicitly acknowledged and incorporated into the Limitations section.\nInconsistencies in reference numbering and cross-references\n→ The authors have stated that they have carefully reviewed and corrected these inconsistencies.\nMinor points:\n• As noted, it would be appropriate to spell out iPTs (injured proximal tubules) at its first appearance in the main text. In fact, the rebuttal document already provides this clarification as “iPTs (injured proximal tubular cells).”\n• FBDM-Sh3 (Fyn-binding domain mutant Shroom3): This abbreviation should be defined at its first occurrence.\n• P2Is (Shroom3-Rock interaction inhibitors): Although the definition is provided in the text, the introduction of this abbreviation is somewhat abrupt. It would be clearer and more reader-friendly to introduce it more explicitly at first mention, for example: “we developed novel small molecule inhibitors of Shroom3-Rock interaction (P2Is).”\n• Several references appear to be incomplete. In particular, references 7, 18, 24, 55, and 79 seem to be missing page numbers.\n• Additional inconsistencies may also exist, for example in references 25–27 (murine TIF models) and 65–67 (recently added AKI-related studies). These should be checked carefully to ensure that full bibliographic details, including page ranges, are provided."}, "author_response": "To our Editor and Reviewers,\nWe would like to thank you for the thorough evaluation of our manuscript and providing us with constructive\nfeedback. We greatly appreciate the opportunity to revise and resubmit our work.\nAll comments from the reviewer have been addressed in the accompanying point-by-point response document.\nRevisions to the manuscript have been clearly highlighted as per journal guidelines.\nWe have ensured full compliance with all editorial policies and requirements, including:\n• Submission of the Editorial Policy Checklist and attached to the Reporting Summary (Similar to first\nrevision).\n• Updated Data Availability section with deposition of datasets in appropriate public repositories (Similar to\nfirst revision).\n• Providing a comprehensive Source Data files for all main figures as ppt and tables in Excel format (Similar\nto first revision).\n• Edited data in the Figure panels and Supplementary information as suggested by the reviewer #4.\n• Consideration and appropriate reporting of sex and gender in line with SAGER guidelines (Similar to first\nrevision).\nWe enclose in the revised submission (i) a marked copy (yellow highlights for changes in revision), (ii) a clean\ncopy of the manuscript file, (iii) Supplementary information: Supplementary figures/legends (Marked and\nUnmarked copies); Reporting summary and Editorial Policy Checklist and (iv) Supplementary datasets 1-4.\nWe confirm that there have been no changes to the author's list.\nWe sincerely hope that the revised version addresses all the concerns raised and meets the expectations of the\nreviewer and editorial board.\nPoint by point response to all reviewers’ comments are listed below:\nReviewer #1 (Remarks to the Author):\nAll my comments have been addressed and the manuscript is suitable for publication\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early\nCareer Researchers who co-review manuscripts.\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #3 (Remarks to the Author):\nThe authors have answered all of my comments. It is Ok for me to accept it now.\nWe appreciate the reviewer’s time and effort and favorable review of our work.\nReviewer #4 (Remarks to the Author):\nThank you for your special efforts to revise this manuscript. I believe it has improved considerably. However, to\npreserve the robustness of the work in Nat Commun, the following points still require attention; I would appreciate\nyour continued consideration.\nWe appreciate the reviewer’s comments and detailed suggestions which have helped us improve our manuscript,\nand appreciate the favorable review of our work.\n1. Contradiction in expression patterns in Figure 1A/S1a\n→ From the dot plots, it appears that at most ~10% of cells within the proximal tubule and myofibroblast/fibroblast\npopulations express the genes in question, and likely at quite low levels. If violin plots are shown, it will probably\nbecome clear that there is essentially no expression. It is not reasonable to discuss genes that are virtually not\nexpressed in the single-cell data, and this likely contributes to the apparent discrepancy between Figure 1A and\nS1a. Even without the single-cell data the manuscript is sufficiently interesting, so it would be better to remove\nthese data.\n-We believe the reviewer is referring to Figures 1a, S1a and S1b (all single cell or single nuclear data related\nfigures), and appreciate the reviewer pointing this issue of Shroom3, Rock1 and Rock2 expression levels.\nOverall, we appreciate this valuable suggestion from the reviewer.\n-To respond to this question raised by the reviewer we generated violin plots from our co-author’s publication 1.\nAs shown below, iPTs during the fibrosis stage show increased expression of Shroom3, Rock1 and Rock2,\nsupporting mechanistic experiments in tubular cell lines. Myofibroblast cluster (which includes myofibroblasts\nand fibroblasts together) robustly express Rock1 and Rock2, but inconsistently express Shroom3 in this IRI\ndataset. As observed in the Li et al dataset, myofibroblasts do have lower expression of Shroom3.\n-We also evaluated recent independent data from a multispecies-integrated Single-Cell Kidney Atlas, by\nKloetzer, Susztak et al 2, which evaluated ScRNA data from healthy and diseased murine kidneys from multiple\npublications (total cells after QC =447499 (Healthy =64948 ,diseased =382551) [located at\nhttps://susztaklab.com/SISKA/mouse]. Here the proportion of iPT cells expressing Shroom3 ranged from 33 to\n45%, and in stromal cells (which includes fibroblasts/ myofibroblasts) Shroom3 expression ranged from 52 to\n53%. These corresponding data for Rock1 were 33-36% in iPTs and 31-32% in stromal cells, and for Rock2\nwere 41-50% in iPTS and 47-56% in stromal cells, respectively. These again reflect variability in cell-type specific\nexpression of these genes in single cell transcriptomic data across different publications, but support their\nexpression in iPTs and stromal cells. We have now included this recent reference in the revision.\nHence, these data and others suggest that there is sufficient expression of these genes in PTs and fibroblast\ncells providing rationale for our sequential mechanistic experiments, which then allowed us to infer the central\nrole of Shroom3-Rock interaction in iPTs (vs fibroblasts) during TIF. As suggested by the reviewer, since these\nderivative data from prior publications are not essential for the manuscript, and to avoid inconsistency between\nfigures, we have now removed these panels from the revised manuscript. We have redrafted the paragraph\nreferring to these data as follows.\n“We previously reported that tubular-specific Shroom3 knockdown alone could mitigate TIF in a UUO\nmodel 8. Recent functional genomics studies evaluating CKD-associated-SNPs using single cell-ATACseq also\nconcluded that single-cell, cis-eqtl effects of Shroom3 intronic variants are identifiable in proximal tubular cells\n(PTs) in human CKD24. These pointed to PTs as key players for TIF from excess Shroom3. We also considered\nthat fibroblasts which are key cells for matrix production during TIF, express Shroom3 and show high Rock1,\nRock2 expression in published data from murine TIF models25, 26, 27, 28. Podocytes show high expression of\nShroom3 at rest and injury but are not expected to play key roles in these TIF models after PT cell injury25, 26, 27.\nHence, we focused on tubular and fibroblast cell lines which were potential sites of Shroom3 interaction with\nRock1 and/or Rock2 during TIF for subsequent mechanistic experiments.”\n2. Insufficient explanation for the contradiction that ASD2Δ-Sh3 suppresses proliferation yet confers\nrenoprotection\n→ Prior reports (e.g., JCI 2019) indicate that tubular cell proliferation is required for renal recovery, but this\napparent contradiction has not yet been addressed.\nWe appreciate this point raised by the reviewer. We have now added a paragraph in the discussion to detail\npotential explanations behind the dichotomous association seen in epidemiologic studies of enhancer Shroom3\nSNPs (ie with more Shroom3-Rock interaction) with improved AKI but consistently increased CKD risk. We\nbelieve that Rock-activation mediated Ccl2-excess from iPTs sets up cross-talk to with immune cells (via Ccr2)\nwhich in turn activate resident stromal cells setting up “fibro-inflammation” and CKD progression. Ccl2 was\nconsistently observed to be inhibited significantly during genetic or pharmacologic inhibition of ASD2-Rock\ninteraction in vitro and in vivo in our data. This is also suggested from the multiple data showing consistent\nbenefit of Rock-inhibitors in AKI 3, 4. We have also accepted as limitation that we have not currently done time-\ncourse studies where we induced Shroom3-Rock interaction inhibition (genetically or pharmacologically) during\nAKI, during AKI recovery or during TIF stage alone.\nTo summarize this in the manuscript, we have added the following to the discussion.\n“It is also important to discuss implications of our work for acute kidney injury (AKI). We note that in contra-\ndistinction to multiple reported adverse associations of Shroom3-SNPs with CKD, a potential protective role for\na linked intronic Shroom3-SNP in AKI in humans was reported recently65, and increased AKI also occurred in\nheterozygous global Shroom3-knockout mice31. Facilitation of Rho-kinases by SHROOM3 could aid in spindle\nformation and cytokinesis during cell division, and inhibiting activation of ROCKs via ASD2 could reduce PT\nproliferation after injury 66, 67, consistent with our observations in vitro in IMCD lines. Since cell proliferation is\nrequired for tubular repair 68, delineating the role of increased Shroom3-Rock interaction during AKI, recovery\nfrom AKI vs TIF/CKD is needed to avoid any potential harm during therapeutic use of P2Is. We acknowledge\nthat we did not conduct such time course experiments. However, we note that ASD2Δ-Sh3 was continuously\nexpressed in our models throughout the AKI phase and we still observed benefit in TIF. Next, in spite of this\npotential adverse effect of inhibiting ROCKs during AKI, Rock-inhibitors have consistently shown benefit in\nmodels of AKI66, 69. A possible explanation is the observation that inhibition of Shroom3-Rock interaction in tubular\ncells alone markedly inhibited Ccl2, a chemokine signal known to be facilitated by rho-kinase activation 32, 70, 71.\nCcl2 mediates immune cell recruitment via its receptor Ccr2 and is a potent facilitator of tubular-inflammatory\ncell crosstalk after injury28. Hence, in spite of potential deleterious effects on tubular cell proliferation during AKI,\nexcess Shroom3-Rock interaction in iPTs overexpressing SHROOM3 would enhance crosstalk between iPTs\nand interstitial - inflammatory and/or stromal -cells, culminating in increased “fibro-inflammation” and progressive\nCKD.”\n3. Mismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ Although the rebuttal compared multiple datasets to explain this, the main text does not explicitly state it.\nPlease incorporate this discussion into the Discussion section.\nWe apologize for not including these lines from the rebuttal statement in the manuscript. We have now included\nthe following in the limitations section of the discussion as follows to address the Reviewers initial concern.\n“From the multiple single cell expression datasets 24, 25, 27 26, it is apparent to us that cell-type specific expression\npatterns of each Rock paralog and Shroom3 are variable and sometimes mismatched between datasets. This\nvariability may have resulted from technical variability between datasets (single cell vs single nuclear, etc). To\nminimize the impact of this in our work, we mined multiple datasets which included multiple injury models and\nmodalities of single cell transcriptome assessment to provide cell-type rationale for our mechanistic studies.”\n4. Other\n→ Because numerous new references were added in the revision, many citation numbers have changed.\nPlease carefully check that numbering and cross-references are consistent.\nWe apologize for this oversight. We cross-checked the references and ran the reference library for the Response\ndocument separate from the main manuscript document.\n1. Xu L, Guo J, Moledina DG, Cantley LG. Immune-mediated tubule atrophy promotes acute\nkidney injury to chronic kidney disease transition. Nat Commun 13, 4892 (2022).\n2. Klotzer KA, et al. Analysis of individual patient pathway coordination in a cross-species single-\ncell kidney atlas. Nature genetics 57, 1922-1934 (2025).\n3. Prakash J, et al. Inhibition of renal rho kinase attenuates ischemia/reperfusion-induced injury. J\nAm Soc Nephrol 19, 2086-2097 (2008).\n4. Liang H, et al. CXCL16/ROCK1 signaling pathway exacerbates acute kidney injury induced by\nischemia-reperfusion. Biomed Pharmacother 98, 347-356 (2018).\nREVIEWERS' COMMENTS\nWe appreciate the reviewers detailed and favorable responses.\nReviewer #4 (Remarks to the Author):\nWith regard to the reviewer’s concerns, the authors have addressed the issues as\nfollows. As a result, the manuscript has become logically coherent, more readily\ncomprehensible, and in my view has been further polished.\nContradiction in expression patterns in Figure 1A/S1a\n→ The authors re-examined the data using violin plots, removed non-essential data in\naccordance with the reviewer’s recommendation, and revised the relevant paragraph in\nthe text.\nContradiction that ASD2Δ-Sh3 suppresses proliferation yet confers renoprotection\n→ A new paragraph has been added to the Discussion, elaborating on the apparent\ndiscrepancy within the context of AKI versus CKD, with additional explanation involving\nCcl2/ROCK signaling.\nMismatch between Shroom3 and ROCK expression patterns (Humphreys dataset)\n→ The variability across datasets has now been explicitly acknowledged and\nincorporated into the Limitations section.\nInconsistencies in reference numbering and cross-references\n→ The authors have stated that they have carefully reviewed and corrected these\ninconsistencies.\n-We appreciate the positive evaluation of our revisions by the reviewer.\nMinor points:\n• As noted, it would be appropriate to spell out iPTs (injured proximal tubules) at its first\nappearance in the main text. In fact, the rebuttal document already provides this\nclarification as “iPTs (injured proximal tubular cells).”\nReply: We thank the reviewer for the correction. We added the expansion for iPTs at the\nfirst occurrence.\n• FBDM-Sh3 (Fyn-binding domain mutant Shroom3): This abbreviation should be\ndefined at its first occurrence.\nReply: We thank the reviewer for pointing out this. We have now expanded the\nabbreviation at the first occurrence.\n• P2Is (Shroom3-Rock interaction inhibitors): Although the definition is provided in the\ntext, the introduction of this abbreviation is somewhat abrupt. It would be clearer and\nmore reader-friendly to introduce it more explicitly at first mention, for example: “we\ndeveloped novel small molecule inhibitors of Shroom3-Rock interaction (P2Is).”\nReply: We appreciate the reviewer’s suggestion and have incorporated it.\n• Several references appear to be incomplete. In particular, references 7, 18, 24,\n55, and 79 seem to be missing page numbers.\nWe appreciate these issues that were pointed out. We have corrected and highlighted in\nmarked copy.\nReference 7 & 11 – we have included page numbers for these references\nReference 18 and 11 were duplicated. Hence in the revision Ref 18 has been deleted,\nand all references below 18 have been pushed up by 1.\nReferences 24, 55, 79 have now become 23, 54 and 78, respectively. We have added in\npage numbers obtained from pubmed and the journal websites.\n• Additional inconsistencies may also exist, for example in references 25–27 (murine\nTIF models) and 65–67 (recently added AKI-related studies). These should be checked\ncarefully to ensure that full bibliographic details, including page ranges, are provided.\nWe checked these references carefully. The endnote program picked these PMIDs from\npubmed and cited these in Nature communications output format. The only potentially\nmissing detail to be added in to the manuscript are the issue numbers – which we have\nnow added (marked copy). These may not be automatically included in Nature\ncommunications but regardless we have now added these references."}]

Decision letter