An epithelial morphogenetic program for maximal urine concentration

Domain: biology
Published: 2026-03-21
Editorial outcome: major_revision

Abstract
Mammals achieve the highest urine concentrations of any vertebrate, a feat that hinges on generating steep osmotic gradients within the renal medulla. Interestingly, the region with the highest osmolality, the inner medulla, is unique to mammals. Among the nephron’s segments, the ascending thin limb (aTL) is the sole element exclusive to this zone and is thought to mediate passive salt reabsorption. However, the architecture and functional impact of the aTL have remained obscure. Here we uncover an unexpected morphogenetic program in the aTL, characterized by extensive apical-junctional interdigitations that greatly increase cell-to-cell contact area. Integrating single-nucleus transcriptomics with high-resolution imaging, we identify claudin-10b, a tight junction protein and paracellular cation pore, as a central driver of this architecture. Inducible deletion of claudin-10b specifically in the aTL abolishes membrane interdigitations and markedly reduces urine-concentrating ability, thereby establishing a direct link between segment-specific epithelial morphology and whole-organ function. Claudin-10b proves necessary for interdigitation formation, acting through transcellular adhesion and interaction with the tight-junction scaffold ZO1. These findings offer definitive evidence that the inner medulla and aTL are essential for maximal urinary concentration, while revealing a non-canonical, morphogenetic role for claudin-10b.

Introduction
The ability to concentrate urine represents one of the most remarkable physiological adaptations in mammalian evolution, enabling survival in terrestrial environments by conserving water while eliminating metabolic waste. This critical function depends on the kidney's capacity to establish and maintain steep osmotic gradients across the renal medulla, with the inner medulla, a specialized compartment unique to mammals, acting as a primary site for achieving maximal urine concentration1,2.

The nephron, the functional unit of the kidney, comprises a series of morphologically and functionally distinct segments that collectively fine-tune solute and water balance. Among these segments, the Loop of Henle (LOH) plays a pivotal role in establishing the corticomedullary osmotic gradient essential for urine concentration (Fig. 1a)2. The thick ascending limb (TAL) in the outer medulla is well-characterized for its active sodium chloride transport, which drives the initial establishment of medullary hypertonicity3,4. In contrast, the inner medullary thin segments operate primarily through passive transport mechanisms, as ATP-driven active transport systems are notably absent in this region5,6.

Within the inner medulla, the ascending thin limb (aTL) of the Loop of Henle stands as the sole nephron segment confined exclusively to this region1. Despite its unique position and presumed role in passive salt reabsorption, the aTL has remained one of the most enigmatic components of renal physiology since Kokko and Rector first proposed passive inner medullary sodium reabsorption in the 1970s7. While chloride transport in the aTL has been demonstrated to occur transcellularly via the chloride channel Clck18-10, the molecular mechanisms governing sodium transport remain poorly understood5,6. The aTL's deep anatomical location within the inner medulla, combined with the historical lack of segment-specific genetic tools11, has severely hampered mechanistic studies of this critical nephron segment. These technical limitations have left fundamental questions unanswered regarding the precise molecular mechanisms underlying aTL function, its contribution to medullary hypertonicity, and the structural features that enable its physiological role.

Here, we report the presence of a unique epithelial architecture in the aTL: extensive apical-junctional membrane interdigitations that dramatically expand cell-to-cell contact area. Through integration of single-nucleus transcriptomics, high-resolution imaging, and functional studies, we identify claudin-10b as a central molecular driver of this unique morphology. We demonstrate that claudin-10b functions through transcellular adhesive interactions and tight junction scaffolding proteins to create these interdigitations, and that this architectural program is essential for normal urinary concentrating ability. Our findings establish direct evidence for aTL-specific contributions to kidney function while definitively demonstrating an in vivo morphogenetic role for claudin proteins that extends beyond their traditional functions in epithelial barrier formation.

Results
Identification of arborized aTL projections
To identify molecular mechanisms underlying inner medullary urine concentration, we focused on the ascending thin limb (aTL) of the Loop of Henle. Previous studies reported expression of Prox1, a transcription factor known as a master regulator of lymphatic development12, in the developing aTL13. Based on this, we hypothesized that the Prox1-CreERT2 knock-in mouse could be used as a genetic entry point to target this nephron segment. To test this, we crossed Prox1-CreERT2 knock-in mice14 with the mTmG reporter line and induced recombination at 4 weeks of age. With mTmG, cells express membrane-tdTomato (mT) before Cre recombination and membrane-GFP (mG) after. We observed a strong GFP signal in the inner medulla, a region that lacks lymphatic vessels (Fig. 1b). Co-localization of the medullary GFP signal with the chloride channel Clck1/2, a marker of the ascending LOH11,15, established that Prox1-CreERT2 drives recombination in the aTL and thick ascending limb (TAL) (Fig. 1b,c). Adjacent medullary tubules, including the collecting duct, the descending thin limb, and the vasculature, do not express Prox1 (Supplementary Fig. 1).

A low dose of tamoxifen administered to Prox1-CreERT2; mTmG mice resulted in sparse GFP labeling of the aTL and TAL (Fig. 1d), which enabled visualization of single aTL cells. Surprisingly, this revealed a striking sunburst morphology characterized by interdigitating, finger-like cell projections that extend laterally between adjacent cells rather than apically like microvilli (Fig. 1d, Supplementary Movies 1-3). Three-dimensional reconstructions of individuals cells via confocal microscopy (Fig. 1e) and Axially-Swept Light-sheet Microscopy (ASLM)16 (Fig. 1f, Supplementary Movie 4) further resolved these projections into high-curvature primary processes with branched secondary processes.

The aTL projections developed postnatally, increasing in number and length from postnatal day 7 (P7) until adulthood (Fig. 1g). Structural analysis revealed that they contained a cortical actin network, microtubules, and mitochondria, supporting their identity as cytoplasmic extensions (Supplementary Fig. 2). Filamentous actin was most enriched at the junctional regions, aligning parallel to the plasma membrane, rather than as a core actin bundle (Supplementary Fig. 2a,b). The discovery of these elaborate projections raised fundamental questions about the molecular mechanisms governing cell morphology, particularly the drivers of interdigitations in the aTL.

Claudin-10 in the developing aTL
To investigate how aTL cells form these interdigitating projections, we conducted a time-course single nucleus (snuc) RNA sequencing experiment at postnatal day 0 (P0), P2, P7, P14, P21 and 6 weeks, capturing the transcriptional landscape of postnatal projection development. At 6 weeks, we identified a distinct aTL cluster that expressed Prox1 and Clck1 but lacked markers of other nephron segments, such as Slc12a1 (NKCC2), a TAL marker (Fig. 2a,b, Supplementary Fig. 3). The identity of the aTL cluster was further validated by demonstrating that Prox1-CreERT2; mTmG GFP signal in the inner medulla colocalized with aTL markers (Prox1, Clck1) (Fig. 2c) but did not overlap with markers of near-by nephron segments (Supplementary Fig. 1b,c). This confirmed that the aTL cluster identified by snucRNA-sequencing corresponds to a distinct Prox1+ epithelial population in the inner medulla.

To uncover molecular regulators of aTL morphology, we performed differential gene expression analysis within the aTL cluster. One intriguing candidate was Cldn10, which encodes the transmembrane tight junction protein claudin-10 (Fig. 2d). Although also expressed in the proximal tubule and TAL (Supplementary Fig. 4a, Supplementary Movies 5-6), Cldn10 was among the top twenty enriched genes in aTL (13th overall) (Fig. 2d). Importantly, Cldn10 transcript levels increased postnatally, coinciding with the emergence of aTL projections (Fig. 2e, 1g). Claudin-10 has been shown to immunolocalize to the aTL17, and was the most highly expressed claudin in the aTL, surpassing others by at least threefold (Supplementary Fig. 4b). Its enrichment in the aTL was corroborated by an independent mouse kidney dataset18, and Cldn10 has been identified as a marker gene in human aTL19.

Immunostaining confirmed that claudin-10 colocalized with the mosaic Prox1-CreERT2; mTmG GFP signal in the inner medulla, specifically at sites of interdigitation along the periphery of aTL projections (Fig. 2f). At the subcellular level, claudin-10 predominantly localized to apical junctions, colocalizing with its interactors ZO1 and ZO2 (Supplementary Fig. 5)20,21.

Claudin-10b is the aTL isoform
Claudin-10 exists as two isoforms, claudin-10a and claudin-10b, which differ only in their first exon, but exhibit distinct functions: claudin-10a forms anion pores, while claudin-10b forms cation pores22,23. To identify which isoform is expressed in the aTL, we performed RT-PCR on RNA isolated from the cortex, outer medulla and inner medulla. As expected, claudin-10a was detected in the cortex, while claudin-10b was expressed in the outer medulla (Fig. 2g), consistent with its known localization in the proximal tubule and TAL, respectively22,23. In the inner medulla, claudin-10b was the isoform detected (Fig. 2g). Notably, since no other tubules in the inner medulla expressed claudin-10 (Supplementary Fig. 4c), these findings confirm that the aTL produces claudin-10b, expanding its known expression profile to this nephron segment.

Claudin-10b regulates epithelial morphology
To investigate the role of claudin-10b in cell morphology, we first turned to an in vitro system amenable to mechanistic analysis. Notably, no existing epithelial cell culture model fully recapitulates the complex interdigitating projections observed in the aTL. However, a related feature, apical-junctional membrane ‘waviness’ can serve as a mechanistically relevant readout for apical-junctional interdigitations.

MDCKII cells, a widely used kidney epithelial cell line, exhibit modest junctional membrane waviness24,25, making them a suitable system in which both gain- and loss-of-function effects on junctional morphology can be readily detected. Importantly, while wild-type MDCKII cells express multiple claudins, including claudin-1, -2, -3, -4, and -7, previous studies have shown that simultaneous knockout of these five major claudins (quinKO or qKO) eliminates membrane waviness24,26.

Strikingly, we found that claudin-10b expression in quinKO cells induced pronounced apical-junctional membrane waviness (Fig. 3a). This phenotype was quantified using an automated ‘waviness index’, a metric comparing the convex hull perimeter to the actual plasma membrane perimeter (Fig. 3b, Supplementary Fig. 6), with higher values indicating increased membrane waviness (see Methods for additional details). Automated measurements correlated well with a manual measure of membrane waviness, underscoring the robustness and reliability of this approach. In addition to its effects on junctional morphology, claudin-10b expression increased transepithelial resistance, decreased conductance, and increased paracellular sodium selectivity in quinKO cells, as assessed by PNa/PCl ratios and dilution potentials (Supplementary Fig. S7), consistent with previous reports24,26.

A dual role for claudin-10b in aTL structure and function in vivo
While our in vitro findings demonstrated that claudin-10b promotes apical-junctional membrane waviness, it remained unclear whether claudin-10b similarly regulates epithelial morphology in vivo, where aTL cells form elaborate interdigitations. In addition, the functional role of claudin-10b in aTL physiology remained unclear. Given claudin-10b’s localization at aTL interdigitations and its function as a paracellular cation pore, we hypothesized that claudin-10b has dual roles: regulating epithelial morphology via interdigitations and contributing to urinary concentrating ability, potentially by influencing sodium handling.

To evaluate the role of claudin-10b in the aTL, we generated a conditional knockout mouse by flanking exons 2 and 3 of Cldn10 with loxP sites (Supplementary Fig. 8). Cldn10f/+ mice were crossed with Cldn10f/+;Prox1-CreERT2; mTmG mice to produce control (Cldn10+/+;Prox1-CreERT2; mTmG) and mutant (Cldn10f/f;Prox1-CreERT2; mTmG) mice, ensuring that both groups carried a fluorescent reporter to visualize aTL projections. Cldn10 mutant mice were healthy, with no differences in weight between genotypes at 10 weeks of age (mean ± SD : CTL, 20.17 ± 3.31g [n=18]; HET, 20.60 ± 2.17g [n=15]; MUT, 20.71 ± 3.85g [n=17]; Kruskal-Wallis Test, p>0.99 for all comparisons).

Immunofluorescence confirmed that recombined GFP+ tubules in Cldn10 mutant mice lacked claudin-10 protein (Fig. 3c,d). Notably, Cldn10 mutant mice displayed a drastic reduction in aTL cell projections, with many cells completely lacking interdigitations. (Fig. 3e). Although plasma membrane surface area was greatly reduced from the lack of interdigitations, the tight junction adaptor protein ZO1 was still appropriately recruited to the plasma membrane at levels comparable to controls (Fig. 3f,g).

The physiologic function of the aTL is poorly understood, particularly its contribution to solute transport and urine concentration. Although the aTL has been implicated in passive sodium and chloride reclamation5,6, its definitive contribution to medullary hypertonicity, which is essential for urine concentration, has not been established. Moreover, the molecular mechanisms governing sodium movement in the aTL remain unresolved. A major barrier to testing claudin-10b’s role in this segment has been the lack of genetic tools capable of selectively manipulating the aTL in vivo. Our discovery that Prox1-CreERT2 induces recombination in the aTL and TAL provided a promising genetic tool, but achieving aTL specificity required further refinement. Single nucleus transcriptomic analysis revealed that Prox1 is expressed in the developing aTL prior to the TAL (Supplementary Fig. 9), with expression in both segments at later developmental timepoints (Supplementary Fig. 9). Consistent with this pattern, neonatal tamoxifen administration from postnatal day 2 (P2) to P7 selectively targeted the aTL (Supplementary Fig. 10a-c), whereas tamoxifen administered at later timepoints resulted in recombination in both the aTL and the TAL (Supplementary Fig. 10d-i). This temporal control of Prox1-CreERT2 therefore enabled genetic analysis of aTL-specific gene function. Claudin-10 is absent from renal lymphatics at all tamoxifen dosing ages analyzed (Supplementary Fig. 11), indicating the observed phenotype is unlikely to arise from lymphatic recombination.

To assess the functional consequences of Cldn10 loss specifically within the aTL, we administered the aTL-selective tamoxifen regimen (P2-P7, hereafter referred to as Cldn10ΔaTL) to control (CTL), heterozygous (HET), and Cldn10 mutant (MUT) mice. Under baseline conditions, Cldn10ΔaTL mutant mice exhibited reduced urine osmolality and increased 24-hour urine volume compared with controls (Fig. 4a,b). To determine if these changes reflected a defect in urinary concentrating ability rather than increased water intake, we subjected mice to 24-hour water deprivation. Under these conditions, urine osmolality increased in all groups; however, Cldn10ΔaTL mutants achieved a significantly lower maximal urine osmolality than control and heterozygous littermates (Fig. 4c; Supplementary Fig. 12). These findings indicate a reduced maximal concentrating capacity after aTL-specific deletion of Cldn10.

Notably, non-recombined aTL cells in mutant mice that were not adjacent to recombined cells exhibited normal projections, indicating that the phenotype is primarily cell-autonomous rather than driven by local osmotic or other microenvironmental changes (Supplementary Fig. 13).

Claudin-10 transcellular adhesion drives projections
While our in vivo findings demonstrated that claudin-10b is essential for aTL projection formation, the molecular mechanism by which claudin-10b promotes membrane interdigitation remained unclear. To address this, we conducted in vitro mutagenesis targeting key functional domains of claudin-10b.

Claudin-10b contains two extracellular loops (ECL1 and ECL2)27, where ECL1 governs sodium permeability, and ECL2 mediates transcellular adhesive interactions (Fig. 5a)28. To dissect the relative contributions of sodium transport and cell-cell adhesion to projection formation, we generated point mutants in ECL1 (D65N) and ECL2 (F146A), both of which have been previously validated and characterized29-31.

Strikingly, expression of the claudin-10b D65N mutant, which disrupts sodium permeability while preserving adhesive interactions, induced apical-junctional projections. In contrast, expression of the claudin-10b F146A mutant, which disrupts transcellular adhesion, failed to induce projections (Fig. 5b,c). These findings suggest that transcellular adhesive interactions, rather than paracellular sodium transport, are critical for interdigitation.

To directly test whether transcellular interactions between neighboring cells are required for interdigitations, we co-cultured quinKO cells with quinKO+Cldn10b-expressing cells and examined the clonal interface (Fig. 5d). Notably, projections were absent at this boundary, despite robust projection formation between adjacent claudin-10b-expressing cells, providing direct evidence that transcellular adhesion is required for claudin-10b-mediated interdigitation.

Based on this observation, we re-examined mutant epithelia in vivo to identify aTL sections with mosaic deletion of claudin-10. Consistent with our in vitro findings, non-recombined claudin-10-expressing cells adjacent to claudin-10-deficient cells failed to form projections (Fig. 5e). Together, these three lines of evidence strongly suggest that claudin-10’s adhesive transcellular interactions, rather than its role in sodium transport, are essential for epithelial interdigitation in the aTL.

Projections require claudin-10b-ZO1 binding
In quinKO+Cldn10b cells, we observed that claudin-10 localizes to both the apical junctions and the lateral membrane (Supplementary Fig. 14a), similarly to some other claudin family members in MDCK cells24,32,33. Notably, membrane waviness was confined to apical junctions where claudin-10 co-localized with ZO1, a tight junction adaptor protein previously shown to promote junctional membrane waviness when expressed at high levels34. Membrane waviness was not observed along lateral membranes lacking ZO1 (Supplementary Fig. 14a).

To determine whether interactions between claudin-10b and tight junction scaffold proteins are required for interdigitation, we expressed claudin-10ΔYV, a C-terminal mutant lacking the PDZ-binding motif necessary for ZO1/2 interaction21, in quinKO cells. In contrast to wild type claudin-10b, claudin-10ΔYV accumulated intracellularly rather than localizing to the plasma membrane and failed to induce membrane projections (Fig. 5f). This suggests that PDZ-mediated interactions are necessary for claudin-10b localization and function, but does not distinguish if ZO1/2 are the key factors.

To distinguish whether loss of projections reflected impaired membrane localization versus loss of scaffold interactions, we engineered claudin-10ΔYV-CAAX, a variant incorporating a CAAX motif to promote plasma membrane association35. Although this modification sporadically restored claudin-10b localization at the plasma membrane, only very slight membrane waviness was observed (Fig. 5f). This indicates that membrane localization of claudin-10ΔYV is not sufficient to rescue projection formation.

To determine whether ZO1 or ZO2 are specifically required for claudin-10b function, we generated single knockouts of ZO1 or ZO2 in quinKO+Cldn10b cells. Due to incomplete knockout efficiency, the resulting cultures contained a mixture of ZO1+ and ZO1– cells (Fig. 5g,h) or ZO2+ and ZO2– cells (Supplementary Fig. 14b), effectively creating co-cultures within individual wells. This enabled direct comparison of membrane morphology between knockout and wild-type cells in the same environment. Loss of either ZO1 or ZO2 did not impair recruitment of claudin-10b to the plasma membrane (Fig. 5g, Supplementary Fig. 14b) and neither knockout impacted the localization of the other (Fig. 5h, Supplementary Fig. 14b), consistent with previous reports34. However, ZO1– cells failed to form projections (Fig. 5g,h), indicating that ZO1 is required for claudin-10b-mediated morphogenesis. Notably, ZO1 was required in both cells for projections to form (Fig. 5g,h arrows).

Loss of ZO2 displayed an intermediate phenotype, with reduced but not absent projections (Supplementary Fig. 14b). These mechanistic insights likely also apply to the in vivo aTL tubule, where claudin-10 co-localizes with ZO1/2 (Supplementary Fig. 5). Together these findings support a model in which both claudin-10b transcellular interactions and claudin-10b-ZO1 interactions are required to drive apical-junctional membrane projections (Fig. 5i).

Methods
All research in this study complies with ethical regulations and guidelines, including UT Southwestern IACUC.

Reagents: All reagents are listed in Supplementary Tables 1-3.

Animals
Mice were housed in a 12-hour light/dark cycle (6am - 6 pm) with the temperature held between 68-79˚F and humidity ranging between 30-70%. Mice were maintained on a mixed C57Bl6/129X1/SvJ genetic background and genotyped by standard PCR. See Supplementary Table 2 for primer information. Cldn10f/f were generated using EasiCrispr52 (Supplementary Fig. 8). Cldn10f/+ females were bred to Cldn10f/+;Prox1-CreERT2; mTmG14 males to obtain control (Cldn10+/+;Prox1-CreERT2; mTmG), heterozygous (Cldn10f/+;Prox1-CreERT2; mTmG), and mutant (Cldn10f/f;Prox1-CreERT2; mTmG) mice. 75 mg/kg tamoxifen was administered orally or via intraperitoneal injections at designated times. Further details are provided in Supplementary Fig. 10. All procedures followed UTSW IACUC-approved guidelines. Male and female mice were used for each experiment (see figure legend for details), and all observations reported are applicable to both sexes. The ages of animals used in each experiment are detailed in the figure legends.

RT-PCR
Cortex, outer medulla and inner medulla of 8-week-old mixed sex mice were dissected. RNA was isolated using the RNeasy Mini Kit and cDNA was synthesized using SuperScript III Reverse transcriptase. Primers sequences are available in Supplementary Table 223.

Single nucleus RNA sequencing
The kidney pairs of two male and two female mice at each age (P0, P2, P7, P14, P21, 6wk) were dissected in cold PBS. Cortical regions were partially removed to enrich for medullary cell types. Kidneys were minced into small cubes, dounced 5 times with the loose pestle in Nuclei Lysis Buffer 1 (NLB1) (4 ml of nuclei EZ lysis buffer, 20 µl of RNasin Plus, 20 µl of SUPERaseIN, 1% BSA, cOmplete ULTRA tablet), and passed through a 200 µm strainer. The sample was then dounced 10 times with the tight pestle, incubated for 1 minutes in NLB1 on ice, passed through a 40 µm strainer, and centrifuged at 500g for 1 minutes at 4˚C. The pellet was resuspended in NLB2 (4 ml of Nuclei EZ Lysis Buffer, 4 µl of RNasin Plus, 4 µl of SUPERaseIN, 1% BSA, cOmplete ULTRA tablet), incubated on ice for 5 minutes, and centrifuged at 500g for 5 minutes at 4˚C. The pellet was resuspended in nuceli suspension buffer (2 ml of calcium/magnesium free PBS, 2 µl of RNasin Plus, 2% BSA) and passed through a 5 µm strainer. The nuclei were counted by hemocytometer and diluted to 1200 nuclei/uL.

Libraries were prepared from 10,000 nuclei per timepoint using the 10x Genomics Chromium Next GEM Single Cell 3’ Kit v3.1 kit. All samples were sequenced on a NovaSeq S4 with 50,000 reads per cell and 28x90 read length.

Single-nucleus RNA-seq preprocessing
Filtered feature-barcode matrices were generating by running the CellRanger count pipeline. Nuclei were called from empty droplets by testing for deviation of the expression profile for each cell from the ambient RNA pool53. Nuclei with low numbers of detected genes or library sizes, as defined as a deviation greater than three median absolute deviations (MADs) below the median, were excluded from further analysis. Nuclei with large mitochondrial proportions, i.e., more than 3 mean-absolute deviations above the median, with a minimum difference between the threshold of 0.5% between the threshold for exclusion and the median mitochondrial transcript count, were removed. Nuclei were pre-clustered and a deconvolution method was applied to compute size factors for all cells54 and normalized log-expression values were calculated.

Single-nucleus RNA-seq analysis
Cells were clustered by building a shared nearest neighbor graph55 on the normalized expression data and executing the Louvain algorithm. Diffusion maps were calculated using an adaption of the algorithm presented in Haghverdi et al56. Normalized expression data was imputed applying Markov Affinity-based Graph Imputation of Cells (MAGIC)57 to the previously constructed diffusion maps. Marker genes for each subset of cells under investigation were calculated by applying the EIGEN algorithm58. Differential expression between pre-specified groups of nuclei was calculated from the Hurdle model implemented in the MAST59 Bioconductor package. Gene set enrichment analysis was performed for sets curated in MsigDb60 using with fgsea61 Bioconductor package. For each set, the enrichment in each cell was calculated using AUCell62. Normalized expression data and marker gene analysis (consensus markers) are available in Supplementary Data 1-7. Expression plots for genes of interest expression were generated using dplyr, tidyr, and ggplot2 packages in RStudio (Version 2025.05.1+513).

Cell culture
MDCKII cells (gift from Keith Mostov) were grown in 10% DMEM low glucose with 2 mM L-glutamine. Cells were grown in 8-well chamber slides and imaged using a Zeiss 880 confocal microscope63,64. Quintuple knockout (quinKO) MDCKII cells (gift from Mikio Furuse) were generated using TALEN vectors24. Wild-type and mutant Cldn10b constructs were synthesized, validated by Sanger sequencing, and cloned into pENTR221 by ThermoFisher GeneArt Services. These constructs were inserted into the inducible lentiviral vector pCW57.1 using Gateway cloning. ZO1 and ZO2 gRNAs65 were cloned into the pLV[2CRISPR]-hCas9:T2A:Bsd-U6 Dual gRNA lentiviral vector by VectorBuilder. ZO1 and ZO2 gRNAs are detailed in Supplementary Table 2. Lentiviruses were packaged in HEK293T (gift from Keith Mostov) using Mirus-TransIT-LT1, and viral supernatant was harvested for transduction after 48 hours. Transduced quinKO cells were selected with puromycin (5 µg/mL) for pCW57.1 or blasticidin (10 µg/mL) for ZO1/ZO2 knock-out. Heterogeneous or clonal populations were used for analysis as indicated. Inducible cell lines were treated with 0.5 to 10 µg/mL doxycycline as indicated for 4-5 days. For immunofluorescence, cells were plated at 6.6x104 cells/well in 8-well chamber slides and cultured for 5-7 days.

In vitro electrophysiology and measurement of dilution potentials
Cells were seeded onto 12 mm Snapwell inserts with a 0.4 µm pore polycarbonate membrane (Corning 3407) at a density of 1x105 and incubated for 7 days with media changes every other day. At 7 days post-seeding, inserts were mounted in modified Ussing chambers (P2300, Physiologic Instruments Inc., Venice, FL, U.S.A.) with an exposed surface area of 1.12 cm2 maintained at 37 °C under symmetrical, current-clamp conditions (VCC MC6; Physiologic Instruments) with Buffer A (120 mM NaCl, 5 mM KCl, 10 mM NaHCO3, 1.2 mM CaCl2, 1 mM MgSO4, 10 mM HEPES, and 10 mM glucose, pH 7.4), which was simultaneously gassed and stirred with 95% O2/5% CO2. Following a 20-minute equilibration, dilution potentials were determined by replacing Buffer A in the basolateral chamber with Buffer B (60 mM NaCl, 120 mM mannitol, 5 mM KCl, 10 mM NaHCO3, 1.2 mM CaCl2, 1 mM MgSO4, 10 mM HEPES, and 10 mM glucose, pH 7.4) and recording transepithelial voltage (Vt) for 5 minutes. Buffer B was then replaced with Buffer A and Vt recorded for a further 5 minutes. The Na+/Cl- permeability ratios (PNa/PCl) were calculated using the mean of the last twelve data points (one minute) recorded during a given measurement period following the buffer change66. Transepithelial resistance was calculated by Ohm’s Law using the deflection in Vt following the application of a 40 µA pulse at 5 second intervals. Electrophysiological measurements were continuously recorded using Acquire & Analyze software (v2.0; Physiologic Instruments).

Immunofluorescence and Microscopy
Kidneys were immersion fixed in 4% PFA/PBS for 4 hours and sectioned using a vibratome (LeicaVT1000S). Samples were permeabilized with 0.3% TritonX-100/PBS (PBS-T) and blocked with 10% donkey serum/PBS-T67. Antigen retrieval was performed with Trilogy, if needed, and tissue clearing performed using CUBIC68. MDCKII cells were fixed, permeabilized with 0.5% PBS-T and blocked with 0.7% fish skin gelatin in PBS containing 0.025% saponin (PFS)67. Tissues and cells were incubated with primary antibodies overnight at 4˚C, and with fluorophore-conjugated secondary antibodies for 2 hours at room temperature. Antibodies were diluted in 2% donkey serum/PBS-T for kidney sections and in PFS for MDCKII cells. See Supplementary Table 3 for antibody list.

Kidney sections were mounted in ProLong Gold. All samples were imaged with a Zeiss LSM880 confocal microscope (ZEN Black 2.3 software) or custom Axially Swept Light-sheet Microscope16. 3D projections and movies were created in Image J using the 3D projection plugin. Images were minimally processed in ImageJ (Version 2.16.0/1.54Sp) and Adobe Photoshop 2026 (27.3.1 Release), including pseudo-coloring to avoid red-green color combinations. Schematics were created in Adobe Illustrator 2026 (Version 30.1) and/or BioRender (see figure legends for details).

Urine studies
Mice were singly housed and acclimated in metabolic cages for 72 hours with food and water ad libitum. A baseline 24-hour urine sample was collected, after which the bladder was emptied and water access removed for 24 hours to obtain a water deprivation urine sample. Spot urines were collected post-deprivation. Urine osmolality was measured using an OsmoPRO micro-osmometer.

Waviness Index Quantification
3D reconstructions of ZO1-stained confocal images were generated in ImageJ using maximum projection, followed by despeckling, segmentation with MorphoLibJ, and image inversion. A convex hull was created for each cell, and the waviness index was calculated as the ratio of the convex hull perimeter to the actual plasma membrane perimeter (Supplementary Fig. 6). Dividing cells were excluded from analysis.

Statistics
All immunofluorescence analysis of mouse tissue was conducted in n ≥ 3 mice. See figure legends for panel specific details. All in vitro experiments were repeated independently at least three times. Images shown are representative. n = 9-12 mice per genotype were used for metabolic urine studies. This sample size allows detection of an effect size of 1.2-1.41 between groups with 80% power at 5% two-sided type I error. All mouse studies were analyzed using Mann-Whitney U test (for two groups) or Kruskal Wallis H test followed by Dunn’s test (> 2 groups). Waviness index measurements were compared using Kruskal-Wallis H test followed by Dunn’s test. In vitro electrophysiology data was analyzed using one-way ANOVA with Tukey’s multiple-comparisons test. Graphed data are presented as mean ± s.d or in box-and-whiskers plots (see legends for details). Data was analyzed using GraphPad Prism (Version 10.3.1), with p-values<0.05 considered significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this work, the authors, identify Claudin10 B as an important regulator of paracellular sodium transport in the ascending\nthin limb of Henley. Using a Prox1-CE mouse, they discover that it can immediate recombination in the ATL and using a\nsparse labeling strategy discover that ATL epithelia are characterized by interdigitating finger like protrusions. Using single\nnucleus RNA-seq they identify Claudin 10 as a marker of a TL and show that it is enriched at the cell membrane of the ATL.\nUsing an in vitro model they demonstrate that Claudin10B is sufficient to drive membrane waviness, a surrogate for these\nfinger like protrusion. They generate a Claudin10B knockout in the ATL and show that Claudin10B is required for urinary\nconcentration. Finally, they perform structure function studies and confirm that Claudin10b interacts with Zo1 to drive these\nmembrane in interdigitation and regulate urine osmolarity.\nOverall, this is a highly novel work that reveals in unexpected epithelial morphology, and provides strong evidence that\nClaudin10b is required for urinary concentration.\n1. Can the authors directly measure paracellular sodium transport using their qKO MDCKII model, perhaps by TEER, to\ndirectly probe the role of Claudin10b, ZO1 and membrane ruffling in sodium transport?", "reviewer_2": "This manuscript characterizes the morphology and functional elements of the aTL, which is a poorly understood segment of\nthe nephron. This is an important advance in our knowledge of renal physiology, which is key for better understanding fluid\nand electrolyte disorders, and potentially even for developing new medications, such as diuretics. The study also offers a\nnew method for specific genetic targeting of the aTL, which will serve as an important tool for further investigations of this\nnephron segment.\nThe work is original and significant, as it elucidates the previously undescribed morphology of aTL cells while providing a\nmechanism for the functional significance of the morphology. The focal point of the study, Claudin-10b, is also expressed in\nthe TAL, and there is a study by Quintanova et al that describes the role of Claudin-10b in producing a similar morphology of\nmembrane infoldings or protrusions in TAL cells – the authors briefly allude to this feature of Claudin-10b and cite the\nQuintanova et al manuscript in the discussion, but they could spend a little more time describing the similarities and\ndifferences between the role of Claudin-10b in TAL vs aTL morphologies.\nThe work does support the conclusion that Claudin-10b is required for normal aTL morphology. However, a few clarifications\nare needed.\n1. In the abstract, the authors state that Claudin-10b “proves both necessary and sufficient” for interdigitation formation –\nwhile they show that it is necessary, they have not provided adequate evidence to claim that it is “sufficient”. In fact, they\nshow that ZO-1 is required for formation of the protrusions in aTL cells – so Claudin-10b is not sufficient. They also perhaps\nmake this claim because Claudin-10b can restore membrane waviness in MDCK cells that lack expression of other claudins\n– however, this is an artificial in vitro system that is a tool to study Claudin-10b function but cannot faithfully recapitulate aTL\nmorphology and function. The ability of Claudin-10b to restore membrane waviness in MDCK cells, therefore, does not\nnecessarily indicate that it is sufficient for protrusion formation in aTL cells.\n2. In the first section of the results, the authors should clearly state that Prox1 expression is very specific to the TAL and aTL\nand is not expressed in other parts of the inner medulla (namely, descending thin limb and inner medullary collecting duct)\nas shown by Kim et al. (the paper cited by the authors) – this will just help highlight the specificity of their Cre recombinase.\n3. In the results section titled “Caludin-10b is the aTL isoform”, the authors state “since no other tubules in the inner medulla\nexpressed claudin-10” – is this claim based on their single nucleus RNA sequencing data?\n4. Why is apical-junctional membrane “waviness” or “ruffling” a good surrogate for lateral protrusion formation? Is it just\nbecause of the morphological similarity? It would be good to also show some molecular similarities that justify this as a\nsurrogate for aTL protrusions.\n5. Why does the induction of Prox1-creERT2 at different time points lead to recombination only in aTL vs TAL? Is Prox1\nexpressed in the aTL earlier than in TAL? Is there evidence of this?\n6. Were the 24 hour urine osmolality and urine volume studies done in Cldn10 cKO mice where induction of Cre expression\nwas limited to aTL by Tamoxifen-mediated induction between P2-P7? This is important to clarify, because of course the\nCldn10 cKO where Cre expression is induced at an older age to target both the aTL and TAL will not be able to differentiate\nbetween the effect of aTL vs TAL on urinary concentrating ability. Maybe the Cldn10 cKO specific to aTL and not TAL can be\nre-named to stress the difference if the studies were done in the aTL-specific Cldn10\ncKO. If the studies were done in the\nolder cKO, then they need to be releated in the aTL-specific cKO.\n7. In the last section of the results, it would be good to show whether ZO1 or ZO2 or both colocalize with Claudin-10b in the\naTL – these studies are all in vitro in MDCK, which helps shed some mechanistic insights on how Claudin-10b interacts with\nother tight junction proteins, but again, it’s not a faithful representation of how Claudin-10b actually behaves in the aTL. This\nis obviously very difficult to replicate in vitro because no kidney cells will behave the same outside the kidney, but some high\nresolution co-staining with ZO1 and ZO2 could help strengthen the in vitro findings.\n8. In the discussion, the authors claim that “this finding resolves a decades-old question by establishing that the inner\nmedulla actively participates in urine concentration, rather than merely serving as a passive conduit for osmotic\nequilibration.” This is an inaccurate statement – Claudin-10b is a cation channel that facilitates passive diffusion of cations\nbut not an active transporter like NKCC2 in the TAL.", "reviewer_3": "This study offers a straightforward and rather elegant explanation for how mammals generate highly concentrated urine for\norganismal fluid balance.\nData summary: The ascending thin limb (aTL) of the nephron is long known to reside in the inner medulla, which manifests\nthe highest osmolality in the kidney. This study validated nephron segment-“quasi-specific” genetic lineage labeling tools to\naddress this question, using the Prox1-CreERT2mTmG reporter. Induction with dilute Tamoxifen concentrations allowed\nvisualization of lineage labeled single cell features with unprecedented clarity, revealing time-dependent development\n(weeks-months) of highly branched, membrane interdigitations, resulting in a ‘sunburst-like’ cell presumably suited for ion-\ntransport function, given the expanded junctional seam area for these cells (Fig. 1). To understand genes/protein that drive\nthis morphological transition, the authors carried out single nuclear transcriptomics across post-natal development (Fig. 2),\nidentifying top differentially expressed genes for the aTL cluster, settling on Claudin 10 as a gene that emerges during the\ntemporal window when membrane interdigitation develops (Fig. 2D,E). Since Claudin 10 comes in two functionally distinct\nisoforms (A,B), the authors showed it was the cation-specific 10b isoform that is expressed the aTL (Fig. 2G), consistent with\nit being a candidate for sodium resorption in the kidney.\nThe authors turned to a reductionist claudin-null MDCK cell system to address whether Cldn10b was sufficient to drive\nsimilar apical junction features seen in the aTL (Fig. 3; Fig. S5). MDCK tight junctions are known to display waviness in\nmature monolayers, a feature previously ascribed to the claudins expressed in MDCK. Using this system, the authors\nshowed that Cldn10b is sufficient (in the MDCK-pan claudin-null background) to promote apical junction waviness, using\nestablished calculation methods. Most remarkably, targeted loss of Claudin10 in mice inhibited the apical junction\nwaviness/interdigitation in aTL cells (Fig. 3C-F). Along with these dramatic morphological differences, the authors show\nfunctional differences in urine osmolarity—where the Cldn10 KO mice show more dilute urine, suggestive of less capacity to\nresorb sodium (Fig. 4). To further refine understanding- the authors use a panel of previously established mutants, to show\nthat it is the transcellular-recognition and ZO-1/ZO-2-binding domains of Cldn10, but not its ion-selective function, that is\nrequired for the wavy/interdigitated morphology (Fig. 5).\nAssessment: Overall, this is a clearly presented well designed study that links a spectacular aTL apical junction/membrane\nform to kidney function/generation of concentrated urine. The notion that Cldns are regulators of epithelial membrane\nstructure- not simply barrier function as previously known, is compelling. I have no major concerns, but have technical\nquestions and minor suggestions regarding terminology (e.g., ruffling, protrusions, lateral interdigitations), which may better\nalign with terminology in the epithelial junction field.\nMajor Questions:\n1. Is it possible that the Cldn10 KO alters the time dependence of the membrane elaborations, but does not completely block\nit? How long out were cell phenotypes characterized in the KO condition? Is it possible that the Cldn10 KO merely delays,\nrather that fully blocks the membrane elaborations? Please elaborate.\n2. Is there an overall viability phenotype with the Prox-Cldn10 KO mouse?\n3. For the urine osmolarity measurements in Fig. 4, did the authors use the lowest dose of TMX to bias Cldn10 removal from\nthe medulla, rather than other segments of the nephron?\nMinor:\n1. Lines 9; 97; Line 297: The authors often refer to the apical junction membrane interdigitations as ‘lateral’ interdigitations. I\nthink this could be confusing. Lateral interdigitations have been described in MDCK (PMID: 34099568), which are much\ntaller than the 1um-thin aTL cells (Fig S1). I might refer to these as apical junction interdigitations for better alignment with\nwork of others in MDCK (PMID: 35389423), and in distinction from true basolateral invaginations (PMID: 34099568).\nAlternatively, if Cldn10b really drives lateral interdigitations as well- please show full x-z stacks for images in Fig 5D and F\n(comparing the Cldn10b WT vs PDZ-binding motif mutant as control) to show that this is really a lateral interdigitation, as in\nPMID: 34099568. Also, I might not refer to the MDCK apical waviness as “membrane ruffling” (line 162), as that term is often\nused to describe a behavior of the true apical membrane (not the apical junction), as in a recent study that generates these\nruffles to release membrane tension and favor micropinocytosis (PMID: 40550809).\n2. It does seem that claudin 10 is not very co-local with ZO-1 in the MDCK cell system (Fig. 5D), but rather more basolateral\nto ZO-1. This somewhat undercuts the suggestion that the apical junction/waviness phenotype is due to a\nconstitutive/stoichiometric interaction between Cldn10b/ZO-1, perhaps favoring a more indirect mechanism?\n3. Figure 1G shows a very nice time course of aTL morphological development. Based on this sequence, and absent live\nimaging- I might not refer to these structures as “protrusions”- since protrusive behavior is not yet known. Indeed, given top\ngene in Fig. 2D indicates high expression of an actin severing component- the morphological changes that occur slowly\nbetween post-natal day 14 and 6 weeks might be through some sort of membrane retraction/sculpting mechanism. This is a\nminor quibble, but it is probably best to use a term that is mechanistically agnostic to how the structures are ultimately\ngenerated. Finger-like ‘projections” might be better.\n4. “ultrastructural analysis” (line 70) historically refers to analysis at the EM level, not light microscopic level."}, "author_response": "Re: Response to reviewers, Manuscript #NCOMMS-25-50790-T\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\nReviewer #1:\n1. Can the authors directly measure paracellular sodium transport using their qKO MDCKII model,\nperhaps by TEER, to directly probe the role of Claudin10b, ZO1 and membrane ruffling in sodium\ntransport?\nThank you for this suggestion. We have now added electrophysiology data reporting the TER,\nconductance, P /P , and dilution potential of wild-type MDCKII, quinKO, and quinKO+Cldn10b\nNa Cl\ncells (NEW Supplementary Fig. 7, see below). We find that the quinKO cells have a “leaky”\nparacellular barrier as indicated by low TER and high conductance, and that expression of\nCldn10b confers sodium selectivity, consistent with previous reports1,2. Prior publications have\nalready shown that loss of ZO1 causes epithelial barrier dysfunction1,3.\nNEW Supplementary Figure 7: Claudin-10b confers paracellular sodium selectivity in vitro.\na, Transepithelial resistance in MDCKII cells, quinKO cells, and quinKO+Cldn10b cells.\nb, Monolayer conductance in MDCKII cells, quinKO cells, and quinKO+Cldn10b cells.\nc, Ratio of sodium permeability to chloride permeability (PNa/PCl) in MDCKII cells, quinKO cells,\nand quinKO+Cldn10b cells.\nd, Dilution potentials measured under NaCl gradients in MDCKII cells, quinKO cells, and\nquinKO+Cldn10b cells, demonstrating increased sodium selectivity with Cldn10b expression.\nStatistical significance was assessed using one-way ANOVA with Tukey’s multiple-comparisons\ntest (F = 64.18 (a), 34.41 (b), 50.46 (c), and 539.2 (d), and df = 16). P-value is noted above\nbracket for each comparison. Data represent mean ± s.d of three independent experiments (n=3),\nwith duplicate measurements averaged within each experiment.\nReviewer #2:\nThe focal point of the study, Claudin-10b, is also expressed in the TAL, and there is a study by\nQuintanova et al that describes the role of Claudin-10b in producing a similar morphology of\nmembrane infoldings or protrusions in TAL cells – the authors briefly allude to this feature of\nClaudin-10b and cite the Quintanova et al manuscript in the discussion, but they could spend a\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\nlittle more time describing the similarities and differences between the role of Claudin-10b in TAL\nvs aTL morphologies.\nThank you for this suggestion. Because Prox1-CreERT2; mTmG also recombines in the TAL (Fig.\n1a-c, NEW Supplementary Videos 5-6), we were able to visualize TAL cells with GFP reporter.\nConsistent with Quintanova et al4, we observed that the TAL has basolateral infoldings that are\npositive for Cldn10 (NEW Supplementary Videos 5-6). However, we also note that Cldn10 is\npresent at apical-lateral (tight) junctions, where its levels are substantially higher than along the\nbasolateral membrane (NEW Supplementary Videos 5-6).\nIn contrast, we found that aTL interdigitations occur specifically at the level of the tight\njunctions, where claudin-10b and ZO1 co-localize along the projections (NEW Supplementary\nFig. 5, see pg. 6). By comparison, TAL basal infoldings are confined to the basolateral membrane,\nwhere tight junctions and ZO1 are absent (NEW Supplementary Videos 5-6). Thus, although\nclaudin-10b is associated with membrane elaborations in both segments, these morphologies\noccur in distinct plasma membrane domains and differ in their organization and molecular\ncomponents. Notably, TAL basolateral infoldings are not thought to interdigitate with neighboring\ncells, distinguishing them from aTL interdigitations.\nWe have now expanded the Discussion to explicitly address these similarities and\ndifferences, emphasizing that claudin-10b can influence epithelial morphology in multiple nephron\nsegments but likely does so through distinct molecular mechanisms that reflect segment-specific\ncellular contexts.\n1. In the abstract, the authors state that Claudin-10b “proves both necessary and sufficient” for\ninterdigitation formation – while they show that it is necessary, they have not provided adequate\nevidence to claim that it is “sufficient”. In fact, they show that ZO-1 is required for formation of the\nprotrusions in aTL cells – so Claudin-10b is not sufficient. They also perhaps make this claim\nbecause Claudin-10b can restore membrane waviness in MDCK cells that lack expression of\nother claudins – however, this is an artificial in vitro system that is a tool to study Claudin-10b\nfunction but cannot faithfully recapitulate aTL morphology and function. The ability of Claudin-10b\nto restore membrane waviness in MDCK cells, therefore, does not necessarily indicate that it is\nsufficient for protrusion formation in aTL cells.\nThank you for bringing this to our attention. We agree and have removed all assertions that\nclaudin-10b is sufficient to drive interdigitation from the manuscript.\n2. In the first section of the results, the authors should clearly state that Prox1 expression is very\nspecific to the TAL and aTL and is not expressed in other parts of the inner medulla (namely,\ndescending thin limb and inner medullary collecting duct) as shown by Kim et al. (the paper cited\nby the authors) – this will just help highlight the specificity of their Cre recombinase.\nThank you for this suggestion. We have now explicitly stated in the Results that Prox1 expression\nis enriched the ascending loop of Henle (aTL and TAL) and is not detected in other inner medullary\ntubules, including the descending thin limb and inner medullary collecting duct. We have added\nnew-single nucleus transcriptomic analysis and immunostaining to support this specificity (NEW\nSupplementary Fig. 1, see below).\nIt is worth noting that single nucleus transcriptomics show moderate Prox1 expression in\nthe DCT at 6 weeks, consistent with previous reports5. However, we did not observe any Prox1-\nCreERT2 recombination activity in the DCT (not shown) (see also Supplementary Fig. 10\nlegend).\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\nNEW Supplementary Figure 1: Prox1 is enriched in the ascending limb of the Loop of\nHenle.\na, Single nucleus transcriptomics of Prox1 expression in nephron epithelia at 6 weeks of age. Dot\nsize and color intensity denote mean log expression level.\nb, Immunofluorescence of Prox1-CreERT2; mTmG reporter with GFP (green) and Aqp2\n(magenta, marks collecting ducts) in the 6-week-old mouse kidney. Dotted line denotes IM/OM\nboundary.\nc, Immunofluorescence of Prox1-CreERT2; mTmG reporter with GFP (green) and Aqp1 (cyan,\nmarks descending thin limb and vasculature) in the 6-week-old mouse kidney. Dotted line denotes\nIM/OM boundary.\nd, Immunostaining of Prox1 (green), Aqp2 (magenta) and Aqp1 (cyan) in the inner medulla at 6\nweeks.\nPanels b-d used mixed sex, 6-week-old mice (n=3).\nScale bars = 100 μm (b,c) and 10 μm (d).\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\n3. In the results section titled “Claudin-10b is the aTL isoform”, the authors state “since no other\ntubules in the inner medulla expressed claudin-10” – is this claim based on their single nucleus\nRNA sequencing data?\nThis conclusion is supported by a combination of RT-PCR, single-nucleus RNA sequencing, and\nimmunofluorescence, rather than by transcriptomics alone. RT-PCR performed on dissected\nkidney regions demonstrates that claudin-10b is the isoform expressed in the inner medulla (Fig.\n2g). Single-nucleus RNA sequencing was then used to identify the nephron epithelial populations\nexpressing Cldn10, revealing that within inner medullary nephron epithelial clusters, Cldn10\nexpression is restricted to the aTL cluster.\nImportantly, this conclusion was independently validated at the protein level by\nimmunostaining. As shown in NEW Supplementary Fig. 4, claudin-10 protein localizes to aTL\ncells in the inner medulla but is absent from adjacent inner medullary nephron segments, including\nthe collecting duct and descending thin limb, as well as from non-nephron structures. We have\nclarified this point in the text accordingly.\nNEW Supplementary Figure 4: Claudin-10 marks a unique aTL gene signature.\na, Single nucleus transcriptomics of Cldn10 expression in nephron epithelia at 6 weeks. Dot size\nand color intensity denote mean raw expression level.\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\na, Immunofluorescence of Cldn10 (magenta) in the GFP+ (green) aTL, Na/K-ATPase+ (green)\nTAL, LTL+ (green) PT, and GFP+ (green) Vegfr3+ (blue) renal lymphatics. Images taken in same\ntissue with identical imaging parameters.\nb, Mean expression of claudins in the 6-week aTL cluster. Cldn10 is most abundant by more than\nthreefold. Cation pore-forming claudins denoted in green.\nc, Immunostaining of Cldn10 (magenta), Aqp1 (green) and Aqp2 (cyan) in the inner medulla at 6\nweeks.\na,c used mixed sex, adult mice (n=3).\nScale bars = 10 μm (a,c).\n4. Why is apical-junctional membrane “waviness” or “ruffling” a good surrogate for lateral\nprotrusion formation? Is it just because of the morphological similarity? It would be good to also\nshow some molecular similarities that justify this as a surrogate for aTL protrusions.\nThank you for this comment. As mentioned in the Results, there is currently no established in vitro\nmodel that fully recapitulates epithelial interdigitation. Claudin-10b-induced membrane waviness\nin MDCK cells offers a reductionist system to study the molecular underpinnings of interdigitation\nformation. While this phenotype does not reproduce the full architecture of aTL interdigitations,\nthe in vivo interdigitations and in vitro waviness share similar features, including a high degree of\nmembrane curvature and apical junctions composition, including claudins and ZO1/2. However,\nwe appreciate your point regarding our language. The term “good surrogate” is too strong, and\nwe have removed this language from the manuscript.\n5. Why does the induction of Prox1-CreERT2 at different time points lead to recombination only\nin aTL vs TAL? Is Prox1 expressed in the aTL earlier than in TAL? Is there evidence of this?\nTranscriptomics show that Prox1 expression is enriched in the developing aTL earlier than in the\nTAL (NEW Supplementary Fig. 9), consistent with early tamoxifen induction selectively targeting\nthe aTL.\nNEW Supplementary Figure 9: Prox1 is expressed in the developing aTL.\nSingle nucleus transcriptomic analysis showing Prox1 expression in the aTL and TAL clusters at\nP2, P7, P14, P21 and 6 weeks. Prox1 expression is enriched in the developing aTL at early\npostnatal stages, preceding robust expression in the TAL, before ultimately being expressed in\nboth segments. Dot size and color intensity denote mean raw expression level.\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\n6. Were the 24 hour urine osmolality and urine volume studies done in Cldn10 cKO mice where\ninduction of Cre expression was limited to aTL by Tamoxifen-mediated induction between P2-P7?\nThis is important to clarify, because of course the Cldn10 cKO where Cre expression is induced\nat an older age to target both the aTL and TAL will not be able to differentiate between the effect\nof aTL vs TAL on urinary concentrating ability. Maybe the Cldn10 cKO specific to aTL and not\nTAL can be re-named to stress the difference if the studies were done in the aTL-specific Cldn10\ncKO. If the studies were done in the older cKO, then they need to be related in the aTL-specific\ncKO.\nYes, the urine studies were conducted in Cldn10; Prox1-CreERT2 mice that received aTL-specific\ntamoxifen induction from P2-P7. We appreciate your suggestion to use clearer nomenclature and\nnow refer to these mice as Cldn10ΔaTL to distinguish them from broader aTL+TAL deletions.\n7. In the last section of the results, it would be good to show whether ZO1 or ZO2 or both\ncolocalize with Claudin-10b in the aTL – these studies are all in vitro in MDCK, which helps shed\nsome mechanistic insights on how Claudin-10b interacts with other tight junction proteins, but\nagain, it’s not a faithful representation of how Claudin-10b actually behaves in the aTL. This\nis obviously very difficult to replicate in vitro because no kidney cells will behave the same outside\nthe kidney, but some high resolution co-staining with ZO1 and ZO2 could help strengthen the in\nvitro findings.\nThank you for this suggestion. We now include high resolution co-localization of claudin-10b with\nZO1 and ZO2 in the ascending thin limb (NEW Supplementary Fig. 5). We have added a\nstatement to the Results to reference this data: “These mechanistic insights likely also apply to\nthe in vivo aTL tubule, where claudin-10 co-localizes with ZO1/2.”\nNEW Supplementary Figure 5: Claudin-10b localizes to aTL apical-lateral tight junctions in\nthe aTL.\na, Immunofluorescence of ZO1 (green) and Cldn10 (magenta) in the aTL shows co-localization.\nb, Immunofluorescence of ZO2 (cyan) and Cldn10 (magenta) in the aTL shows co-localization.\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\nEach experiment used mixed sex, adult mice (n=3).\nScale bars = 10 μm (a,b)\n8. In the discussion, the authors claim that “this finding resolves a decades-old question by\nestablishing that the inner medulla actively participates in urine concentration, rather than merely\nserving as a passive conduit for osmotic equilibration.” This is an inaccurate statement – Claudin-\n10b is a cation channel that facilitates passive diffusion of cations but not an active transporter\nlike NKCC2 in the TAL.\nThank you for bringing this to our attention. We did not intend to imply a mechanism of transport\nand have removed this wording to improve clarity.\nReviewer #3:\n1. Is it possible that the Cldn10 KO alters the time dependence of the membrane elaborations,\nbut does not completely block it? How long out were cell phenotypes characterized in the KO\ncondition? Is it possible that the Cldn10 KO merely delays, rather that fully blocks the membrane\nelaborations? Please elaborate.\nThank you for this question. Cldn10-deficient aTL cells still lack interdigitations at 1 year of age\n(NEW Rebuttal Fig. 1, see below). This persistent absence of interdigitations at a time point well\nbeyond normal developmental maturation argues against a temporal delay and instead indicates\nthat Cldn10 is required for interdigitation.\nRebuttal Fig. 1 Cldn10-deficient tubules lack interdigitation at 1 year of age.\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\nImmunostaining for GFP (green), Cldn10 (magenta), and ZO1 (cyan) in 1-year-old control (CTL)\nand Cldn10 cKO (MUT) aTL tubules. MUT tubules lack interdigitation, as evidenced by persistent\nsimplification of ZO1 staining. n=2 mice per genotype.\n2. Is there an overall viability phenotype with the Prox-Cldn10 KO mouse?\nWe did not observe a viability defect in the Cldn10; Prox1-CreERT2 mutant mice. The Cldn10ΔaTL\nmice appear healthy despite urinary concentrating defects. To bolster this statement, we have\nadded a sentence to the Results stating that there is no difference in body weight between control,\nheterozygous and Cldn10; Prox1-CreERT2 mutant mice at 10 weeks of age. Although we did not\nperform a Kaplan-Meier survival analysis, a previous report using broader deletion of Cldn10\nusing Ksp-Cre reported that mutant mice are viable and fertile with no obvious growth defects6,\nconsistent with our observations.\n3. For the urine osmolarity measurements in Fig. 4, did the authors use the lowest dose of TMX\nto bias Cldn10 removal from the medulla, rather than other segments of the nephron?\nYes, the urine studies were conducted in Cldn10; Prox1-CreERT2 mice that received the aTL-\nspecific tamoxifen dosing from P2-P7, now denoted as Cldn10ΔaTL for increased clarity.\nMinor:\n1. Lines 9; 97; Line 297: The authors often refer to the apical junction membrane interdigitations\nas ‘lateral’ interdigitations. I think this could be confusing. Lateral interdigitations have been\ndescribed in MDCK (PMID: 34099568), which are much taller than the 1um-thin aTL cells (Fig\nS1). I might refer to these as apical junction interdigitations for better alignment with work of others\nin MDCK (PMID: 35389423), and in distinction from true basolateral invaginations (PMID:\n34099568). Alternatively, if Cldn10b really drives lateral interdigitations as well- please show full\nx-z stacks for images in Fig 5D and F (comparing the Cldn10b WT vs PDZ-binding motif mutant\nas control) to show that this is really a lateral interdigitation, as in PMID: 34099568. Also, I might\nnot refer to the MDCK apical waviness as “membrane ruffling” (line 162), as that term is often\nused to describe a behavior of the true apical membrane (not the apical junction), as in a recent\nstudy that generates these ruffles to release membrane tension and favor micropinocytosis\n(PMID: 40550809).\nThank you for these recommendations. We initially used the term “lateral” to distinguish aTL\ninterdigitations from apical projections such as microvilli and stereocilia; however, we agree that\nthis terminology is confusing. We have therefore revised the manuscript to refer to the in vivo\nstructures as “apical-junctional interdigitations,” as suggested.\nWe have also updated our terminology for the in vitro membrane phenotype, as the\nclaudin-10b-induced waviness occurs specifically at apical junctions and does not occur along\nthe basolateral membrane (see 2. below). Accordingly, we now refer to this phenotype as “apical-\njunctional membrane waviness”.\nWe also appreciate your clarification regarding the term “membrane ruffling.” To avoid\nconfusion with apical membrane ruffles described in other contexts, we now refer to the in vitro\nmembrane morphology as “waviness” rather than “ruffling”.\n2. It does seem that claudin 10 is not very co-local with ZO-1 in the MDCK cell system (Fig. 5D),\nbut rather more basolateral to ZO-1. This somewhat undercuts the suggestion that the apical\nBioRender, Created in BioRender. Felan, B. (2026) https://BioRender.com/kqhmxig\nRe: Response to reviewers, Manuscript #NCOMMS-25-50790-T\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\njunction/waviness phenotype is due to a constitutive/st o ichiometric interaction between\nCldn10b/ZO-1, perhaps favoring a more indirect mechanism?\nThank you for this comment. Fig. 5d is a 3D projection because the MDCK monolayer is not\nentirely flat, and a projection allows visualization of the full apical plane of the epithelium.\nIn MDCK cells, several claudins, including claudin-10, localize to both the apical-lateral (tight\njunction) domain AND the lateral membranes7,8. Importantly, claudin-10b-induced waviness is\nrestricted to the apical-lateral junctions where claudin-10b co-localizes with ZO1, and not at the\nlateral membrane where only claudin-10b is present (NEW Supplementary Fig. 12a). Thus, the\ndata supports, rather than undercuts, the necessity of claudin-10b-ZO1 interaction for membrane\nwaviness. We have added this clarification to the legend of Supplementary Fig. 12a.\nNEW Supplementary Figure 12: ZO1, but not ZO2, is required for Cldn10-mediated\nmembrane interdigitation.\na, Immunostaining for ZO1 (green) and Cldn10 (red) in quinKO+Cldn10b cells at the apical-lateral\n(tight junction) domain (a’) and lateral membrane (a’’). Claudin-10 localizes to both tight junctions\nand lateral membranes while ZO1 is restricted to tight junctions. Membrane waviness only occurs\nat the tight junctions where claudin-10 and ZO1 co-localize (a’, a’’). a’’’ shows a schematic of the\napical-lateral (tight) junctions and the lateral membrane.\nb, Co-culture of quinKO+Cldn10b cells and quinKO+Cldn10b+ZO2 KO cells. Immunostaining for\nZO1 (green), ZO2 (magenta) and Cldn10 (cyan). White boxes denote insets. Insets 1 and 2\nindicate junctions between quinKO+Cldn10b cells and quinKO+Cldn10b+ZO2 KO cells,\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\nrespectively. Loss of ZO2 (Inset 2) results in mildly reduced, but not absent, apical-junctional\nwaviness. Cldn10 is still recruited to the membrane in ZO2-deficient cells.\nAll data represent 3 independent experiments (n=3).\nScale bars = 10 μm (a,b), 5 μm (Insets of b).\n3. Figure 1G shows a very nice time course of aTL morphological development. Based on this\nsequence, and absent live imaging- I might not refer to these structures as “protrusions”- since\nprotrusive behavior is not yet known. Indeed, given top gene in Fig. 2D indicates high expression\nof an actin severing component- the morphological changes that occur slowly between post-natal\nday 14 and 6 weeks might be through some sort of membrane retraction/sculpting mechanism.\nThis is a minor quibble, but it is probably best to use a term that is mechanistically agnostic to\nhow the structures are ultimately generated. Finger-like ‘projections” might be better.\nThank you for this suggestion. We now refrain from using the word “protrusions”, substituting\n“projections” and “interdigitations” instead.\n4. “ultrastructural analysis” (line 70) historically refers to analysis at the EM level, not light\nmicroscopic level.\nThank you for bringing this to our attention. We replaced “ultrastructural analysis” with “high\nresolution imaging.”\nWarshaw et al., “An Epithelial Morphogenetic Program for Maximal Urine Concentration.”\nReferences\nepithelial polarity. J Cell Biol 218, 3372-3396 (2019).\nhttps://doi.org/10.1083/jcb.201812157\nsubtypes in epithelial cells. Cell Struct Funct 48, 1-17 (2023).\nhttps://doi.org/10.1247/csf.22068\njunction solute barrier through coupling to the perijunctional cytoskeleton. Mol Biol\nCell 20, 3930-3940 (2009). https://doi.org/10.1091/mbc.e09-04-0320\ninfoldings of the thick ascending limb. Ann N Y Acad Sci 1517, 266-278 (2022).\nhttps://doi.org/10.1111/nyas.14882\ndistal convoluted tubule causes hypomagnesemia via reduced expression of TRPM6\nand NCC. Pflugers Arch 473, 79-93 (2021). https://doi.org/10.1007/s00424-020-\n02491-1\nimpairs paracellular sodium permeability and leads to hypermagnesemia and\nnephrocalcinosis. Proc Natl Acad Sci U S A 109, 14241-14246 (2012).\nhttps://doi.org/10.1073/pnas.1203834109\ndecreases paracellular conductance through a selective decrease in sodium\npermeability. J Clin Invest 107, 1319-1327 (2001). https://doi.org/10.1172/JCI12464\nclaudins but not occludin are added to the basal side of the tight junction. Mol Biol\nCell 30, 1406-1424 (2019). https://doi.org/10.1091/mbc.E19-01-0008\n11"}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is an excellent contribution to the literature and the authors are to be congratulated.\nBen Humphreys", "reviewer_2": "I would like to thank the authors for their thorough responses to the previous round of review. The revisions have\nsignificantly improved the clarity of this important manuscript. However, upon reading the revised version of the manuscript,\nadditional questions have emerged that require further clarification before the manuscript is suitable for publication.\n1) The authors refer to the Cldn10 deletion mouse as a conditional knockout. Since the deletion is induced by Tamoxifen-\nmediated translocation of the Cre recombinase to the nucleus, this is technically an inducible knockout rather than a\nconditional knockout. This terminology should be corrected for technical accuracy.\n2) What ages are the mice whose kidneys are shown in Figures 3c-g? Can the authors also provide more clarity regarding\nthe mouse ages in every figure? For instance, “adult” can indicate quite a broad age range. It would be preferred if they can\nprovide ages in weeks or months for each figure.\n3) At what age was Cldn10 deletion induced in the mice in Figure 3? They note in Supplemental Figure 10 that deletion was\ninduced at P2-P7, P5-P13, and P28-P32, but they do not specify which of these deletion timepoints was used for Figure 3.\n4) The authors note using mixed sex mice for each experiment – are they able to comment on whether they noted any\ndifferences in male mice versus female mice?\n5) At what age was Cldn10 expression checked in the lymphatics by immunostaining in supplemental Figure 4a? It would\nbe good to ensure that Cldn10 is not expressed in lymphatics at the timepoints used for all Cldn10 deletion experiments (P2-\nP7, P5-P13, and P28-P32) - this will help eliminate the possibility of the phenotype resulting from off-target effects of the\nProx1-CreERT2 on the lymphatics.\nThis is especially important, as areas of recombination are seen as GFP expression in between tubules in Supplemental\nFigure 10 (specifically in the cortex and OSOM) – these areas are presumably the lymphatics that are known to express\nProx1 as the authors point out at the beginning of the Results section. However, if Cldn10 is not expressed in the lymphatics,\nProx1-CreERT2 expression in the lymphatics should not affect the phenotype in these mice. Therefore, showing the\nabsence of Cldn10 expression in the lymphatics at each deletion timepoint is very important.\n6) Since the same mice were used for both baseline 24 hour urine studies and the subsequent water deprivation test, can\nthe authors provide a graph of the change in urine osmolality between normal conditions and water deprivation for each\nmouse? In Figures 4A and 4C, it looks like the urine osmolality in mutants does increase (< 2000 mOsm/kg to > 2000\nmOsm/kg) after water deprivation, so they don't seem to have complete inability to concentrate urine. It would be good to see\nthe delta or change in urine concentration between normal and water deprivation conditions in mutants vs controls. Can the\nauthors also provide the urine volume in 24 hrs with the water deprivation if these data are available as well? Would expect\nthe volume to decrease much more significantly in the control/het mice compared to the mutants.\n7) In the discussion, the authors claim that the urinary concentrating function of the aTL “is dependent on a unique\nmorphological adaptation” and that “this loss of claudin-10b-mediated aTL interdigitation causes a urinary concentration\ndefect, underscoring how intricate cell architectures enable unique physiological capabilities”.\nHowever, I do not believe that the authors have sufficient evidence to claim that the urinary concentrating defect due to\nCldn10 deletion is a direct result of the loss of interdigitations. They have clearly shown that Cldn10b is needed for formation\nof interdigitations/membrane waviness and the ability to optimally concentrate urine (most likely via the facilitation of\nparacellular sodium transport), but they have not shown that the sodium transport is dependent on the interdigitations.\nEven in cell culture, they showed that expression of the F146A mutant abolishes transcellular adhesions and membrane\nwaviness. However, they have not presented data showing an effect of this mutation/loss of membrane waviness on the\nsodium permeability of the cells. Thus, both the mouse model and the cell culture model provide insufficient evidence to link\nthe aTL’s interdigitations to its urinary concentrating ability.", "reviewer_3": "Very responsive to all of my questions/concerns."}, "author_response": "Warshaw et. al., An Epithelial Morphogenetic Program for Maximal Urine Concentration. #NCOMMS-25-50790A. CONFIDENTIAL.\nRESPONSE TO REVIEWERS\nReviewer #1 (Remarks to the Author)\nThis is an excellent contribution to the literature and the authors are to be congratulated.\nBen Humphreys\nReviewer #2 (Remarks to the Author)\nI would like to thank the authors for their thorough responses to the previous round of\nreview. The revisions have significantly improved the clarity of this important\nmanuscript. However, upon reading the revised version of the manuscript, additional\nquestions have emerged that require further clarification before the manuscript is\nsuitable for publication.\n1) The authors refer to the Cldn10 deletion mouse as a conditional knockout. Since the\ndeletion is induced by Tamoxifen-mediated translocation of the Cre recombinase to the\nnucleus, this is technically an inducible knockout rather than a conditional knockout.\nThis terminology should be corrected for technical accuracy.\nThank you for pointing out this technical inaccuracy. We now only use the term\n“conditional knockout” in reference to the insertion of lox-p sites flanking exons 2 and 3\nof Cldn10. We refer to inducible knockout mice (Cldn10f/f;Prox1-CreERT2;mTmG mice)\nas Cldn10 mutant mice, or MUT for short.\n2) What ages are the mice whose kidneys are shown in Figures 3c-g? Can the authors\nalso provide more clarity regarding the mouse ages in every figure? For instance,\n“adult” can indicate quite a broad age range. It would be preferred if they can provide\nages in weeks or months for each figure.\nThe mice used in Figures 3c-g are 10 weeks old. We have added this information to the\nfigure legend of Figure 3. The specific age (in weeks or months where relevant) is now\nstated for each figure panel in the legends.\n3) At what age was Cldn10 deletion induced in the mice in Figure 3? They note in\nSupplemental Figure 10 that deletion was induced at P2-P7, P5-P13, and P28-P32, but\nthey do not specify which of these deletion timepoints was used for Figure 3.\nMice received tamoxifen every other day from P5-P13 for the experiments shown in\nFigure 3. This is now stated in the figure legend.\n4) The authors note using mixed sex mice for each experiment – are they able to\ncomment on whether they noted any differences in male mice versus female mice?\nNo, we did not observe any differences between male and female mice in our\nexperiments. We simply note that we used mixed sex mice for each experiment to\nacknowledge that the reported observations (i.e. disrupted interdigitation in Cldn10\nmutant mice) are applicable to both male and female mice. We added the following\nstatement to the “Animals” section of the methods for increased clarity: “Male and\nfemale mice were used for each experiment (see figure legend for details), and all\nobservations reported are applicable to both sexes.”\n5) At what age was Cldn10 expression checked in the lymphatics by immunostaining in\nsupplemental Figure 4a? It would be good to ensure that Cldn10 is not expressed in\nlymphatics at the timepoints used for all Cldn10 deletion experiments (P2-P7, P5-P13,\nand P28-P32) - this will help eliminate the possibility of the phenotype resulting from off-\ntarget effects of the Prox1-CreERT2 on the lymphatics.\nThis is especially important, as areas of recombination are seen as GFP expression in\nbetween tubules in Supplemental Figure 10 (specifically in the cortex and OSOM) –\nthese areas are presumably the lymphatics that are known to express Prox1 as the\nauthors point out at the beginning of the Results section. However, if Cldn10 is not\nexpressed in the lymphatics, Prox1-CreERT2 expression in the lymphatics should not\naffect the phenotype in these mice. Therefore, showing the absence of Cldn10\nexpression in the lymphatics at each deletion timepoint is very important.\nThank you for this suggestion. Supplemental Fig. 4a used 2-month-old mice. We have\nnow added a NEW Supplementary Fig. 11 to include immunostaining for claudin-10 in\nthe renal lymphatics (marked by LYVE-1) at P3, P10, and P29, which are timepoints\nrepresentative of each tamoxifen regimen. At each of these ages, claudin-10 is absent\nfrom the lymphatics, therefore eliminating the possibility of off-target lymphatic\nphenotypes. We now clarify this point in the text.\nNEW Supplementary Figure 11: Cldn10 does not co-localize with LYVE-1, a\nmarker of renal lymphatics.\nImmunostaining for Cldn10 and lymphatic marker LYVE-1 at P3, P10 and P29. White\ndashes outline LYVE-1-positive regions. Images were acquired from the renal cortex in\nareas adjacent to Cldn10⁺ cortical TAL tubules.\nImages representative of n=4 mixed sex mice analyzed per age. Scale bar = 10 µm.\n6) Since the same mice were used for both baseline 24 hour urine studies and the\nsubsequent water deprivation test, can the authors provide a graph of the change in\nurine osmolality between normal conditions and water deprivation for each mouse? In\nFigures 4A and 4C, it looks like the urine osmolality in mutants does increase (< 2000\nmOsm/kg to > 2000 mOsm/kg) after water deprivation, so they don't seem to have\ncomplete inability to concentrate urine. It would be good to see the delta or change in\nurine concentration between normal and water deprivation conditions in mutants vs\ncontrols. Can the authors also provide the urine volume in 24 hrs with the water\ndeprivation if these data are available as well? Would expect the volume to decrease\nmuch more significantly in the control/het mice compared to the mutants.\nWe appreciate this suggestion. Because baseline and water deprivation studies were\nperformed sequentially in the same mice, we have now added paired analyses showing\nthe urine osmolality for each animal under baseline condition and after 24-hour water\ndeprivation (NEW Supplementary Fig. S12). As expected, these analyses show that\nmutant mice retain the ability to increase urine osmolality in response to dehydration,\nhowever they fail to achieve the same maximal urine osmolality reached by control and\nheterozygous mice. We have revised the text to clarify that this represents a reduced\nmaximal concentrating capacity rather than a complete inability to concentrate urine.\nNEW Supplementary Fig. 12. Urine concentrating ability at baseline and after 24-\nhour water deprivation in individual animals.\na, Urine osmolality at baseline (BL) and following 24-hour water deprivation (DEP) in\nindividual control (CTL, green) (n=12), heterozygous (HET, blue) (n=9) and Cldn10\nmutant (MUT, purple) (n=9) mice. Each line represents one animal.\nb, Grouped data from panel a showing mean urine osmolality at BL and DEP for CTL\n(n=12), HET (n=9) and MUT (n=9) mice. Data is shown as mean ± s.d.\nUrine osmolality is the most sensitive and physiologically relevant readout of renal\nconcentrating capacity in water deprivation studies and therefore was the primary\nendpoint in our analysis. We did quantify 24-hour urine volume during water deprivation\nand observed a trend toward higher urine volume in Cldn10 mutant mice compared with\ncontrol and heterozygous littermates following 24-hour water deprivation (Rebuttal Fig.\n1a). However, urine volume measurements under dehydration conditions are\nsubstantially more variable than urine osmolality measurements (Manuscript Fig. 4,\nRebuttal Fig. 1a,b), which likely contributes to the lack of statistical significance. This is\na common occurrence with 24-hour urine collections as metabolic cages are susceptible\nto technical variability, including urine droplets sticking to the collecting funnel and\nevaporation of collected samples. Thus, the spot urine osmolality measured at the end\nof the 24-hour deprivation period is the most reliable indicator of urine concentrating\nability. Importantly, mutant mice have reduced maximal urine osmolality (Manuscript\nFig. 4c), consistent with a reduction in maximal concentrating capacity.\nRebuttal Fig. 2. 24-hour water deprivation urine volume.\na, 24-hour water deprivation urine volume in control (CTL, green) (n=12), heterozygous\n(HET, blue) (n=9) and Cldn10 mutant (MUT, purple) (n=9) mice. Box-and-whiskers plot\nshow the interquartile range, the mean, and the minimum and maximum data points,.\nStatistics are the results of a Kruskal Wallis test with Dunn’s multiple comparisons test.\nb, Tabulated values from panel a shown as mean ± s.d.\n7) In the discussion, the authors claim that the urinary concentrating function of the aTL\n“is dependent on a unique morphological adaptation” and that “this loss of claudin-10b-\nmediated aTL interdigitation causes a urinary concentration defect, underscoring how\nintricate cell architectures enable unique physiological capabilities”.\nHowever, I do not believe that the authors have sufficient evidence to claim that the\nurinary concentrating defect due to Cldn10 deletion is a direct result of the loss of\ninterdigitations. They have clearly shown that Cldn10b is needed for formation of\ninterdigitations/membrane waviness and the ability to optimally concentrate urine (most\nlikely via the facilitation of paracellular sodium transport), but they have not shown that\nthe sodium transport is dependent on the interdigitations.\nEven in cell culture, they showed that expression of the F146A mutant abolishes\ntranscellular adhesions and membrane waviness. However, they have not presented\ndata showing an effect of this mutation/loss of membrane waviness on the sodium\npermeability of the cells. Thus, both the mouse model and the cell culture model provide\ninsufficient evidence to link the aTL’s interdigitations to its urinary concentrating ability.\nWe agree and thank the reviewer for this comment. We have modified the manuscript\nand discussion to avoid implying that the urinary concentrating defect is a direct result of\nthe loss of interdigitation. Specifically, we now state “Here, we report that the aTL is\nnecessary for urinary concentration and identify tight junction protein claudin-10b as a\ndual regulator of aTL form and function.” and “Furthermore, loss of claudin-10b-\nmediated aTL interdigitation is associated with a urinary concentration defect, raising\nimportant questions regarding how intricate cell architectures may enable unique\nphysiological capabilities.”\nReviewer #3 (Remarks to the Author)\nVery responsive to all of my questions/concerns.\n6"}]

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