Energy-sensing molecule RORγ regulates cholesterol metabolism and immune signaling in diabetic kidney disease and aging

Domain: chemistry
Published: 2026-02-18
Editorial outcome: major_revision

Abstract
Aging is a major risk factor for diabetic kidney disease (DKD), with both conditions exhibiting similar renal pathology. We identify the energy-sensing molecule Retinoic acid-related orphan receptor γ (RORγ) as significantly downregulated in diabetic and aged kidneys. Tubule-specific RORγ deficiency exacerbates kidney injury, whereas its overexpression protects. Mechanistically, RORγ stabilizes insulin-induced gene 1 (INSIG1) by upregulating the deubiquitinase YOD1 and enhancing AMPK activity via CAB39, which together promote INSIG1 phosphorylation and subsequent stabilization. Stabilized INSIG1 potently blocks the ER-to-Golgi transport and activation of SREBP2 (cholesterol synthesis) and STING (inflammatory signaling). In diabetes, RORγ itself is suppressed transcriptionally by CTCF and functionally by impaired AMPK/SIRT1 signaling, which hinders its activation. Importantly, administration of a RORγ agonist or RORγ-enriched exosomes effectively alleviates diabetic kidney injury. Thus, RORγ emerges as a key regulatory node that mitigates DKD and renal aging by co-regulating AMPK-mediated metabolic and STING-driven innate immune pathways through INSIG1 stabilization.

Introduction
Lipid metabolism disorders, including dyslipidemia, are prevalent across all stages of diabetic kidney disease (DKD)1. Research has demonstrated a positive correlation between renal lipid accumulation and the extent of tubulointerstitial damage in DKD2–4. Proposed mechanisms leading to lipid accumulation in proximal tubules include increased lipid uptake and synthesis and diminished β-oxidation and cholesterol efflux5–7. Previous studies suggest that disulfide-bond A oxidoreductase-like protein protects against lipid-induced renal damage in DKD through enhanced triglyceride breakdown and decreased cholesterol synthesis3. Intracellular cholesterol sensing is mainly regulated via sterol regulatory element-binding protein (SREBP and its known isoforms SREBP-1a, SREBP-1c, and SREBP-2), an endoplasmic reticulum (ER) residents. Newly synthesized SREBPs are held in an inactive state on the ER membrane by associating with SREBP-cleavage activating protein (SCAP)8. When cellular sterol levels are sufficient, SCAP restrains SREBP on the ER membrane by interacting with insulin-induced genes (INSIGs)9,10. Elevated SREBP expression contributes to kidney damage in obesity-related diabetes and in mice fed on a high-fat diet (HFD)6,11–13. Correspondingly, SREBP isoforms inhibition attenuates renal phenotypes such as albuminuria or mesangial expansion in age-related renal disease and DKD11,14–16. Although the role of SREBP in exacerbating renal pathology is increasingly recognized, the precise molecular mechanisms underlying its abnormal activation in renal tubules remain poorly defined.

In DKD progression, immunopathology is initiated by the abnormal release of self-DNA derived from the nuclei or mitochondria of podocytes. This release activates cyclic GMP-AMP synthase (cGAS) to produce cyclic GMP-AMP (cGAMP)17. cGAMP then directly activates the stimulator of interferon genes (STING) signaling, which is a critical player in metabolic inflammation18. Activation of STING in a cGAS- and cGAMP-independent manner is also possible and has been described previously in studies of STING gain-of-function mutants19–22. Both ligand-dependent and -independent STING activation require STING translocation from the ER to the ER-Golgi intermediate compartment and Golgi23. Mitochondrial dysfunction and the subsequent activation of the mtDNA-cGAS-STING pathway in tubular cells are critical regulators of kidney injury24. Cisplatin induced mtDNA leakage into the cytosol in tubules, with subsequent activation of the cGAS-STING pathway, thereby triggering inflammation and acute kidney injury progression, which is improved in STING-deficient mice24. A recent study showed that the enrichment of m6A in the cGAS-STING pathway led to increased mRNA stability for inflammatory genes cGAS and STING1, which in turn, induced a sterile inflammatory response and kidney fibrosis25. The activation of the cGAS-STING pathway may exacerbate renal aging, particularly in renal tubular epithelial cells, as evidenced by the diminished expression of senescence-associated secretory phenotypes with the use of the STING inhibitor, suggesting its potential role in cellular aging26. Despite the importance of STING in modulating inflammation, little is known about the master regulator of STING in renal tubules that operates in a cGAS- and cGAMP-independent manner and how this regulator controls STING signaling.

Aging is a significant risk factor for DKD27. High glucose stimulation has been shown to accelerate cellular senescence in several types of kidney cells28. The aging kidney also shares many features of DKD, including glomerulosclerosis, tubulointerstitial fibrosis, and tubular atrophy27. The pathophysiology of age-associated renal injury is multifactorial and includes the progressive loss of nephrons, cell senescence, inflammation, lipid accumulation, and dysfunctional mitochondria with ROS production29. These findings suggest that integrative research on aging and DKD is important for the development of novel diagnostic and therapeutic strategies. To explore the shared pathogenic mechanisms of DKD and aging, we performed comparative transcriptomic analyses of renal tissues from diabetic mice, aged mice, and human patients with diabetes. Through the intersection analysis of these datasets, we observed a significant downregulation of the transcription factor Retinoic acid-related orphan receptor gamma (RORγ) in all three datasets. RORγ and the related RORα and RORβ constitute a subfamily of the nuclear receptor superfamily of transcription factors that are attractive therapeutic targets for metabolic and autoimmune diseases30. RORγ exists in two subtypes, namely RORγ1 (RORγ) and RORγ2 (commonly referred to as RORγt), the latter of which is the primary transcription factor for the cytokine IL-17 and is mainly expressed in Th17 cells, γδ T cells, and type 3 innate lymphocytes. Research on RORγ typically concentrates on immune cells31–33, but its role in kidney function is relatively unknown. By interrogating the role of RORγ in the development of DKD, here we identify RORγ as a positive feedback regulator of AMP-activated protein kinase (AMPK), which senses the cellular energy status and thereby limits the excessive intracellular cholesterol synthesis mediated by SREBP2 and inhibit the STING signaling pathway induced by the diabetic milieu. Therefore, our findings define RORγ as a previously unsuspected master regulator of cholesterol biosynthesis and inflammation in DKD development and renal aging, and identify it as an attractive therapeutic target.

Results
RORγ deficiency in kidneys of DKD mouse models and patients
To explore the shared pathogenic mechanisms of DKD and aging

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Xiang et al. investigate the mechanism of RORγ-STING axis activation in DKD and aging models. Using a sophisticated combination of ex vivo, in vitro, in vivo experiments, and in silico analysis, the authors elegantly demonstrate that in tubular cells under DKD conditions:\n1) CTCF downregulates RORγ transcription, suppressing YOD1 expression and leading to increased INSIG1 ubiquitination. This destabilizes INSIG1, promoting the trafficking of STING and SREBP2 from the ER to the Golgi and facilitating STING activation.\n2) Reduced CAB39 levels suppress AMPK activity, inhibiting INSIG1 phosphorylation and promoting its ubiquitination. This also impairs RORγ phosphorylation and nuclear translocation, reducing its transcriptional activity.\n3) Reduced SIRT1 activity increases RORγ acetylation, impairing its DNA binding and transcriptional activity.\nThe authors also demonstrate the protective effects of RORγ overexpression against DKD. Furthermore, the therapeutic candidate identified by the authors targets the same mechanisms and has shown promising effects in treating DKD.\nAlthough SREBP/SCAP-STING activation has been previously demonstrated (Nature. 2021 Aug;596(7873):570-575. iScience. 2023 Apr 8;26(5):106613.), this study explores upstream regulators, targets new organs and diseases, and identifies therapeutic candidates highlighting the study's clinical significance, providing solid conceptual advances and comprehensive data. The study is groundbreaking and well-completed at a high level.\nBelow are some comments for improvement:\nMajor Comments:\nComment 1. Validation data on aging are limited, which makes the story somewhat unclear.\nComment 2. Fig. 1F and G1: The control of MCD with nephrotic-range proteinuria raises concerns about the comparison.\nComment 3. STING expression changes in RORγ KO/overexpression mice, GMNT, and exoRORγ treatment are confusing. Since STING downregulation appears beneficial for kidney protection, how do the authors explain these observations?\nComment 4. Expression of key molecules such as INSIG1 and YOD1 should be validated in human datasets.\nComment 5. Fig. 7D: The fold changes in ChIP-qPCR are small. Adding ChIP-seq data would strengthen the conclusions.\nComment 6. The authors should consider showing DNA methylation changes upon 5-aza treatment.\nComment 7. The explanation of the models in the section \"K37R mutation potentiates, while S510A mutation diminishes, the Anti-DKD activity of RORγ\" is insufficient, making it difficult to understand. Please improve this.\nMinor Comments:\nComment 1. A graphical abstract in figures would greatly aid readers in understanding the extensive data and mechanisms presented.\nComment 2. Several sentences are redundant and should be simplified (e.g., Lines 256-268).\nComment 3. Line 600: \"RORC\" is a typographical error. Additionally, Fig. 1C refers to 18 hours, not 24 hours.\nComment 4. Line 643: The conclusion of this section would benefit from emphasizing that these phenomena occur during the early phase.\nComment 5. Include brief descriptions of pharmacological agents used (e.g., actinomycin D, MG132, TSA, NAM) to assist readers.\nComment 6. Consider moving the description of exoRORγ to the section on RORγ overexpression for better logical flow.", "reviewer_2": "A. Summary of the key results\nThis study investigates the role of Retinoic acid-related orphan receptor γ (RORγ) in regulating inflammation and its impact on diabetic kidney disease (DKD) progression. Using in vitro and in vivo mouse models, the authors report that RORγ deficiency accelerates diabetes or aging-induced kidney injury via STING-dependent but cGAS-independent inflammation. They propose a mechanism where RORγ deficiency suppresses STING signaling through the expression and activation of the deubiquitinase YOD1 and the Calcium-binding protein 39 (CAB39)-AMPK complexes. These proteins stabilize INSIG1 in the ER, blocking STING translocation from the ER to the Golgi, which is essential for STING activation. The authors also suggest that under diabetic conditions, RORγ expression is downregulated, and reduced SIRT1 activity increases RORγ acetylation, impairing its DNA binding and transcriptional activity.\nB. Originality and significance:\nWhile the findings are considerably novel and interesting, however, the mechanistic aspects are scattered, making the study overwhelming and confusing. Focusing on one mechanism in greater depth would be more effective than attempting to elucidate multiple mechanisms superficially. \nC. Data & methodology:\nThe data and methodological approaches seem appropriate for the most part. I have provided specific suggestions on areas of improvement. The data is packed and overwhelming to go through. As noted above, it would have been more appropriate if the authors investigated one mechanism in depth rather than several superficially. Sometimes less is more.\nD. Appropriate use of statistics:\nThe statistics seem appropriately applied.\nE. Conclusions;\nMost of the conclusions seem reliable. As noted below in my suggestions, I am entirely convinced that increased STING signaling in the absence of RORy is due to increased ER to Golgi translocation of STING or increased expression or stability. Also, it is unclear how STING signaling is initiated independently of cGAMP/cGAS and how this culminates in the induction of IFN genes independently of IRF3.\nF. Suggested improvements:\nComment 1. The claim of cGAS-independent STING activation lacks clarity. The authors should explain how STING-driven inflammation occurs independently of the cGAMP/cGAS axis and how this leads to the induction of IFNs and inflammatory genes.\nComment 2. The study lacks data on phosphorylated forms of STING and the downstream transcription factor IRF3. These should be included in relevant figures (e.g., Fig. 3B,D,E,H, Fig. 4A,B,F,I,J,K) to support the proposed mechanism.\nComment 3. The proposed mechanism of RORγ suppressing STING signaling by affecting its translocation from the ER to the Golgi requires verification through microscopy, preferably live-cell imaging, to clearly demonstrate this effect.\nComment 4. The authors consistently show increased STING levels in the absence of RORγ. If indeed RORγ only impedes the translocation of STING to the Golgi and not its stability, how does this lead to the increase in STING levels? The authors should provide data on the effect of RORγ the expression or stability of STING.\nComment 5. The analysis of RORγ's impact on INSIG1 ubiquitination (Fig. 5 and Fig. S4A,B) is superficial. More robust methods, such as TUBE and UbiCrest assays (e.g. as in PMID: 30405132), should be employed to define the ubiquitin linkages involved in RORγ-YOD1 mediated control of INSIG1.\nComment 6. The manuscript is replete with many grammatical errors and typos. They should very carefully weed them out.", "reviewer_3": "The report by Xiang, Yang, and Ma et al is a wide-ranging study that isolates a new role for the transcription factor RORy in kidney disease. Previous work has shown that STING signaling is regulated by SREBP2/Scap trafficking from the ER to Golgi. Scap is inhibited by its co-factor Insig, which forms a sterol-mediated complex with Scap. Here, the authors propose that loss of RORy drives STING signaling by reducing the stability of Insig and thereby increasing SREBP transport. Two mechanisms are proposed: First, RORy increases expression of a DUB (Yod1) that acts on Insig-1; and second, RORy increases expression CAB39, a scaffold protein that leads to increased AMPK activity, which phosphorylates Insig-1 and reduces its interaction with its E3 ligase, GP78. My evaluation focused on this Insig work. Strengths of the paper include the use of established mutants of Insig and SREBP (Fig, 4 G,H,K) and the beautiful cell fractionation result for Scap/Insig from WT and RORy KO cells (Fig 4B). My major concern, detailed below, is that the authors have not sufficiently established that Insig degradation mediates the robust RORy-KO effect. If the authors can address this point and the other suggestions, I think this paper would be suitable for NCB.\nMajor concern:\nComment 1. The proposed mechanisms for RORy on SREBP processing is control post-translational degradation of Insig-1, but the critical CHX and MG132 experiments on Insig stabilization/degradation (e.g. Figs. 4J, 5A,B) do not show a robust phenotype, raising concern that the detailed work (e.g. Figs 5-6), which overall is well done, may be pursuing the wrong mechanism.\nComment 2. The authors must test whether the CAB39 and Yod1 mechanisms synergize to recapitulate the RORγ KO phenotype on SREBP processing in the same cultured cells used for the other mechanistic studies. The animal studies along these lines (e.g. Fig 6R-T) are indirect and would need to be controlled with genetic deletion of SREBP to isolate the effect of Yod1 and CAB39 deletion, which undoubtedly affect many pathways.\nComment 3. Overall, the ubiquitination assays are more robust than the cell-based protein turnover assays. Are the authors sure that Insig ubiquitination is the regulated step? Other precedents exist – for example, Insig recruits the E3 ligase GP78 to ubiquitinate HMGCR, but the extraction of HMGCR from ER membranes is also regulated by metabolites (reviewed in PMID 35940903)\nOther points that need to be addressed:\nComment 1. Fatostatin is a non-specific small molecule inhibitor of Scap. The Espenshade group has shown that it inhibits cell growth of both WT and Scap-deficient cells equally (PMID 27324795) and, of specific concern to this manuscript, the Radhakrishnan group showed that fatostatin impairs ER to Golgi trafficking generally (PMID 38330014). I suggest the authors remove the experiments with fatostatin (Fig. 4C-F) or else revise their interpretations given these concerns.\nComment 2. The following important papers need to be cited and discussed\nComment 2a. Previous demonstration of Insig1 ubiquitination at the two Lys residues identified here (PMID 16399501) – please cite this one around lines 420 and 427 where these ubiquitination sites are re-identified.\nComment 2b. Whether the serine they identified connects to this previous report of differential regulation of Insig-1 and Insig-2 by serines flanking the sites of ubiquitination (PMID 16549805)\nComment 2c. The original paper showing ubiquitination and degradation of Insig-1 by GP78 (PMID 17043353)\nComment 2d. Regulation of Insig-2 but not Insig-1 by a phosphorylation mechanism (PMID 39145929)\nComment 3. 5L and the other docked models: I would prefer to see these removed; they don’t add credibility to the story because programs like HDOCK will form a complex with anything you ask them, and they should be considered wrong until proven right. If the models are included, please correct the methods to explain in detail how the input models were obtained (what PDBs, what homology models, what softwares were used?). Define the colors, label the active site of Yod1, is it near the putative ubiquitins on Insig? Please show all side chains for labeled residues and include the electrostatic surface of the interacting protein to show if there is surface complementarity. Why would Yod1 bind Insig1 when it isn’t ubiquitinated? Is that substrate specificity precedented? Above all, mutagenic interaction studies based on the predictions must be included to justify use of these models.\nComment 4. 6O – needs a Yod1+ cell line to justify the Yod1 antibody exposure.\nComment 5. Over-expression plasmids are not suitably defined. Please include the source or method of generation for all. I noticed the SREBP2 dbHLH construct is not defined because I was surprised this deletion protein migrated at the full-length size in Fig 4G.\nComment 6. All blots with SREBP2 need to show both the precursor and cleaved product to be interpretable (i.e. Fig. 4D, F).\nComment 7. AMPK activity reducing the interaction between Insig-1 and GP78 should also reduce the degradation of HMGCR, have the authors tested HMGCR stabilization in their RORγ KO cells?\nComment 8. Line 402 referencing comparison of Insig1 and Insig2 in RORy KO cells, - this important data should be shown and could be discussed in context with some of the references listed above.\nComment 9. Some typos: a. Line 385 should be Sterols are b. The sentence in lines 386-9 is not clear and I think says the opposite of what the authors mean? – inactivation of Insig releases SREBP2 for trafficking\nComment 10. I understand the journal may have different space considerations, but I would prefer to see the methods for the cull culture work included in the main text, or else move all the methods to supplement. I did not understand the logic for only including the animal work in the main text for a study that is trying to get at cellular mechanisms.", "reviewer_4": "The authors have substantially improved the manuscript, and the revisions adequately address this reviewer's concerns."}, "author_response": "Response to Reviewers\n\n[...Author rebuttal text provided in the prompt...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have substantially improved the manuscript, and the revisions adequately address this reviewer's concerns.", "reviewer_2": "The authors have addressed the points I raised on the previous review. I recommend this paper be accepted and I have only a few minor suggestions.\nComment 1) In Fig 4D, are the labels for WT and KO switched? I thought there was more correlation between STING/ERGIC in the RORy KO cells.\nComment 2) Both of the quantifications for the CHX and MG132 experiments shown in Reviewer Fig R3 (corresponding to the replicates of Fig 5A and 5B) should be added to the main text figures. This would increase confidence in the phenotype.\nComment 3) Regarding the response to comment #6, I do think it is necessary to include the full SREBP2 blots showing the precursor and nuclear form in the manuscript and not just the reviewer figures. They could be shown in a supplement.\nComment 4) There are several references to data not shown, which I believe contradicts policies for Nature Communications and will need to be included."}, "author_response": "Response: We sincerely appreciate this careful observation. The labels for WT and KO in Fig. 4D were indeed reversed, and this error has now been corrected in the revised manuscript.\nResponse: Thank you for this constructive suggestion. To strengthen the confidence in the reported phenotype, the quantifications of replicate experiments for the CHX and MG132 assays (corresponding to Fig. 5A and 5B) have been added to the supplementary materials as Fig. S4H and S4I.\nResponse: We thank the reviewer for this valuable comment. As recommended, the full western blot images of SREBP2 (including both precursor and nuclear forms) have been incorporated into the supplementary materials as Fig. S14.\nResponse: We apologize for this oversight. All data previously referred to as “not shown” have now been included in the supplementary materials as Fig. S4J and S3P, and the relevant descriptions in the manuscript have been revised accordingly to comply with the policies of Nature Communications."}]

Decision letter