BACKGROUNDYouth with type 2 diabetes (T2D) and severe obesity face high risk of diabetic kidney disease, which metabolic bariatric surgery (MBS) can mitigate. This study explores structural and molecular changes in kidneys after vertical sleeve gastrectomy (VSG), a form of MBS.METHODSWe performed paired analyses, including metabolic profiling, kidney volume assessment, histological evaluation, and single-cell RNA sequencing (scRNA-seq), on kidney biopsies from 5 youth with T2D and obesity pre- and 12 months post-VSG in the IMPROVE-T2D (Impact of Metabolic surgery on Pancreatic, Renal and cardiOVascular hEalth in youth with T2D) cohort. Circulating proteomics with kidney transcriptomics were linked using data from an independent cohort of youth with obesity, with or without T2D, undergoing MBS in Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS, n = 64).RESULTSAfter VSG, participants lost weight and had improvements in insulin sensitivity and metabolic parameters. Kidney changes included reduced renal hyperfiltration, total kidney volume, mesangial matrix area, and microalbuminuria. scRNA-seq in proximal tubule (PT) and thick ascending limb cells indicated repression of glycolysis, gluconeogenesis, and tricarboxylic acid (TCA) cycle genes, with upregulation of AMP-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). Decreased metabolic signaling aligned with reduced ribosomal phosphorylated S6K (pS6K), suggesting attenuated mTORC1 activity. Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation in PT was diminished, correlating with lower circulating ligands from Teen-LABS proteomic data.CONCLUSIONMBS/VSG prompts kidney molecular adaptations, providing potential targets for nonsurgical interventions against obesity- and diabetes-associated kidney disease.FUNDINGUniversity of Washington with the American Diabetes Association, University of Michigan with Chan Zuckerberg Initiative, and Breakthrough T1D.

Transcriptional reprogramming has an important role in kidney glomerular disease. Using in vivo murine models of podocyte injury, we studied the roles of the FOXC2 and WT1 transcription factors (TFs) in podocyte injury. Podocytes are a crucial cell type of glomeruli, the filtration units of each nephron. Podocyte injury is often the incipient event leading to chronic kidney disease. It is well established that the TFs FOXC2 and WT1 are required in podocytes to maintain the glomerular filtration barrier. Their role in the response to injury is less well understood. Here, we tested the hypothesis that FOXC2 and WT1 act together to mediate transcriptional reprogramming in response to podocyte injury. Similarly to that of WT1, genome-wide FOXC2 binding to target genes is dynamic during the course of injury, initially increasing, but late in injury there is a dramatic decrease in FOXC2 expression and in its binding to target genes. Podocyte-specific inactivation of FoxC2 or Wt1 in adult mice limits the transcriptional response to injury. Correlating FOXC2 and WT1 ChIP-seq analyses demonstrated that they co-bind many genes expressed in podocytes. Thus, reprogramming the transcriptome involves dynamic changes in the binding of FOXC2 and WT1 to their target genes during a reparative injury response.

Idiopathic nephrotic syndrome (INS) is a podocyte disease triggered by immune-derived factors. Endothelial activation occurs in this context, but whether the activated endothelium contributes to podocyte injury is unknown. We tested the hypothesis that CD93, a protein primarily expressed in the endothelium, is a contributory factor of podocyte injury. We studied 460 patients with INS and 150 with other podocytopathies. CD93 was analyzed in kidney tissue, urine, and serum samples. We tested the efficacy of CD93 blockade in vitro and in vivo and investigated the relationship between soluble CD93 and clinical outcomes in human INS. CD93 was highly expressed by glomerular endothelial cells (GEnCs) in human INS, and INS sera stimulated cultured human GEnCs to release CD93. Mechanistically, soluble CD93 mediated podocyte activation via β1 integrin/FAK signaling in cultured human podocytes. CD93 blockade mitigated the activation of cultured human podocytes and albumin permeability in human GEnC-podocyte cocultures as well as albuminuria, glomerulosclerosis, and podocyte loss in two models of nephrotic syndrome: podocyte-specific transforming growth factor-β1 signaling (PodTgfbr1) mice and adriamycin-treated mice. Cd93 knockout mice showed less proteinuria and glomerulosclerosis, compared with controls, after adriamycin injection. In patients with INS, soluble CD93 was high in urine in ~90% and 50% of patients in relapse and remission, respectively. High urinary CD93 was associated with faster decline in kidney function and slower response to immunosuppression. Soluble and glomerular CD93 was also elevated in other podocytopathies. We conclude that soluble CD93 contributes to podocyte injury.

Sodium-glucose co-transporter 2 inhibitors (SGLT2-i) slow progression of kidney disease but therapeutic mechanisms remain elusive. Here we report the beneficial effect of dapagliflozin on hypoxia-mediated kidney tubular epithelial cell injury, a contributing factor to kidney disease progression, using a human pluripotent stem cell (hPSC)-derived hypoxic kidney organoid model. Hypoxic organoids showed increased expression of Hypoxia Inducible Factor (HIF)-associated transcriptional targets, decreased tricarboxylic acid (TCA) cycle metabolites and mitochondrial β-oxidation protein expression, and activated unfolded protein response. A transcriptional signature derived from hypoxic organoids 1) identified a subgroup of individuals whose kidney disease subsequently progressed, and 2) correlated with worse tubular injury. Dapagliflozin enhanced mitochondrial stress response resulting in reversed hypoxia-induced tubular epithelial cell apoptosis, reactive oxygen species (ROS) accumulation, and organoid fibrosis. These results indicate that dapagliflozin may contribute to improved kidney disease outcomes by attenuating hypoxia-induced metabolic stress-mediated tubular epithelial cell injury.

INTRODUCTIONBK polyomavirus (BKV) infection is associated with injury and subsequent graft loss due to the extent of injury or rejection. However, the molecular mechanisms driving injury and subsequent adverse outcomes remain poorly understood.METHODSIn a cross-sectional study, single-cell RNA-seq from kidney allograft biopsies was used to assess cell type-specific responses between uninfected controls and 2 distinct phases of BKV infection: peaking (increasing viral blood titers) and resolving (decreasing viral titers following immunosuppression reduction).RESULTSGenes upregulated in BK viral nephropathy (BKVN) were enriched for polyomavirus infection hallmarks, including ribosome biogenesis, translation, and energy restructuring. Additionally, enriched pathways included wound healing, cellular stress, antigen presentation and immune signaling. Even without BKVN (peaking BK viremia alone), epithelial cells expressed signatures for wound healing, cellular stress, and extracellular matrix remodeling. In vivo tubular cell responses at single-cell resolution were validated against single cell transcriptomic data of BKV-infected cells in a cell culture model. Despite similarities, in vivo tubular cells underwent metabolic adaptation favoring fatty acid oxidation and proinflammatory responses not observed in culture models, likely due to an absent innate and adaptive immune system. Despite lymphopenia and immunosuppressive therapies, the proportion of recipient-derived intrarenal adaptive immune cells was increased in biopsies associated with peaking viremia alongside activation of innate immune responses. Adaptive immune cells exhibited persistent inflammatory signaling and remodeling of energy metabolism during the resolving phase of infection.CONCLUSIONThese not previously reported insights into BKV-associated injury may have implications for clinical management and improved allograft outcomes.

INTRODUCTION:Increased endothelin-1 (ET1) and endothelin receptor A (ETA) signaling have been implicated in the pathogenesis of focal segmental glomerulosclerosis (FSGS). Previous studies have suggested that crosstalk between activated podocytes and glomerular endothelial cells (GECs) could contribute to the pathogenesis of FSGS. METHODS:To examine this, we developed mouse lines with endothelial cell-targeted and conditional deletion of ETA using the Cre-LoxP system (Scl:Cre-ETAfl/fl), as well as targeted deletion of ET1 in podocytes (Nphs2:Cre-ET1fl/fl). RESULTS:The absence of endothelial ETA in mice was protective in adriamycin-induced glomerular injury, as evidenced by decreased albuminuria and reduced podocyte depletion. RNA-seq of ET1-treated mouse glomerular endothelial cells showed activation of cellular signaling pathways and alteration of matrix component deposition programs via ETA. Endothelin-1 expression was detected in glomerular cells in patient biopsy samples and mice with FSGS. Compared to adriamycin-treated mice, podocyte-specific ET1 knockout mice treated with adriamycin had reduced glomerular injury, albuminuria, and podocyte depletion. Furthermore, canonical transforming growth factor (TGF)β signaling mediates ET1 release by podocytes, and Edn1 knockout in podocytes with inducible TGFβ receptor-1 signaling (Nphs2:Cre-ET1-Nphs1:TgfbrI) abrogated glomerular injury and albuminuria upon TGFβ receptor-1 activation. Ultrastructural changes and podocyte depletion were completely prevented in these mice, and there was no increase in GEC-associated ETA expression. Mathematical modelling supports rapid bidirectional diffusion of ET1 across the glomerular basement membrane. CONCLUSIONS:Our studies provide in vivo evidence that crosstalk of podocyte-derived ET1 with activation of GEC ETA contributes to glomerular injury in FSGS.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors slow diabetic kidney disease progression, but their intrarenal mechanisms remain incompletely understood, particularly in type 1 diabetes (T1D), where kidney protection has not been definitively established. ATTEMPT (NCT04333823) was a placebo-controlled trial in which 98 youth (ages 12 to 21) with T1D and hyperfiltration were randomized 1:1 to dapagliflozin (5 milligrams) or placebo for 16 weeks. Participants underwent sequential kidney biopsies, multiparametric kidney MRI, and plasma and urine proteomics. The sequential research kidney biopsies were performed on adults 18 years or older at one of three sites (baseline n = 16, follow-up n = 11). Single-cell RNA sequencing of 214,415 cells across 27 biopsies revealed coordinated transcriptional shifts across nephron, vascular, and immune compartments. In the proximal tubule, the primary site of SGLT2 expression, dapagliflozin down-regulated glycolysis, gluconeogenesis, and oxidative stress markers. Endothelial cells showed reduced profibrotic and inflammatory gene expression with increased protective factors. Podocytes demonstrated enhanced cytoskeletal reinforcement and suppressed interferon signaling. These molecular changes paralleled clinical improvements, including attenuation of hyperfiltration, improved glycemic control, and normalization of medullary oxygenation. Trajectory analyses revealed dapagliflozin shifted tubular cells from injury-prone toward healthier phenotypes. Cross-cohort comparison against healthy controls showed that more than 55% of dapagliflozin-responsive genes shifted toward healthy control expression patterns. Urine proteomics mirrored tissue changes with decreased injury markers and increased protective proteins. These convergent molecular mechanisms, metabolic reprogramming, dampened inflammation, and normalized oxygen handling provide hypothesis-generating mechanistic insights into potential kidney-protective mechanisms of SGLT2 inhibitor therapy in youth with T1D.

To map transcriptional programs in rare glomerular diseases, single-nucleus RNA sequencing (snRNAseq) on kidney biopsies (N=120) from the Nephrotic Syndrome Study Network were integrated with snRNAseq and single-cell sequencing (scRNAseq) of reference kidney tissue (N=50) to create the Omnibus of CElls And Nuclei (OCEAN). Unsupervised analysis of multi-cellular programs identified that JAK-STAT pathway in podocytes and endothelial cells was associated with clinical measures of disease severity. JAK-STAT pathway activity was strongly correlated with apolipoprotein1 (APOL1) mRNA transcript expression and high risk APOL1 variant genotype, a major risk factor for FSGS. These findings were confirmed in an independent study of Black participants where loss of APOL1 function decreased JAK-STAT pathway activation in patient derived podocyte ex vivo models. The findings are consistent with a feed forward loop regulating the JAK-STAT-APOL1 driven tissue damage, providing mechanistic support for the JUSTICE Phase II trial targeting JAK activation in APOL1-mediated kidney disease.

DNA repair is essential for preserving genome integrity. Podocytes, postmitotic epithelial cells of the kidney filtration unit, bear limited regenerative capacity, yet their survival is indispensable for kidney health. Podocyte loss is a hallmark of the aging process and of many diseases, but the underlying factors remain unclear. We investigated the consequences of DNA damage in a podocyte-specific knockout mouse model for DNA excision repair protein Ercc1 and in cultured podocytes under genomic stress. Furthermore, we characterized DNA damage-related alterations in mouse and human renal tissue of different ages and patients with minimal change disease and focal segmental glomerulosclerosis. Ercc1 knockout resulted in accumulation of DNA damage and ensuing albuminuria and kidney disease. Podocytes reacted to genomic stress by activating mTOR complex 1 (mTORC1) signaling in vitro and in vivo. This was abrogated by inhibiting DNA damage signaling through DNA-dependent protein kinase (DNA-PK) and ataxia teleangiectasia mutated (ATM) kinases, and inhibition of mTORC1 modulated the development of glomerulosclerosis. Perturbed DNA repair gene expression and genomic stress in podocytes were also detected in focal segmental glomerulosclerosis. Beyond that, DNA damage signaling occurred in podocytes of healthy aging mice and humans. We provide evidence that genome maintenance in podocytes is linked to the mTORC1 pathway and is involved in the aging process as well as the development of glomerulosclerosis.

The molecular pathophysiology of nephrotic syndrome remains largely elusive in pediatric patients. While most children with minimal change disease (MCD) show favorable responses to immunosuppressive therapy, those with focal segmental glomerulosclerosis (FSGS) often exhibit poorer treatment responses, with many experiencing either partial remission or no remission of proteinuria. The need for reliable glomerular disease biomarkers to predict treatment response and understand molecular pathways governing responsiveness and resistance is a critical unmet need in pediatric nephrology. In this study, we sought to characterize urine proteomes in children with MCD and FSGS to identify biomarkers distinguishing disease activity and associated molecular pathways. Using quantitative proteomics, urine proteins from children with MCD and FSGS in the CureGN Study were identified and correlated with disease onset and activity. Unbiased cluster analyses of nephrotic urine proteomes demonstrated a cluster with relatively increased immune response and complement proteins, suggesting important distinctions in disease characteristics within the nephrotic subgroups. These analyses yielded patient subpopulations with proteinuria and distinct urine proteome differences associated with 116 proteins exerting cluster separation in the multivariate analyses. These findings highlight the potential of unsupervised clustering to identify disease subgroups and provide insights into the underlying molecular heterogeneity within nephrotic syndrome, paving the way for more tailored therapeutic strategies and improved patient management.

The expression and location of proteins in tissues represent key determinants of health and disease. Although recent advances in multiplexed imaging have expanded the number of spatially accessible proteins 1–3 , the integration of biological layers (that is, cell structure, subcellular domains and signalling activity) remains challenging. This is due to limitations in the compositions of antibody panels and image resolution, which together restrict the scope of image analysis. Here we present pathology-oriented multiplexing (PathoPlex), a scalable, quality-controlled and interpretable framework. It combines highly multiplexed imaging at subcellular resolution with a software package to extract and interpret protein co-expression patterns (clusters) across biological layers. PathoPlex was optimized to map more than 140 commercial antibodies at 80 nm per pixel across 95 iterative imaging cycles and provides pragmatic solutions to enable the simultaneous processing of at least 40 archival biopsy specimens. In a proof-of-concept experiment, we identified epithelial JUN activity as a key switch in immune-mediated kidney disease, thereby demonstrating that clusters can capture relevant pathological features. PathoPlex was then used to analyse human diabetic kidney disease. The framework linked patient-level clusters to organ disfunction and identified disease traits with therapeutic potential (that is, calcium-mediated tubular stress). Finally, PathoPlex was used to reveal renal stress-related clusters in individuals with type 2 diabetes without histological kidney disease. Moreover, tissue-based readouts were generated to assess responses to inhibitors of the glucose cotransporter SGLT2. In summary, PathoPlex paves the way towards democratizing multiplexed imaging and establishing integrative image analysis tools in complex tissues to support the development of next-generation pathology atlases.

Introduction and Objective: Metabolic bariatric surgery (MBS) offers therapeutic benefit in youth-onset type 2 diabetes (Y-T2D) but its impact on DKD pathophysiology is poorly understood. We aimed to evaluate immune, fibrotic, and senescent changes in DKD and effects of MBS. Methods: Kidney biopsies from lean controls (LC; n=10), Y-T2D at baseline (n=8) and 12-months post-MBS (n=5) were analyzed using scRNA-seq and Visium. Pseudobulk differential expression was performed. Cell-type deconvolution, spatial gene-set scoring and cell-cell interaction analysis characterized kidney microarchitecture. Results: At baseline, Y-T2D exhibited significant immune activation (e.g., HLA class I/II upregulation) and cell senescence, with myeloid infiltration and T cell activation compared to LC. Fibroblast interactions were prominent, indicating a pro-inflammatory, pro-fibrotic environment. Post-MBS, T cell activation was reduced but senescence and myeloid infiltration persisted with no significant changes in key cell types (e.g., PT-S1/S2, fibroblasts; p > 0.2) (Figure 1). Conclusion: DKD in Y-T2D is characterized by myeloid and T cell activation, cellular senescence and fibrosis. MBS provides partial resolution of inflammation but not senescence or myeloid infiltration, which may potentially be addressed by cell therapy and senotherapy. L. Yuan: None. Y. Choi: None. F. Alakwaa: None. N. Nguyen: None. P. Narongkiatikhun: None. J. Goodrich: None. K.L. Tommerdahl: None. K.J. Nadeau: None. J.R. Ryder: Board Member; Calorify. Research Support; Boehringer-Ingelheim, Eli Lilly and Company, Recordati. T. Inge: Consultant; Mediflix, UpToDate, Teleflex, Medtronic, Eli Lilly and Company, BrainStorm Cell Therapeutics, Standard Bariatrics. J.B. Hodgin: None. J.A. Schaub: Consultant; Klick. Research Support; AstraZeneca, Roche Genetech, Novo Nordisk, Eli Lilly and Company. A. Naik: None. P.J. McCown: None. F.C. Brosius: Consultant; MAKSCIENTIFIC. R.G. Nelson: None. M. Kretzler: Research Support; AstraZeneca, Boehringer-Ingelheim, Chinook Therapeutics, Inc, Eli Lilly and Company, Moderna, Inc, Janssen Pharmaceuticals, Inc, National Institutes of Health, European Union. Advisory Panel; Novartis Pharmaceuticals Corporation. Research Support; Novo Nordisk. Advisory Panel; Otsuka America Pharmaceutical, Inc. Research Support; Regeneron Pharmaceuticals, Renalytix, Roche Pharmaceuticals, Sanofi, Travere, Certa, Dimerix. P. Bjornstad: Consultant; AstraZeneca, Bayer Pharmaceuticals, Inc, Lilly USA LLC, Novo Nordisk. L. Pyle: None. NIDDK P30 DK081943NIDDK (P30 DK116073); JDRF (2-SRA-2019-845-S-B)

BACKGROUND In type 1 diabetes (T1D), impaired insulin sensitivity may contribute to the development of diabetic kidney disease (DKD) through alterations in kidney oxidative metabolism.METHODS Young adults with T1D (n = 30) and healthy controls (HCs) (n = 20) underwent hyperinsulinemic-euglycemic clamp studies, MRI, 11C-acetate PET, kidney biopsies, single-cell RNA-Seq, and spatial metabolomics to assess this relationship.RESULTS Participants with T1D had significantly higher glomerular basement membrane (GBM) thickness compared with HCs. T1D participants exhibited lower insulin sensitivity and cortical oxidative metabolism, correlating with higher insulin sensitivity. Proximal tubular transcripts of TCA cycle and oxidative phosphorylation enzymes were lower in T1D. Spatial metabolomics showed reductions in tubular TCA cycle intermediates, indicating mitochondrial dysfunction. The Slingshot algorithm identified a lineage of proximal tubular cells progressing from stable to adaptive/maladaptive subtypes, using pseudotime trajectory analysis, which computationally orders cells along a continuum of states. This analysis revealed distinct distribution patterns between T1D and HCs, with attenuated oxidative metabolism in T1D attributed to a greater proportion of adaptive/maladaptive subtypes with low expression of TCA cycle and oxidative phosphorylation transcripts. Pseudotime progression associated with higher HbA1c, BMI, and GBM, and lower insulin sensitivity and cortical oxidative metabolism.CONCLUSION These early structural and metabolic changes in T1D kidneys may precede clinical DKD.TRIAL REGISTRATION ClinicalTrials.gov NCT04074668.FUNDING University of Michigan O’Brien Kidney Translational Core Center grant (P30 DK081943); CROCODILE studies by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (P30 DK116073), Juvenile Diabetes Research Foundation (JDRF) (2-SRA-2019-845-S-B), Boettcher Foundation, Intramural Research Program at NIDDK and Centers for Disease Control and Prevention (CKD Initiative) under Inter-Agency Agreement #21FED2100157DPG.

Glucocorticoids and calcineurin inhibitors (CNI) are part of KDIGO guidelines for treatment of steroid resistant glomerular diseases. Despite widespread use, predicting whether a patient will respond to therapy remains elusive. Given toxicities associated with prolonged CNI use, identifying patients who are both likely and unlikely to respond to these therapies presents an opportunity to improve overall patient care.