Introduction and Objective: In REMODEL, semaglutide improved endothelial cell (EC) health in adults with type 2 diabetes (T2D) and chronic kidney disease (KD), but intrarenal mechanisms of glucagon-like peptide-1 receptor agonists (GLP-1RA) are unclear. We identified intrarenal genes and pathways responsive to GLP-1RA use in KD. Methods: scRNA-seq analysis was done in kidney biopsy tissue from 11 participants with KD (UACR ≥30 mg/g or eGFR <90 mL/min/1.73m2; Fig A). Genes expressed in ≥5% of cells in at least 1 major cell type were included, and ~118k cells remained after quality control (Fig B). NEBULA was used for cell type specific differential gene expression (DEG) between GLP-1RA users/non-users, followed by gene set enrichment analysis. Results: Figs D-K summarize findings in immune lymphoid, cortical thick ascending limb 2 (C-TAL-2), EC/vascular smooth muscle cells (VSMC), and immune myeloid cells, which had the highest numbers of DEGs (Fig C). Angiogenesis was enriched in immune cells, whereas hypoxia and TNFα-NFκB signaling were suppressed in immune myeloid cells. Glycolysis was enriched in EC/VSMC, whereas hypoxia and multiple metabolic pathways, including fatty acid metabolism, glycolysis, and oxidative phosphorylation, were suppressed in immune and C-TAL-2 cells. Conclusion: GLP-1RA use in early KD is associated with distinct vascular, immune, and tubular pathway signatures, extending prior observations of EC effects to the intrarenal cellular level. Disclosure Y. Choi: None. H. Hampson: None. J.D. Weissenkampen: None. J. Kanter: None. T. Vaisar: None. K. Sulek: Employee; Current; Novo Nordisk A/S. K. Bornfeldt: Advisory Panel; Current; ESPERION Therapeutics, Inc. J.A. Schaub: Consultant; Ended; Klick. D. van Raalte: Consultant; Current; AstraZeneca. Research Support; Current; AstraZeneca. Consultant; Current; Boehringer Ingelheim International GmbH. Research Support; Current; Boehringer Ingelheim International GmbH. Consultant; Current; Eli Lilly and Company. Research Support; Current; Eli Lilly and Company. Consultant; Current; Merck & Co., Inc. Research Support; Current; Merck & Co., Inc. Consultant; Current; Novo Nordisk. Research Support; Current; Novo Nordisk. K.J. Nadeau: None. S. Eddy: Research Support; Current; AstraZeneca, Eli Lilly and Company, Genentech, Inc., Sanofi, Novo Nordisk A/S, Travere Therapeutics. Other - Royalties administered through University of Michigan; Current; AstraZeneca. Research Support; Current; Certa Therapeutics, Vera Therapeutics, Dimerix. V. Nair: None. P. McCown: None. A.S. Naik: None. M.O. Alaba: None. F. Alakwaa: None. N. Nguyen: None. C. Smith: None. J.B. Hodgin: None. R. Nelson: None. K. Tuttle: Consultant; Ended; Alnylam Pharmaceuticals, Inc. Consultant; Current; AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, GlaxoSmithKline plc., Novo Nordisk, Lilly, ProKidney. Consultant; Ended; Roche Diabetes Care. Research Support; Ended; Travere. P.E. Ladd: None. E. Zeitler: Consultant; Current; Novo Nordisk. A. Karihaloo: None. M. Kretzler: Research Support; Current; AstraZeneca, Boehringer Ingelheim International GmbH, Bayer AG, Dimerix, Eli Lilly and Company, F. Hoffmann-La Roche Ltd., European Union, JDRF, National Institutes of Health, Novo Nordisk, Sanofi, vera therapeutics, Regeneron Pharmaceuticals Inc., Travere. P. Bjornstad: Consultant; Current; Bayer AG, Boehringer Ingelheim International GmbH, Lilly, Novo Nordisk. Funding Children's Hospital of Colorado

Identifying mechanisms of kidney disease commonly involves comparing diseased samples with healthy reference tissues; however, the effects of variability in tissue procurement, storage, and donor characteristics remain underexplored. In this study, we systematically evaluated 3 reference tissue types — tumor nephrectomy (TN), pretransplant biopsies from living donors (LD), and percutaneous biopsies from healthy control volunteers (HC) — to determine their impact on differential gene expression across 3 diabetic kidney disease states. We observed distinct injury markers, cell state proportions, and gene signatures associated with procurement method, sex, and donor age. Adjustment for these confounding factors significantly influenced pathway analysis results. Specifically, correcting for age and sex eliminated significant enrichment of IFN-γ response when comparing the diabetes mellitus–resilient group and HC group. Processes related to biological aging were enriched in older reference tissues, potentially confounding disease-specific interpretations. Importantly, TNF signaling via NF-κB remained enriched in LD and TN samples relative to HC, even after accounting for confounders. These results underscore the critical importance of selecting appropriate control tissues and rigorously adjusting for confounding variables to reliably discern the molecular mechanisms underlying kidney diseases.

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.

Poor outcomes in proteinuric kidney diseases are challenging to successfully manage therapeutically due to the heterogeneity of underlying disease pathogenesis and associated risk for progression. The role of cytoskeleton-associated proteins, including the scaffolding protein Anillin (ANLN), are of specific interest in kidney disease given the importance of actin dynamics in the kidney's specialized epithelial cell types. In this study, we identify the prevalence of genetic variants in ANLN , the gene encoding ANLN, in a cohort of deeply phenotyped individuals with non-diabetic proteinuric kidney disease. Thirty-one individuals (of 864 genotyped) harbor heterozygously expressed variants in ANLN ; 7 unrelated individuals shared the same variant (I1109V) in the C-terminal pleckstrin homology (PH) domain, a region necessary for interaction with the plasma membrane. Kidney organoids generated from I1109V induced pluripotent stem cells from 1 of these individuals showed increased epithelial cell mitogen-activated protein kinase 8 network activity and apoptosis, which was enhanced by tumor necrosis factor alpha (TNF-α) and phenocopied by actin polymerization inhibition. TNF-α-treated I1109V organoids also exhibited tubular lumen expansion. Knockdown and re-expression of the analogous ANLN variant in Xenopus laevis embryonic epithelia resulted in defects in cell-cell junction dynamics including wavy cell membranes exhibiting increased transverse movements as well as abnormal junctional F-actin remodeling in response to mechanical stress and leaky barrier function. Taken together, these results indicate that enhanced tubular epithelial cell death, perturbed cell-cell contacts and barrier function defects are associated with a novel ANLN variant discovered in individuals with non-diabetic proteinuric kidney disease. ONE SENTENCE SUMMARY:Enhanced tubular epithelial cell death and perturbed cell-cell junction integrity and barrier function are associated with a novel Anillin coding variant discovered in a cohort of individuals with proteinuric kidney disease.

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.

Chronic kidney diseases (CKDs) are a global health concern, necessitating a comprehensive understanding of their complex pathophysiology. This study explores the use of 2 complementary multidimensional -omics data integration methods to elucidate mechanisms of CKD progression as a proof of concept. Baseline biosamples from 37 participants with CKD in the Clinical Phenotyping and Resource Biobank Core (C-PROBE) cohort with prospective longitudinal outcome data ascertained over 5 years were used to generate molecular profiles. Tissue transcriptomic, urine and plasma proteomic, and targeted urine metabolomic profiling were integrated using 2 orthogonal multi-omics data integration approaches, one unsupervised and the other supervised. Both integration methods identified 8 urinary proteins significantly associated with long-term outcomes, which were replicated in an adjusted survival model using 94 samples from an independent validation group in the same cohort. The 2 methods also identified 3 shared enriched pathways: the complement and coagulation cascades, cytokine-cytokine receptor interaction pathway, and the JAK/STAT signaling pathway. Use of different multiscalar data integration strategies on the same data enabled identification and prioritization of disease mechanisms associated with CKD progression. Approaches like this will be invaluable with the expansion of high-dimension data in kidney diseases.

Building upon a foundational Human Kidney resource, we present a comprehensive multi-modal atlas that defines spatially resolved versus unresolved repair states and mechanisms in human kidney disease. Homeostatic interactions between injured kidney epithelium and its surrounding milieu determine successful repair outcomes, while pathogenic signaling promotes unresolved inflammation and fibrosis leading to chronic disease. We integrated multiple single-cell and spatial modalities across ~700 samples from >350 patients (~250 research biopsies), analyzing ~1.7 million cells alongside complementary mouse multi-omic profiles spanning acute-to-chronic injury and aging (>300,000 cells) and spatial transcriptomic analysis of >150 human biopsies. This cross-species atlas delineates functional pathways and druggable targets across the nephron and defines gene regulatory networks and chromatin landscapes governing tubular, fibroblast, and immune cell transitions from injury to either recovery or failed repair states. We identified distinct cellular states associated with specific pathological features that show dynamic distributions between acute kidney injury (AKI) and chronic kidney disease (CKD), organized within unique spatial niches that reveal progression mechanisms from early injury to unresolved disease. Gene regulatory analyses prioritized key transcription factor activities (SOX4, SOX9, NFKB1, REL, KLFs) and their target networks establishing disease states and tissue microenvironments. These regulatory programs were directly linked to clinical outcomes, identifying molecular signatures of recovery and secreted biomarkers predictive of AKI-to-CKD progression, providing a key resource for therapeutic development and precision medicine approaches in kidney disease.

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.

Key Points Baseline CKD, even mild, is associated with a higher risk of long coronavirus disease (COVID) in patients with acute severe acute respiratory syndrome coronavirus infection. Among those without CKD at baseline, Long COVID is associated with a higher risk of developing new CKD and faster kidney function decline. Associations between Long COVID and CKD/kidney function decline persist after matching, adjustment, and accounting for the competing risk of death. Background Among patients with acute coronavirus disease-19 (COVID-19), the association of CKD and Long COVID has not been reported in large multicenter cohorts. Methods This study used data from 59 health care systems across the United States, in the National Clinical Cohort Collaborative COVID enclave, to analyze the relationship between CKD and Long COVID among adults diagnosed with acute COVID-19 between October 2021 and September 2023. We conducted two main analyses. First analysis : we tested if baseline CKD (eGFR <60 ml/min per 1.73 m 2 or diagnostic code) or baseline ESKD are risk factors for Long COVID (identified using ICD-10-CM code U09.9). We secondarily assessed associations between baseline mild CKD (Stage 3a, eGFR 45–59 ml/min per 1.73 m 2 ) and Long COVID. Second Analysis : among patients without baseline CKD/ESKD, we examined if incident CKD/ESKD and eGFR decline (≥20% in 1 year) were associated with Long COVID. We used propensity score matching for demographics and data contributing site, with models adjusted for risk factors and competing risk of death. All outcomes were evaluated within a 365-day follow-up period from the onset of acute COVID-19. Results First analysis : From an unmatched cohort of 2,385,20 patients with acute COVID-19, those with baseline CKD/ESKD had a higher risk of Long COVID (adjusted subdistribution hazard ratio [sHR], 1.13; 95% confidence interval [CI], 1.09 to 1.18) after matching. A similar risk was noted even among those with mild CKD (sHR, 1.15; 95% CI, 1.05 to 1.25). Second Analysis: Among patients with acute COVID-19 and without baseline CKD/ESKD, Long COVID was associated with incident CKD/ESKD (sHR, 1.65; 95% CI, 1.51 to 1.81) and 20% or greater eGFR decline (sHR, 1.21; 95% CI 1.04 to 1.40) within 1 year. Conclusions CKD, even mild, was associated with an higher risk of Long COVID. Among those without baseline CKD, Long COVID was associated with incident CKD and eGFR decline.

The dynamics of transcriptional elongation influence many biological activities, such as RNA splicing, polyadenylation, and nuclear export. To quantify the elongation rate, a typical method is to treat cells with drugs that inhibit RNA polymerase II (Pol II) from entering the gene body and then track Pol II using Pro-seq or Gro-seq. However, the downstream data analysis is challenged by the problem of identifying the transition point between the gene regions inhibited by the drug and not, which is necessary to calculate the transcription rate. Although the traditional hidden Markov model (HMM) can be used to solve it, this method is complicated with its hidden variable and many parameters to be estimated. Hence, we developed the R package proRate, which identifies the transition point with a novel least sum of squares (LSS) method and calculates the elongation rate accordingly. In addition, proRate also covers other functions frequently used in transcription dynamic study, including metagene plotting, pause index calculation, gene structure analysis, etc. The effectiveness of this package is proved by its performance on three Pro-seq or Gro-seq datasets, showing higher accuracy than HMM. proRate is freely available at https://github.com/yuabrahamliu/proRate or https://github.com/FADHLyemen/proRate.

Many data resources generate, process, store, or provide kidney related molecular, pathological, and clinical data. Reference ontologies offer an opportunity to support knowledge and data integration. The Kidney Precision Medicine Project (KPMP) team contributed to the representation and addition of 329 kidney phenotype terms to the Human Phenotype Ontology (HPO), and identified many subcategories of acute kidney injury (AKI) or chronic kidney disease (CKD). The Kidney Tissue Atlas Ontology (KTAO) imports and integrates kidney-related terms from existing ontologies (e.g., HPO, CL, and Uberon) and represents 259 kidney-related biomarkers. We have also developed a precision medicine metadata ontology (PMMO) to integrate 50 variables from KPMP and CZ CellxGene data resources and applied PMMO for integrative kidney data analysis. The gene expression profiles of kidney gene biomarkers were specifically analyzed under healthy control or AKI/CKD disease states. This work demonstrates how ontology-based approaches support multi-domain data and knowledge integration in precision medicine.

Background Among patients with acute coronavirus disease-19 (COVID-19), the association of CKD and Long COVID has not been reported in large multicenter cohorts. Methods This study used data from 59 health care systems across the United States, in the National Clinical Cohort Collaborative COVID enclave, to analyze the relationship between CKD and Long COVID among adults diagnosed with acute COVID-19 between October 2021 and September 2023. We conducted two main analyses. First analysis: we tested if baseline CKD (eGFR <60 ml/min per 1.73 m(2) or diagnostic code) or baseline ESKD are risk factors for Long COVID (identified using ICD-10-CM code U09.9). We secondarily assessed associations between baseline mild CKD (Stage 3a, eGFR 45-59 ml/min per 1.73 m(2)) and Long COVID. Second Analysis: among patients without baseline CKD/ESKD, we examined if incident CKD/ESKD and eGFR decline (>= 20% in 1 year) were associated with Long COVID. We used propensity score matching for demographics and data contributing site, with models adjusted for risk factors and competing risk of death. All outcomes were evaluated within a 365-day follow-up period from the onset of acute COVID-19. Results First analysis: From an unmatched cohort of 2,385,20 patients with acute COVID-19, those with baseline CKD/ESKD had a higher risk of Long COVID (adjusted subdistribution hazard ratio [sHR], 1.13; 95% confidence interval [CI], 1.09 to 1.18) after matching. A similar risk was noted even among those with mild CKD (sHR, 1.15; 95% CI, 1.05 to 1.25). Second Analysis: Among patients with acute COVID-19 and without baseline CKD/ESKD, Long COVID was associated with incident CKD/ESKD (sHR, 1.65; 95% CI, 1.51 to 1.81) and 20% or greater eGFR decline (sHR, 1.21; 95% CI 1.04 to 1.40) within 1 year. Conclusions CKD, even mild, was associated with an higher risk of Long COVID. Among those without baseline CKD, Long COVID was associated with incident CKD and eGFR decline.