Mesenchymal stromal cells (MSCs) possess therapeutic potential largely reliant on intact mitochondrial function to maintain reparative function. However, obesity compromises MSC metabolism and reparative capacity. MOTS-c, a mitochondria-derived peptide, is known to regulate cellular metabolism, but its role in human MSC biology remains unclear. We hypothesized that restoring MOTS-c signaling rescues the impaired functionality of adipose-derived MSCs from individuals with obesity. MSCs isolated from abdominal fat of patients with obesity (BMI ≥ 30 kg/m2) and lean donors (BMI < 30 kg/m2) (n = 6/group) were assessed in vitro for changes in proliferation, senescence (p16, p21) TNF-α, and antioxidant gene expression following MOTS-c co-incubation. In vivo, the effects of MOTS-c pre-treatment on the reparative capacity of obese MSC were tested in stenotic mouse kidneys. Basal MOTS-c expression was lower in obese vs. lean MSCs. Nevertheless, although exogenous MOTS-c restored intracellular levels and activated AMPK signaling in obese MSCs, it reduced proliferation, increased expression of senescence-associated genes (p16, p21), and upregulated TNF-α. In vivo, in a murine model of renal artery stenosis, MOTS-c-pretreated MSCs failed to improve renal perfusion, fibrosis, or tubular injury, while pretreatment also blunted the reparative efficacy of lean MSCs. These findings reveal that restoration of mitochondrial metabolic signaling is insufficient to reverse obesity-induced MSC dysfunction and may paradoxically exacerbate senescence and inflammation. These results suggest a dissociation between metabolic activation and functional stemness, underscoring context-dependent effects of mitochondrial-derived peptides in MSC biology.

Aim: Obesity may induce renal microvascular injury, particularly in peritubular capillaries (PTCs). Early detection of these changes may help patient management. We tested the hypothesis that levels of PTC-derived urinary extracellular vesicles (uEVs) reflecting early renal microvascular alterations would be elevated in obese individuals with preserved kidney function. Methods: Urinary samples were collected from 22 obese and 11 lean subjects. uEVs were characterized using flow cytometry for markers of PTC (cluster of differentiation 31 [CD31], plasmalemma vesicle-associated protein [PLVAP], and cluster of differentiation 144 [CD144]), angiogenesis (delta-like-ligand-4 [DLL4]), and inflammation (monocyte chemoattractant protein-1 [MCP-1]). For confirmation, PTC density was quantified and correlated with clinical parameters in kidney biopsies from additional matched groups. Results: Obese individuals had higher body mass index (BMI), glucose, insulin, urinary protein, and urinary MCP-1 levels than lean controls (P < 0.05), indicating metabolic and inflammatory changes, despite no significant difference in serum creatinine or estimated glomerular filtration rate (eGFR). They also exhibited elevated levels of CD31-/PLVAP+/CD144- PTC-derived uEVs expressing DLL4+ and/or MCP-1+ (P < 0.05 each). Levels of angiogenic PTC-derived DLL4+ uEVs correlated with BMI and systemic insulin, vascular endothelial growth factor-A, and angiopoietin-2 levels in our cohorts, whereas MCP-1+ uEVs derived from inflamed PTC correlated with BMI, glucose, blood pressure, and urinary neutrophil gelatinase-associated lipocalin. PTC density was also increased in kidney biopsies from obese individuals, correlating with BMI and glucose. Conclusion: DLL4+/MCP-1+ PTC-derived uEVs levels reflecting early renal microvascular changes are elevated in obese individuals, despite preserved kidney function. These findings may indicate that microvascular remodeling is dissociated from renal dysfunction in patients with obesity.

Introduction: Although progress has been made toward elucidating cellular pathways related to initial cystogenesis in autosomal dominant polycystic kidney disease (ADPKD), the mechanisms that contribute to disease progression and the timing of transitions remain largely unclear. We hypothesized that the predominant kidney biological processes in Pkd1RC/RC and other ADPKD models are highly dynamic throughout the disease, providing insights into the resulting kidney phenotype and conform well to those observed in human ADPKD. Methods: Kidney volume changes by class and age were determined in a large, well-characterized cohort of individuals with ADPKD and long follow-up. Similarly, Pkd1RC/RC and wild-type (WT) mice were studied longitudinally, and their kidney volume changes and function analyzed. Kidney mRNA profiles (mRNA-sequencing [mRNA-seq]) of Pkd1RC/RC mice were investigated at early, mid, and late stages, compared with those reported in other ADPKD models and humans with ADPKD. Results: In most individuals with ADPKD, kidney volume continues to increase; however, the rate of growth differs between classes and within severe classes by age. Kidney volume and function changes in Pkd1RC/RC mice conform well to the kidney volume changes in class 1C patients during adulthood. The kidney transcriptomic profile in Pkd1RC/RC mice evolves over the course of the disease, underscoring their highly dynamic kidney phenotype, presents several commonalities with other ADPKD models, and is relevant to human ADPKD. Conclusion: Our study suggests that, in ADPKD, different therapeutic strategies might be beneficial at different disease stages and identifies target candidate pathways for biomarker discovery that could be further investigated in humans.

Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells that repair other damaged kidney cells. STCs may be damaged and rendered ineffective by renovascular disease (RVD), but the underlying mechanisms remain unknown. We hypothesized that RVD induces changes in methylated (5mC) and hydroxymethylated (5hmC) DNA and modulates the transcriptomic profile and functional properties of swine STCs. CD24+/CD133+ STCs were harvested from pig kidneys after 10 wk of RVD or sham (n = 6 each), and their 5mC and 5hmC profiles of individual peaks were examined by immunoprecipitation sequencing (MeDIP-/hMeDIP-seq, respectively, n = 3 each). Integrated (MeDIP/hMeDIPseq/mRNA-seq) analysis was performed, followed by functional analysis of overlapping differentially expressed (DE) genes. STC-protective effects were assessed in vitro before and after epigenetic (Bobcat339) modulation. MeDIP-seq analysis identified 1,362 hypermethylated and 1,432 hypomethylated peaks in RVD-STCs compared with Normal-STCs, which correlated with 80 upregulated and 55 downregulated genes in RVD-STCs. hMeDIP-seq revealed 1,447 hyper-hydroxymethylated and 765 hypo-hydroxymethylated peaks in RVD-STCs vs. Normal-STCs, which correlated with 80 genes upregulated and 53 downregulated in RVD-STCs. Overlapping upregulated genes were mainly implicated in the regulation of oxidative phosphorylation, apoptosis, and lipid metabolism (e.g., STAT6), whereas overlapping downregulated genes were mainly involved in cell proliferation. Importantly, RVD increased STAT6 protein expression and impaired the proliferative capacity of STCs, which were partially reversed by treatment with Bobcat339, which also enhanced the ability of RVD-STCs to promote the viability of injured tubular epithelial cells. Renal ischemia induces locus-specific epigenetic alterations, associated with transcriptomic changes and impaired reparative function of swine STCs. These observations may contribute to developing novel approaches to preserve the reparative capacity of STCs in individuals with RVD.NEW & NOTEWORTHY Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells that repair other damaged kidney cells. This study shows that renovascular disease (RVD) induces site-specific 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) modifications in DNA associated with transcriptional changes and impaired proliferative capacity of swine STCs, which could be mitigated in RVD-STCs treated with an epigenetic modulator. Therefore, our observations may contribute to the development of novel approaches to preserve the reparative capacity of STCs in individuals with RVD.

Background/Objective Obesity imposes dysfunction of the endogenous cellular reparative system, which may manifest as impaired adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) function or altered characteristics of circulating endothelial progenitor cells (EPCs). However, whether both systems are abnormal in patients with obesity remains unclear. We hypothesized that human obesity induces impairment of MSCs and EPCs that would be reversed after weight-loss surgery (WLS). Methods Abdominal adipose tissue and peripheral blood mononuclear cells were collected to harvest MSCs and EPCs, respectively, from patients with obesity (n = 8) before and 9-12 months after WLS. MSCs mitochondrial function and EPCs number and surface markers were compared to those collected from healthy controls (HC). Results Patients with obesity had a higher basal body mass index compared to both HC (P < 0.0001) and post-WLS (P < 0.001). Compared to HC, MSC proliferative and differentiation capacity was preserved (P > 0.05), but they showed at baseline increased mitochondrial oxidative stress, and cytochrome-c release (P < 0.05), with reduced membrane potential and matrix density, which mostly improved after WLS. The percent of circulating CD34(+)KDR(+)CD133(+) and CD34(+)KDR(+) EPCs was elevated in patients with obesity (P < 0.05), as were EPC fractions expressing the inflammatory marker VAP-1 or pro-calcinogenic marker OCN-1, yet neither fell after WLS (P > 0.05). Conclusion Obesity impairs MSC mitochondrial function and increases the percent of circulating, but also potentially injurious EPCs. WLS largely reverses MSC mitochondrial injury and but not circulating EPC characteristics. Therefore, restoration of the endogenous tissue-resident and circulating cellular regenerative systems in the same patients with obesity may require different strategies or timeframes.

CKM syndrome entails pathophysiological interactions among the heart, the kidneys, and metabolic derangements. The specific cell populations implicated in renal injury have yet to be identified. We leveraged a new model of CKM syndrome in aging swine and single-nucleus RNA sequencing (snRNA-seq) to investigate the renal cell-specific transcriptomic profile and set the stage for future testing of potential new targets. Aging pigs with CKM syndrome were studied for 14 weeks. Kidneys were harvested, nuclei isolated, snRNA-seq performed, and UMAP plots generated using default parameters. Expression profiles of marker genes were visualized to define renal cell types, ranked by the number of differentially expressed genes (DEGs), and factored by the total number of cells per cell type. Top DEGs/cell type were visualized and their protein expression assessed in confirmatory studies. A total of 52,367 nuclei were analyzed. Thirty clusters were identified and filtered by canonical gene markers, revealing 20 renal cell types. Subsequent analysis of CKM syndrome kidneys showed that proximal tubular (PT) and immune (IM) cell subpopulations at 49.1% and 45.8% cells with DEGs, respectively, which were mainly implicated in microtubule organization and age-related inflammation (IL2-STAT5 signaling). Protein expression of 2-tubulin was downregulated in PT cells, whereas expression of STAT5 was upregulated in IM cells. The renal snRNA-seq transcriptomic landscape of a translational swine model of CKM syndrome identified PT and IM cells with the largest numbers of DEGs. Our observations may guide future studies aimed at modulating specific age-related renal abnormalities in CKM syndrome to slow disease progression.

BACKGROUND:Obesity is a major risk factor for kidney dysfunction, with cellular senescence and senescence-associated secretory phenotype (SASP) activation contributing to early kidney injury. Urinary extracellular vesicles (uEVs) provide a non-invasive approach to assess cellular stress. We hypothesised that obesity induces podocyte senescence detectable by podocyte-derived uEVs. METHODS:We recruited 28 obese (OB) individuals and 16 healthy volunteers (HV). Of these, uEVs from 21 OB (OB-1) and 10 HV (HV-1) were analysed for tubular-derived (urat1+, uromodulin+, or prominin+) or podocyte-specific (PODXL+) uEVs bearing markers of senescence (p16) and SASP (MCP-1) using flow cytometry. Their correlations with metabolic and urinary renal injury markers (kidney injury molecule-1 [KIM-1], neutrophil gelatinase-associated lipocalin [NGAL], tumour necrosis factor-alpha [TNF-α], proteinuria) were assessed. Furthermore, kidney biopsies obtained from comparable OB-2 (n = 7) and HV-2 (n = 6) subjects were examined for tissue podocyte senescence using immunofluorescence. Podocytes were also counted in a subset of HV (n = 5) and OB (n = 4) urine samples. FINDINGS:OB had elevated body mass index (BMI) but preserved kidney function. OB-1 exhibited significantly elevated P16+MCP-1+PODXL+ uEVs fractions compared to HV-1, which correlated with metabolic dysfunction (BMI, insulin resistance) and renal injury markers. Tubular-derived uEVs showed no differences between the groups. Kidney biopsies confirmed increased podocyte senescence (P16+ PODXL+ immunoreactivity) in OB-2 vs. HV-2, and numbers of urinary podocytes increased in OB. INTERPRETATION:Human obesity induces in persons with normal kidney function podocyte senescence, which is detectable non-invasively by uEVs. FUNDINGS:This study was partly supported by NIH grant numbers: DK120292, DK122734, HL158691, AG062104 (all LOL), and AG076537 (LJH). KDIGO provided support to Dr. Kukla to the 2024 Obesity and Chronic Kidney Disease Controversies Conference.

KEY POINTS:Experimental autosomal dominant polycystic kidney disease is associated with longitudinal changes in the kidney micro-RNA and mRNA profiles. Alterations in cellular transport, a feature of CKD, and a common biological process within polycystic kidney disease models and human autosomal dominant polycystic kidney disease, is present from early stages. Early versus late micro-RNA expression changes in autosomal dominant polycystic kidney disease could be targeted for a more tailored therapeutic intervention in the disease. BACKGROUND:Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development and enlargement of bilateral kidney cysts, which often leads to kidney failure. This study comprehensively characterized the expression profile of micro-RNAs (miRNAs) and their target genes in Pkd1RC/RC mice and further compared them with other murine models of polycystic kidney disease (PKD) and a model of CKD and individuals with ADPKD. METHODS:Pkd1RC/RC and wild-type (WT) mice ( n =10 each, five males and five females) were studied at 1, 6, and 12 months. At each time point, kidney volume was determined in vivo (magnetic resonance imaging), followed by ex vivo histomorphometric analyses. In randomly selected Pkd1RC/RC and WT mouse kidneys ( n =5/genotype, at 1 and 12 months), miRNA-sequencing (seq) was performed, followed by mRNA-seq, integrated (miRNA-seq/mRNA-seq analysis), and functional analysis of miRNA target genes. Venn diagrams were constructed to identify overlapping and novel differentially expressed (DE) miRNAs in Pkd1RC/RC and other commonly used murine models of PKD, diabetic kidney disease, and individuals with ADPKD. RESULTS:miRNA-seq analysis identified 41 and 181 miRNAs DE in Pkd1RC/RC versus WT kidneys at 1 and 12 months, respectively, which were confirmed by quantitative PCR. Target genes of miRNAs DE in Pkd1RC/RC at early stages encoded for proteins mainly implicated in cell proliferation and α -ketoglutarate ( α -KG) transport, whereas those DE at late stages encoded for transport proteins involved in proinflammatory and metabolic processes. Urine α -KG concentration ( 1 H nuclear magnetic resonance spectroscopy), its fractional excretion, and tissue levels were higher in Pkd1RC/RC during the entire course of the disease and associated with decreased protein expression of α -KG transporters sodium-coupled dicarboxylate transporter 3 and organic anion transporter 1. We further identified common DE miRNAs among murine models of PKD, diabetic kidney disease, and previous reports in individuals with ADPKD, as well as several novel miRNAs DE in early and late Pkd1RC/RC kidneys, which have not been previously reported in either other murine models of PKD or patients with ADPKD. CONCLUSIONS:Our study demonstrates that the renal miRNA expression profile changes longitudinally with the progression of the disease and might suggest that the post-transcriptional regulation of α -KG transport could represent a novel early feature of the disease.

Background: Cardiovascular-Kidney-Metabolic (CKM) syndrome entails pathophysiological interactions leading to multiorgan dysfunction and a high rate of cardiovascular morbidity and mortality, largely driven by the development of heart failure (HF). Mechanisms leading to HF in CKM syndrome are unknown. We created a model of CKM syndrome in swine and showed that cardiac abnormalities associate with cardiac microvascular (MV) rarefaction and decreased expression and availability of VEGF. Emerging research focuses on micro-RNAs (miRNAs) and their role in the pathophysiology of HF, but whether they play a role in the development of cardiac injury in CKM syndrome via VEGF modulation has not been investigated. We aimed to identify specific miRNAs that can target VEGF to test the hypothesis that their direct cardiac inhibition will rescue cardiac VEGF, improve angiogenic signaling, and cardiac hemodynamics in CKM syndrome. Methods: CKM (bilateral renal artery stenosis+high-cholesterol/high-carbohydrate feeding) and normal pigs (n=4 each, 2 males/2 females) were studied for 14 weeks. Unbiased micro-RNA sequencing identified 26 miRNAs differentially expressed in CKM pig hearts, of which only miR-452-5p can target VEGFA, which was confirmed by pulldown analysis and qPCR. In addition, CKM (n=4, 2 males/2 females) were treated after 6 weeks with a single intracoronary (IC) administration of a miR-452-5p inhibitor (antagomiR). Cardiac hemodynamics (echocardiography), MV architecture (3D micro-CT) and cardiac expression of VEGF and miR-452-5p (RT-qPCR) were quantified before IC miR-452-5p inhibition, and again at the end of the study (14 weeks). Results: Pigs with CKM syndrome showed blunted diastolic relaxation, increased cardiac remodeling, and reduced subendocardial MV density and cardiac VEGF expression, accompanied by higher cardiac expression of miR-452-5p compared to normal pigs. Notably, most of these parameters were improved after in vivo IC miR-452-5p inhibition (single dose). Blood pressure was unchanged ( Table 1 ). Conclusions: Our study supports a role for VEGF in preserving cardiac MV integrity in CKM syndrome. Also, it identifies a targeted post-transcriptional regulation of VEGF via miR-452-5p and supports the feasibility and efficacy of its mechanistic modulation in vivo. The predictive power of our model conceptually supports the potential of a novel targeted intervention to protect the heart in CKM syndrome. [Table: see text] Support: NIH AG084154 (ARC), and DK129240 (AE) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The mechanisms contributing to progressive kidney damage in autosomal dominant polycystic kidney disease (ADPKD) remain unclear. Renal microvascular (MV) rarefaction plays an important role in kidney disease, but its natural history, underlying mechanisms, and contributions to renal disease progression in ADPKD remain unknown. We hypothesized that intrarenal MV rarefaction is present early on and is preceded by vascular transcriptional and metabolic changes. Pkd1RC/RC and wild type (WT) mice (n = 16 each) were studied at 1, 6, and 12 mo. Total kidney volume (TKV) was measured in vivo (MRI), whereas renal MV architecture [three-dimensional (3-D) micro-computed tomography, 3-D micro-CT], capillary density, perivascular fibrosis, and histomorphometric parameters were assessed ex vivo. In randomly selected Pkd1RC/RC and WT kidneys (n = 5, each/time point), mRNA-sequencing was performed to identify differentially expressed vasculature-related genes (differentially expressed genes, DEGs). Next, in young humans with ADPKD and matched controls (n = 10 each), plasma cellular energy metabolites were determined (LC-MS/MS), validated in an extended cohort (n = 32 and n = 16, respectively), and correlated with markers of disease severity and progression. Gene-metabolite interaction networks were generated to integrate DEGs in Pkd1RC/RC at 1 mo with metabolites dysregulated in individuals with ADPKD, which were further quantified in WT and Pkd1RC/RC kidneys. Renal MV density was preserved at 1 mo but progressively decreased at 6 and 12 mo, associated with capillary loss and perivascular fibrosis. A total of 110, 48, and 201 DEGs were identified at 1, 6, and 12 mo, respectively. Plasma gamma-aminobutyric acid (GABA), homocysteine (Hcy), and asymmetric dimethyl arginine (ADMA) levels were higher in humans with ADPKD vs. controls, interacted with DEGs implicated in inflammatory and innate immune response and Hcy metabolism, and correlated with TKV and renal blood flow. Our data demonstrate that intrarenal MV abnormalities present early in ADPKD and are preceded by vascular transcriptional and metabolic changes. The renal microcirculation may constitute an important therapeutic target in ADPKD, and its underlying biomarkers may serve to monitor its progression.NEW & NOTEWORTHY We provide the first longitudinal and most comprehensive analysis of the intrarenal microvasculature in a slowly progressive model of autosomal dominant polycystic kidney disease (ADPKD) and integrate the findings with studies in a young cohort of individuals with ADPKD. We identified vasculature-related pathways that could be targeted for therapeutic interventions and contribute promising, noninvasive biomarkers in patients with ADPKD. Alterations of the intrarenal microcirculation may affect drug delivery; a better understanding of its longitudinal changes may aid in treatment management.

Immune checkpoint inhibitors (ICIs) can cause immune-related adverse events (irAEs), with acute interstitial nephritis (ICI-AIN) being the most common irAE. While the exact mechanism remains unclear, upregulation of IFN-γ and TNF-α pathways has been implicated. This study used a humanized chimeric PD-1/PD-L1 mouse model to assess renal effects of ICIs, alone or combined with proinflammatory cytokines, and to test if selective TNF-α blockade could prevent ICI-AIN. Mice were randomly divided into 4 experimental groups: Control, ICI-Only, ICI-Cytokines (ICI-Cyt), and ICI-Block (ICI-TNF-α blockade). Renal function and cytokine profiles were assessed, while kidney tissue was analyzed using microscopy and single-cell RNA-seq. Histology revealed increased renal infiltration of CD4 + /CD8 + T cells in ICI-treated groups and decreased TNF-α expression following TNF-α blockade. Additionally, kidney tissue ELISA demonstrated reduced IFN-γ levels following TNF-α blockade. Plasma IL-6, MCP-1, and TNF-α were lower in ICI-Block mice. Single-cell RNA-seq revealed shifts in immune cell populations and genes of interest including Bcl2a1 , Icos , Il18r1 , Ccr2 , and Jaml . This humanized model replicates ICI-AIN key features, revealing a synergistic role of ICIs and proinflammatory cytokines. TNF-α blockade demonstrated protective effects, supporting its potential role in mitigating the risk of ICI-AIN.

IMPORTANCE:Mesenchymal stromal cells (MSCs) possess therapeutic properties that mediate repair. Obesity impairs MSC functionality and therapeutic efficacy, possibly by eliciting dynamic modifications of epigenetic markers, like 5-hydroxymethylcytosine (5hmC). OBJECTIVE:We hypothesized that human obesity alters the 5hmC landscape and anti-inflammatory capacity of adipose tissue-derived MSCs to activate the prominent inflammatory signaling mediator Interleukin (IL)-1β. DESIGN, SETTING, PARTICIPANTS, INTERVENTION:Adipose tissue samples were collected from obese and lean individuals (body mass index ≥30 or <30 kg/m2, respectively, n = 11 each) during weight-loss or kidney donation surgery. MAIN OUTCOMES AND MEASURES:MSCs were harvested and analyzed for 5hmC profiles (MeDIP-seq) and mRNA expression (RNA-seq) (n = 5 each). Subsequently, MSCs or a vehicle were injected into mice, (n = 6 each) and two-weeks later, kidneys were evaluated using in-vivo magnetic resonance imaging and ex vivo studies. The role of IL-1β was then studied in-vitro in MSC-induced immunomodulation using siRNA in macrophages. RESULTS:Compared to MSC from lean patients, obese-MSC genes showed 2087 differential 5hmC modifications and 175 differential mRNA expression. Among them, 14 genes with overlapping alterations were involved in regulation of cytokine production, prominently IL-1β. Injecting obese MSCs elevated renal expression of IL-1β and M1 macrophage count but lowered kidney perfusion. Silencing IL-1β in obese-MSCs in vitro reduced M1 phenotype switching in co-incubated macrophages. CONCLUSIONS AND RELEVANCE:Obesity induces epigenetic and gene expression changes in MSCs, particularly in IL-1β, associated with impaired anti-inflammatory functionality of MSCs. Targeting IL-1β could be a useful therapeutic approach to modulate the decline in MSC functionality resulting from obesity.

Scattered tubular-like cells (STCs) are renal tubular cells that survive episodes of renal injury and acquire progenitor-like characteristics to repair other damaged kidney cells. STCs release proangiogenic factors in culture and induce microvascular proliferation in injured murine kidneys in vivo. Renovascular disease (RVD) compromises the reparative capacity of STCs, but the underlying mechanisms remain unknown. We hypothesized that RVD alters the expression of vasculature-related genes in swine STCs and impair their vasculoprotective properties. CD24+/CD133+ STCs were harvested from female pig kidneys after 10 wk of RVD or sham (n = 6 each), and the mRNA profiles of vasculature-related genes were analyzed using mRNA and microRNA seq (n = 3/group). STC expression of candidate differentially expressed (DE) genes and their capacity to induce human umbilical endothelial cells (HUVECs) to form tube-like networks were subsequently assessed in vitro before and after micro-RNA (miRNA) modulation (n = 6 each). mRNA-seq identified 67 upregulated and 42 downregulated vasculature-related genes in RVD-STCs. Four miRNAs were upregulated and 12 downregulated in RVD-STCs and found to target 31.3% to 40.5% of DE vasculature-related genes. Modulation in vitro of representative miRNAs decreased RVD-STC expression of anti-angiogenic and increased expression of proangiogenic target genes, respectively. Furthermore, this restored the ability of STCs to induce HUVEC tube formation on Matrigel that was impaired in RVD. Chronic renal ischemia alters the expression of vasculature-related genes in swine STCs, likely through posttranscriptional mechanisms, impairing their proangiogenic activity. These observations may contribute to develop novel approaches to preserve the reparative capacity of STCs in individuals with RVD.NEW & NOTEWORTHY The intrinsic reparative capacity of the adult mammalian kidney is restricted to the ability of scattered tubular-like cells (STCs) to repair damaged kidney cells. Our study provides evidence that chronic renal ischemia alters the mRNA/miRNA profile of angiogenic/vascular development genes of swine STCs, limiting their potential to repair injured tubular cells. Our observations may assist in developing new therapies to improve renal repair in individuals with chronic renal ischemia.

Almost 36 million US adults (1 in 7, 14%) suffer from chronic kidney disease (CKD), which is more prevalent in the elderly, affecting up to 34% of adults ≥ over 65. CKD has limited therapeutic options, partly due to the lack of large animal models that allow the translational study of the impact of age on CKD. This work reports the first preclinical model of CKD in naturally aged swine. CKD was induced in naturally aged (6-9 years old) and young (3 months old) domestic pigs (bilateral renal artery stenosis + high-cholesterol/high-carbohydrate diet), followed for 14 weeks, and compared to normal controls (n = 4–5/group). Renal hemodynamics were quantified in vivo by multidetector CT. Metabolic parameters, renal microvascular (MV) architecture (3D-micro-CT), and morphometric analysis (staining) were investigated ex vivo. Both young and old pigs developed CKD, but the renal and metabolic phenotype was accentuated in aging animals. We found that superimposition of aging on CKD resulted in higher triglycerides, glucose levels, insulin resistance, and adipose tissue accumulation (subcutaneous, visceral, perirenal) (Fig. 1A and B), accompanied by a more severe renal dysfunction (C), cortical MV rarefaction, and reduced peritubular capillary density (D and E), and more severe structural damage disclosed by increased glomerulosclerosis, tubule-interstitial fibrosis, and tubular injury (F). Notably, the severity of renal MV rarefaction in aging CKD correlated with functional and structural abnormalities (simple linear regression analysis, G). Our study establishes the first large animal model of aging CKD, allowing the investigation of age as a biological variable in kidney-metabolic diseases. Age exacerbates metabolic and renal abnormalities, and our data support the critical role of renal MV disease in aging CKD and suggest that its exacerbation is a potential key player in its progression. This novel translational platform could foster new studies toward the elucidation of novel mechanisms of age-related deterioration of renal function in CKD and for developing therapeutic interventions.

INTRODUCTION:CD24+/CD133+ scattered tubular-like cells (STCs) are surviving renal cells that acquire progenitor-like characteristics to repair other damaged kidney cells. Renal artery stenosis (RAS) impairs the reparative capacity of STCs, but the underlying mechanisms remain unknown. STCs contain abundant endoplasmic reticulum (ER), but its capacity to fold proteins could become saturated (ER stress), leading to STC dysfunction. We hypothesized that RAS alters the expression of genes implicated in ER stress in swine STCs. METHODS:STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n = 6 each) and expression of ER stress genes was assessed using mRNA-seq (n = 3 each). To elucidate mechanisms regulating ER stress genes in RAS-STCs, integrated mRNA-seq/microRNA (miRNA)-seq and transcription factor (TF) prediction analysis were performed. STC ER stress was assessed in vitro using Western blotting, serial block-face electron microscopy, and mass spectrometry. The involvement of ER stress in regulating the STC-protective effects was also assessed in vitro by their capacity to improve viability of injured human tubular epithelial cells. RESULTS:RAS pigs developed significant renal dysfunction. mRNA-seq identified 25 ER stress genes upregulated and 30 downregulated in RAS-STCs versus normal-STCs. miRNAs were found to target over a third of all differentially expressed ER stress genes, and almost half of genes encoding for the top 50 TFs involved in regulation of ER stress genes were dysregulated in RAS-STCs. RAS-STCs exhibited higher ER stress compared to normal-STCs, reflected in significant ER dilation and formation of ER-mitochondria contacts and increased levels of ER stress-related amino acids. Importantly, ER stress inhibition improved the reparative capacity of RAS-STCs in vitro. CONCLUSION:Renal ischemia alters expression of ER stress-related genes in swine STCs, likely through post-transcriptional- and TF-regulatory mechanisms, which induces ER stress and impairs their reparative potency. These alterations may limit the potential of STCs to repair damaged kidneys in subjects with RAS.

Chronic kidney disease (CKD), heart failure with preserved ejection fraction (HFpEF), and the recently defined cardiovascular-kidney-metabolic (CKM) syndrome are important challenges for the health care system, associated with a staggering increase in morbidity and mortality rates. Unfortunately, the complex pathophysiology of these processes remains to be fully elucidated, imposing hurdles to prevent their development and decelerate their progression. Large animal models are critical for understanding cardio-renal pathophysiology and for the discovery of new therapies. Among them, pig models offer significant translational power to grasp cardio-renal pathophysiology and to test the feasibility and efficacy of new strategies. We recently developed and characterized swine models of CKD, HFpEF, and CKM syndrome that closely recapitulate human disease and allow for the study of the impact of biological variables, such as sex or age, on these conditions. Furthermore, data from these studies revealed key mechanisms implicated in the pathogenesis of CKD, HFpEF, and CKM syndrome and identified potential targets for intervention. This focused mini review highlights the main features of these novel swine models and discusses ongoing and future research aimed at developing novel therapies.

Renal artery stenosis (RAS) is the leading cause of secondary hypertension worldwide. However, current medical and surgical treatment modalities provide minimal benefits for kidney injury. Recent preclinical RAS models have demonstrated promising potential of human mesenchymal stem cells (MSC) and their daughter extracellular vesicles (EV) in improving murine renal function and attenuating inflammation. However, the extent and mechanisms underlying immune rejection of xenogeneic MSCs or EVs are yet undetermined. Therefore, adipose tissue was harvested from adult healthy patients. Adipose-derived MSCs were extracted and cultured, and EVs were isolated from their supernatants via ultra-centrifugation. Then, mice randomly assigned to RAS or sham surgery were divided into 6 groups: sham surgery, RAS, sham + MSC, RAS + MSC, sham + EV, and RAS + EV. Two weeks after intra-aortic injection of MSCs (5 × 105) or EVs (20 µg protein), we compared the intrarenal T-cell and macrophage accumulation, splenic B-cell numbers, circulating cytokines and anti-human antibodies levels among the groups. MSCs and EVs did not influence intrarenal immune cell infiltrations. However, MSCs significantly increased circulating anti-human antibodies. In the spleen, RAS + EV mice showed higher memory IgM+ B-cells but reduced CD19+ B-cells compared to RAS + MSC. In vitro T-cell recall assay showed that both MSCs and EVs exhibited reduced IFN-γ release upon re-stimulation, indicating an immunosuppressive effect. Therefore, xenogeneic MSCs induced a greater humoral response in mice, while EVs triggered a splenic cellular response, but neither elicits discernible kidney rejection. Our results provide key insights into the immunomodulatory mechanisms of MSCs and EVs and immune mechanisms underlying xenograft rejection.

Background: CD24+/CD133+ scattered tubular-like cells (STCs) are surviving renal cells that acquire progenitor-like characteristics to repair other damaged kidney cells. Renal artery stenosis (RAS) impairs the reparative capacity of STCs, but the underlying mechanisms remain unknown. STCs contain abundant endoplasmic reticulum (ER), but its capacity to fold proteins could become saturated (ER-stress), leading to STC dysfunction. We hypothesized that RAS alters the expression of genes implicated in ER-stress in swine STCs. Methods: STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each) and expression of ER-stress genes was assessed using mRNA-seq (n=3 each). To elucidate mechanisms regulating ER-stress genes in RAS-STCs, integrated mRNA-seq/microRNA (miRNA)-seq and transcription factor (TF) prediction analysis were performed. STC ER-stress was assessed in vitro using western blotting, serial block-face electron microscopy, and mass-spectrometry. The involvement of ER-stress in regulating the STC-protective effects was also assessed in vitro by their capacity to improve viability of injured human tubular epithelial cells. Results: RAS pigs developed significant renal dysfunction. mRNA-seq identified 25 ER-stress genes upregulated and 30 downregulated in RAS-STCs versus Normal-STCs. miRNAs were found to target over a third of all differentially expressed ER-stress genes, and almost half of genes encoding for the top 50 TFs involved in regulation of ER-stress genes were dysregulated in RAS-STCs. RAS-STCs exhibited higher ER-stress compared to Normal-STCs, reflected in significant ER dilation and formation of ER-mitochondria contacts, and increased levels of ER-stress-related amino acids. Importantly, ER-stress inhibition improved the reparative capacity of RAS-STCs in vitro. Conclusion: Renal ischemia alters expression of ER-stress-related genes in swine STCs, likely through post-transcriptional- and TF-regulatory mechanisms, which induces ER-stress and impairs their reparative potency. These alterations may limit the potential of STCs to repair damaged kidneys in subjects with RAS.