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.

We evaluated kidney fibrosis in renovascular disease (RVD) using quantitative (q) and semi-quantitative magnetization-transfer (MT) magnetic resonance imaging (MRI). Ten RVD patients and 22 healthy volunteers (HVs) were prospectively enrolled. Of HVs, 10 (HV-1) underwent imaging studies whereas plasma samples were collected from 12 others (HV-2). The qMT-derived bound-pool fraction (f) was compared to MRI-based MT imaging (MTI)-derived MT ratio (MTR), blood oxygenation-level-dependent (BOLD) R2*, and diffusion-weighted imaging (DWI) apparent diffusion coefficient (ADC). MTR was assessed at offset frequencies of 600 Hz and 1000 Hz, and RVD qMT and MTI on both 1.5T and 3.0T MRI. Additionally, we measured plasma and urinary levels of fibrogenic cytokines and micro-RNAs, and stenotic kidney (STK) perfusion and volume with multidetector computed-tomography (MDCT). At 3.0T-MRI, STK cortex and medulla qMT-f were higher in RVD vs. HV-1 (p = 0.01, p = 0.05, respectively), as was MTR-600 Hz, whereas BOLD-R2* and the DWI ADC were not different. MTR and f measured at 1.5T were comparable to those obtained at 3.0T. STK blood flow was decreased vs. the contralateral kidney (CLK) (p = 0.033) but plasma and urinary fibrogenic indices were unchanged in RVD vs. HV-2. Both f and MTI at 3.0T-MRI may be useful for noninvasive assessment of STK fibrosis, independent of magnetic-field strength. MTI is potentially more sensitive than fibrogenic cytokine levels for detecting mild RVD-related fibrosis changes.

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.

Background Renal artery stenosis (RAS) can trigger the development of renal damage and poststenotic dilatation (PSD). However, the clinical relevance and mechanisms are ill-characterized. Purpose To characterize the mechanisms underlying PSD development distal to the site of RAS in humans. Materials and Methods Forty-nine individuals with RAS were prospectively consecutively recruited from October 2013 to September 2024. The characteristics of the stenosis, including the dilatation-diameter ratio (DDR), and single-kidney volumes, perfusion, and glomerular filtration rate (GFR) were measured at three-dimensional multidetector abdominal CT. Additional characteristics included stenotic kidney (STK) oxygenation (blood oxygen level-dependent MRI), cytokine levels in the STK renal vein (RV) and inferior vena cava (blood sampling), and measured GFR (iothalamate clearance). Seven participants subsequently underwent percutaneous transluminal renal angioplasty (PTRA). Data were analyzed with Spearman correlation and linear regression. Results Of 49 participants with RAS (median age, 73 years [IQR, 67-75 years]; 28 male participants), age, blood pressure, measured GFR, and STK oxygenation were comparable between participants with PSD (n = 31) and participants without PSD (n = 18), but participants with PSD had a lower median STK cortical volume. DDR positively correlated with the STK RV levels of neutrophil gelatinase-associated lipocalin, interferon-γ, and monocyte chemoattractant protein-1 (MCP-1), which were elevated in participants with PSD versus participants without PSD or in the STK RV versus the IVC (all P < .05). The regression coefficient between STK RV MCP-1 level and DDR was 0.813 (95% CI: 0.328, 1.298; P = .002) in an unadjusted model and 0.889 (95% CI: 0.246, 1.532; P = .009) after multivariable adjustment for potential confounders; that is, each 1-ng/mL increase in STK RV MCP-1 level was independently associated with a 0.889-unit DDR increase. DDR also correlated positively with urinary protein level (r = 0.33; P = .04) and negatively with estimated GFR (r = -0.51; P = .004), but there was no evidence of a correlation of DDR with renal hemodynamics, arterial morphologic features, or blood flow velocity at Doppler US. PTRA did not improve estimated GFR but did induce PSD regression, the extent of which correlated negatively with STK RV MCP-1 level. Conclusion PSD, as evaluated at abdominal CT, may be associated with STK damage and the release of MCP-1. Clinical trial registration nos. NCT02266394 and NCT04508049 © RSNA, 2026 Supplemental material is available for this article.

Objective: This study aims to develop a contrast-free, high-sensitivity ultrasound method, denoted as Radon transform-based flow measurement (R-Flow), for in vivo mapping of microvascular flow vectors and for establishing R-Flow-derived vector-field metrics to noninvasively quantify microcirculatory patterns in liver cirrhosis. Impact Statement: R-Flow enables robust, contrast-free imaging of microvascular dynamics and demonstrates translational feasibility in the human liver. Its direction-aware indices offer pilot in vivo quantification of flow redistribution and remodeling, providing unique insights into hepatic flow dynamics. Introduction: Microvascular dysfunction is a hallmark of many diseases, yet noninvasive visualization and quantitative assessment of abnormal microcirculation remain limited. Methods: R-Flow leverages Radon transform to decode red blood cell dynamics from the spatiotemporal domain of ultrasound flow signals, reconstructing velocity vectors at microvascular scale. From these vector maps, unique direction-aware indices are further derived to characterize flow distribution and heterogeneity. Results: Validated across simulations, phantoms, and in vivo studies, R-Flow provides robust velocity estimation across a wide range (1 to 60 mm/s). Notably, it enables high-sensitivity microvascular flow vector mapping of human liver, showing strong agreement with references (r > 0.9). In a rat model, these direction-aware indices revealed a shift from healthy multipath perfusion in control livers to directionally biased vascular pattern in cirrhotic livers, demonstrating significant correlation with pathological indicators. Conclusion: R-Flow enables noninvasive, contrast-free mapping of microvascular blood flow velocity and offers a promising approach for high-resolution assessment of microvascular flow characteristics.

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.

OBJECTIVES:To evaluate the efficacy of quantitative magnetization transfer (qMT) in quantifying interstitial fibrosis in kidney transplantation (KT) recipients, and to assess its dependence on MRI field strength (1.5T vs 3.0T), in comparison to conventional magnetization transfer imaging (MTI) performed at both 600 Hz and 1000 Hz offset frequencies. MATERIALS AND METHODS:This cross-sectional study involved 20 patients, 4 to 10 years post-KT, and 31 healthy volunteers (HV). Using 3.0T-MRI, we assessed KT and HV kidneys using MTI, qMT, blood oxygenation-level-dependent (BOLD), and diffusion-weighted imaging (DWI), and at 1.5T-MRI, we also assessed in KT renal fibrosis with MTI and qMT. In addition, we measured fibrogenic cytokine levels in KT and HV using ELISA and assessed fibrosis and cytokine expression in KT biopsy samples. RESULTS:KT showed renal dysfunction and higher circulating collagen-IV, MCP-1, IL-6, and NGAL than HV. At 3.0T-MRI, both the qMT index f and the MTI index magnetization-transfer ratio (MTR) measured at either 1000 Hz or 600 Hz demonstrated greater KT cortex and medulla fibrosis compared with HV, whereas DWI and BOLD signals showed no difference. Cortical and medullary MTI-MTR-600 Hz and f -qMT were comparable between 1.5T and 3.0T-MRI, whereas MTI-MTR-1000 Hz was not. Inter- and intra-observer reproducibility of qMT and MTR showed consistently high reliability across the cortex and medulla. Histologic cortical fibrosis in KT [11.12% (8.02, 15.32)] correlated directly with f -qMT at 3.0T-MRI (Spearman, P < 0.0001) but not at 1.5T-MRI, and correlated modestly with cortical and medullary MTR at 600 Hz and 1000 Hz at 1.5T but not at 3.0T-MRI. In addition, f correlated with years post-KT. CONCLUSIONS:Both MTI-600 Hz at 1.5T-MRI and qMT at 3.0T-MRI are promising noninvasive tools for evaluating kidney allograft fibrosis, and the choice between them may depend on machine availability. Furthermore, their fidelity between 1.5T and 3.0T-MRI may facilitate clinical translation by affording comparison of renal fibrosis measured on different MRI machines.

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.

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.

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.

Cellular senescence participates in the pathophysiology of post-stenotic kidney damage, but how it regulates tissue remodeling is incompletely understood. Macrophage-myofibroblast transition (MMT) contributes to the development of tissue fibrosis. We hypothesized that cellular senescence contributes to MMT and renal fibrosis in mice with renal artery stenosis (RAS). INK-ATTAC mice expressing p16INK-4a and green fluorescent protein in senescent cells were assigned to control or unilateral RAS, untreated or treated with AP20187 (an apoptosis inducer in p16INK-4a-expressing cells) for 4 weeks. Renal perfusion was studied in vivo using micro-MRI, and kidney morphology, senescence, and MMT ex vivo. Cellular senescence was induced in human renal proximal tubular epithelial cells (HRPTEpiC) in vitro, and interferon-induced transmembrane protein-3 (IFITM3), a cellular senescence vector, was silenced (siRNA) or over-expressed (plasmid). HRPTEpiC were then co-incubated with macrophages with silenced integrin-3 (ITGB3), a regulator of mesenchymal transitions. CD68/p16INK-4a/α-SMA co-expression and senescence markers were studied. Murine RAS kidneys showed increased expression of p16INK-4a and MMT markers (F4/80, α-SMA) vs. controls, which decreased after AP20187, as did renal fibrosis and plasma creatinine, whereas renal perfusion increased. IFITM3 and ITGB3 expression were upregulated in senescent HRPTEpiC or co-cultured macrophages, respectively. MMT markers and TGF-β/Smad3 expression also rose in these macrophages and decreased after IFITM3 or ITGB3 silencing. p16INK-4a-expressing macrophages may regulate interstitial fibrosis in RAS via MMT. This process is associated with elevated expression of ITGB3 and TGF-β/Smad3 pathway activation through neighboring senescent cell-derived IFITM3. These findings may implicate MMT as a therapeutic target in ischemic kidneys.

Introduction:MOTS-c (mitochondrial open reading frame of the 12S rRNA type-c) is a mitochondrial-derived peptide and regulator of metabolic homeostasis. Although its role in glucose and lipid metabolism is emerging, changes in circulating MOTS-c with obesity remain unclear. We hypothesized that circulating MOTS-c concentrations would be altered in obese vs. lean adults in associations with altered metabolic and inflammatory markers. Methods:Circulating MOTS-c levels, metabolic parameters, and inflammatory markers were compared between 22 lean controls and 32 obese participants scheduled for bariatric surgery. Longitudinal changes in weight, MOTS-c levels, and metabolic markers were also analyzed in 10 of the obese patients before and 6 months after bariatric surgery. Additionally, adipose tissue MOTS-c expression was assessed by immunofluorescence in lean kidney donors (n = 6) and obese (n = 14) subjects. Results:Circulating MOTS-c levels were significantly higher in obese compared to lean individuals (273 ± 56 vs. 223 ± 50 pg/mL; P < 0.01). BMI and HOMA-IR independently predicted elevated MOTS-c levels (P = 0.035 and P = 0.032, respectively). MOTS-c showed a biphasic relationship with HOMA-IR, rising sharply above HOMA-IR of ∼ 6.6 mmol/L×µU/mL. Adipose tissue MOTS-c did not differ between the groups or correlate with circulating MOTS-c. Despite significant BMI improvements post-surgery (P < 0.001), circulating MOTS-c levels remained unchanged (P = 0.913). Conclusion:Circulating MOTS-c levels are elevated in obesity, exhibiting a nonlinear relationship with BMI and insulin resistance. MOTS-c may represent a compensatory metabolic response in obesity and insulin-resistant states, highlighting its potential as a clinical biomarker. This preliminary exploratory study warrants validation in larger and independent cohorts.

Microvascular imaging has advanced significantly with ultrafast data acquisition and improved clutter filtering, enhancing the sensitivity of power Doppler imaging to small vessels. However, the image quality remains limited by spatial resolution and elevated background noise, both of which impede visualization and accurate quantification. To address these limitations, this study proposes a high-resolution cross-correlation Power Doppler (HR-XPD) method that integrates spatial radiality weighting with Doppler signal coherence analysis, thereby enhancing spatial resolution while suppressing artifacts and background noise. Quantitative evaluations in simulation and in vivo experiments on healthy human liver, transplanted human kidney, and pig kidney demonstrated that HR-XPD significantly improves microvascular resolvability and contrast compared to conventional PD. In vivo results showed up to a 2 to 3-fold enhancement in spatial resolution and an increase in contrast by up to 20 dB. High-resolution vascular details were clearly depicted within a short acquisition time of only 0.3 s-1.2 s without the use of contrast agents. These findings indicate that HR-XPD provides an effective, contrast-free, and high-resolution microvascular imaging approach with broad applicability in both preclinical and clinical research.

Mesenchymal stromal cells (MSCs) possess therapeutic properties, which can be blunted by obesity. Autophagy, a cellular recycling process, is essential for MSC function. We investigated the mechanisms by which obesity affects the properties of MSCs, with a focus on autophagy. Adipose tissue was obtained from kidney donors [body mass index (BMI) <30 kg/m 2 , non-obese] or individuals undergoing weight loss surgery (BMI ≥30 kg/m 2 , obese) for MSC harvesting ( n = 11 each); samples were randomized to sequencing (seq; n = 5 each) or functional studies ( n = 6 each). MSCs were sequenced to determine their epigenetic (5-hydroxymethylcytosine) and transcriptomic profiles across autophagy-related genes using hydroxymethylated DNA immunoprecipitation sequencing and mRNA-seq, respectively. Genes with shared trends in both datasets underwent Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) validation. During functional studies, 2-h starvation was used to induce autophagy in vitro , enabling detection of changes in the protein expression of microtubule-associated protein 1A/1B-light chain-3 and in autophagic flux. Obesity amplified a starvation-induced reduction in autophagic flux in MSCs while promoting earlier generation of new autophagosomes during autophagy initiation. Integrated analysis of the two sequencing datasets revealed 124 differentially hydroxymethylated genes and 30 differentially expressed mRNAs. Among six overlapping autophagy-related genes, three exhibited same-direction trends. Of these, STX12 and SLC25A4 may be implicated in the impact of obesity on autophagic changes in MSCs. Therefore, human obesity may alter autophagy in adipose tissue–derived MSC, and thereby their metabolism and function.

Ultrasound localization microscopy (ULM) enables microvascular imaging at spatial resolutions beyond the acoustic diffraction limit, offering significant clinical potentials. However, ULM performance relies heavily on microbubble (MB) signal sparsity, the number of detected MBs, and signal-to-noise ratio (SNR), all of which vary in clinical scenarios involving bolus MB injections. These sources of variations underscore the need to optimize MB dosage, data acquisition timing, and imaging settings in order to standardize and optimize ULM of microvasculature. This pilot study investigated temporal changes in MB signals during bolus injections in both pig and human models to optimize data acquisition for clinical ULM. Quantitative indices were developed to evaluate MB signal quality, guiding selection of acquisition timing that balances the MB localization quality and adequate MB counts. The effects of transmitted voltage and dosage were also explored. In the pig model, a relatively short window (approximately 10 seconds) for optimal acquisition was identified during the rapid wash-out phase, highlighting the need for real-time MB signal monitoring during data acquisition. The slower wash-out phase in humans allowed for a more flexible imaging window of 1-2 minutes, while trade-offs were observed between localization quality and MB density (or acquisition length) at different wash-out phase timings. Guided by these findings, robust ULM imaging was achieved in both pig and human kidneys using a short period of data acquisition, demonstrating its feasibility in clinical practice. This study provides insights into optimizing data acquisition for consistent and reproducible ULM, paving the way for its standardization and broader clinical applications.