Lerman, Lilach O

Key Points Activin A is implicated in profibrotic and prosenescent kidney injury and correlates with kidney injury markers in animals and humans. Follistatin, through activin A antagonism, reduces senescence burden, macrophage infiltration, and proinflammatory pathway activation in murine diabetic kidney disease. Follistatin and other antagonists of activin A signaling pathways may be promising, novel therapeutics for diabetic kidney disease. Background Circulating activin A, an inflammatory mediator implicated in profibrotic kidney injury and cellular senescence-induced adipose tissue dysfunction, is increased in human diabetic kidney disease (DKD) and directly correlates with kidney dysfunction. We tested the hypothesis that activin A increases kidney injury, senescent cell abundance, and macrophage infiltration in DKD and antagonism through follistatin (FS) therapy diminishes these effects. Methods An accelerated nephropathy type 2 diabetes (db/db) mouse model was generated by implantation of angiotensin II-loaded osmotic minipumps resulting in increased albuminuria and glomerular and tubular injury. Kidney repair effects of FS (5 µ g intraperitoneal; two doses) were assessed through markers of kidney injury, fibrosis, inflammation, cellular senescence, and macrophage infiltration. In vitro studies examined antiactivin effects of FS on high glucose-exposed human monocytes, renal fibroblasts, and renal tubule epithelial cells. Results Activin A antagonism with FS reduced senescence (p19), proinflammatory (including senescence-associated secretory phenotype), and profibrotic markers including activin A. FS improved kidney morphology, restored podocyte markers (nephrin and Wilms tumor-1), and reduced kidney injury biomarkers, albuminuria and kidney fibrosis. FS decreased kidney macrophage and leukocyte infiltration and absent in melanoma 2 inflammasome activation. FS seemed to suppress inflammation through the toll-like receptor-4/NF kappa-light-chain-enhancer of activated B cells pathway in vivo further supported in human macrophages in vitro . In addition, FS reduced hyperglycemia-induced renal fibroblast activation and renal tubule epithelial cell senescence in vitro . Conclusions Activin A is a mediator of kidney injury through macrophage-associated inflammation in murine DKD. FS acts through senomorphic activities which inhibit profibrotic, proinflammatory, and prosenescence signaling by activin A. Hence, antiactivin targeting may aid in the development of a promising, novel therapeutic for DKD.

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.

Metabolic syndrome (MetS) is associated with low-grade inflammation, which can be exacerbated by renal artery stenosis (RAS) and renovascular hypertension, potentially worsening outcomes through pro-inflammatory cytokines. This study investigated whether mesenchymal stem/stromal cells (MSCs) could reduce fat inflammation in pigs with MetS and RAS. Twenty-four pigs were divided into Lean (control), MetS, MetS + RAS, and MetS + RAS + MSCs. In the MSC-treated group, autologous adipose-derived MSCs (107 cells) were injected into the renal artery six weeks after RAS induction. After four weeks, fat volumes and inflammatory markers were assessed. MSC treatment reduced levels of pro-inflammatory cytokines (MCP-1, TNF-a, IL-6) in the renal vein blood and in perirenal fat. The MSCs also decreased fat fibrosis, restored adipocyte size, and altered adipogenesis-related gene expression, particularly in the perirenal fat. These effects were less pronounced in subcutaneous fat. The MSC therapy attenuated fat inflammation and improved metabolic outcomes in pigs with MetS + RAS, suggesting that adipose-derived MSCs may offer a promising therapeutic approach for metabolic disorders.

Key Points Renovascular disease impairs the capacity of human adipose tissue–derived mesenchymal stem/stromal cells to repair ischemic murine kidneys. miR-378h modulated the capacity of renovascular disease adipose tissue–derived mesenchymal stem/stromal cells to repair ischemic kidneys in vivo . Background Renovascular disease leads to renal ischemia, hypertension, and eventual kidney failure. Autologous transplantation of adipose tissue–derived mesenchymal stem/stromal cells (MSCs) improves perfusion and oxygenation in stenotic human kidneys, but associated atherosclerosis and hypertension might blunt their effectiveness. We hypothesized that renovascular disease alters the human MSC transcriptome and impairs their reparative potency. Methods MSCs were harvested from subcutaneous abdominal fat of patients with renovascular disease and healthy volunteers ( n =3 each), characterized and subsequently injected (5×10 5 /200 μ l) into mice 2 weeks after renal artery stenosis or sham surgery ( n =6/group). Two weeks later, mice underwent imaging and tissue studies. MSCs from healthy volunteers and in those with renovascular disease were also characterized by mRNA/microRNA (miRNA) sequencing. Based on these, MSC proliferation and mitochondrial damage were assessed in vitro before and after miRNA modulation and in vivo in additional renal artery stenosis mice administered with MSCs from renovascular disease pretreated with miR-378h mimic ( n =5) or inhibitor ( n =4). Results MSCs engrafted in stenotic mouse kidneys. Healthy volunteer MSCs (but not renovascular disease MSCs) decreased BP, improved serum creatinine levels and stenotic-kidney cortical perfusion and oxygenation, and attenuated peritubular capillary loss, tubular injury, and fibrosis. Genes upregulated in renovascular disease MSCs versus healthy volunteer MSCs were mostly implicated in transcription and cell proliferation, whereas those downregulated encoded mainly mitochondrial proteins. Upregulated miRNAs, including miR-378h, primarily target nuclear-encoded mitochondrial genes, whereas downregulated miRNAs mainly target genes implicated in transcription and cell proliferation. MSC proliferation was similar, but their mitochondrial structure and reparative function both in vivo and in vitro improved after miR-378h inhibition. Conclusions Renovascular disease impaired the reparative capacity of human MSCs, possibly by dysregulating miR-378h that targets mitochondrial genes. Podcast This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2024_08_21_ASN0000000000000440.mp3

Background Coronary microvascular dysfunction (CMD) represents an early functional characteristic of coronary vascular aging. Klotho (α‐klotho) is a circulating protein inversely linked to physiological aging. We examined low klotho as a potential marker for vascular aging in patients with CMD and no coronary artery disease. Methods and Results Patients undergoing nonurgent angiogram for chest pain who had no coronary artery disease underwent invasive coronary microvascular and endothelial function testing. CMD was defined by ≤50% increase in coronary blood flow (percentage change in coronary blood flow) in response to intracoronary acetylcholine or coronary flow reserve ≤2. Fresh arterial whole blood was used to analyze circulating endothelial progenitor cells with flow cytometry. Stored arterial plasma was used for klotho analysis by ELISA. Participants with CMD (n=62) were compared with those without CMD (n=36). Those with CMD were age 55±10 years (versus 51±11 years; P =0.07) and 73% women (versus 81%; P =0.38). Traditional risk factors for coronary artery disease were similar between groups. Patients with CMD had less klotho (0.88±1.50 versus 1.75±2.38 ng/mL; P =0.03), and the odds of low klotho in CMD were significant in a logistic regression model after adjusting for traditional cardiovascular risk factors (odds ratio [OR], 0.80 [95% CI, 0.636–0.996]; P =0.05). Higher klotho was associated with higher numbers of endothelial progenitor cells with vascular regenerative potential (CD34 + and CD34 + CD133 + KDR + ). Among a subgroup of patients with atherosclerotic cardiovascular disease risk <5% (n=58), CMD remained associated with lower klotho (OR, 0.80 [95% CI, 0.636–0.996]; P =0.047). Conclusions Klotho may be a biomarker for CMD and may be a therapeutic target for groups of patients without significant traditional cardiovascular risk.

The single-cell platform mass cytometry (CyTOF) enables detection of significant changes in one million of renal cells, especially in subsets of immune cells in ischemic human kidneys distal to renal artery stenosis (RAS). We found that pro-repair cell types such as scattered tubular-progenitor-like cells, aged neutrophils, and regulatory B cells show a compensatory increase in RAS. Immune cell phenotype changes may reflect ongoing inflammation and impaired immune defense capability in the kidneys.

Abstract Background: Scattered tubular-like cells (STCs) are differentiated renal tubular cells that during recovery from ischemic injury dedifferentiate to repair other injured renal cells. Renal artery stenosis (RAS), often associated with chronic inflammatory injury, compromises the integrity and function of STCs, but the underlying mechanisms remain unknown. We hypothesized that RAS alters the transcriptomic and epigenetic profile of inflammatory genes in swine STCs. Methods: STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each). STC mRNA profiles of inflammatory genes were analyzed using high-throughput mRNA-sequencing (seq) and their DNA methylation (5mC) and hydroxymethylation (5hmC) profiles by DNA immunoprecipitation and next-generation sequencing (MeDIP-seq) (n=3 each), followed by an integrated (mRNA-seq/MeDIP-seq) analysis. STC protein expression of candidate differentially expressed (DE) genes and common proinflammatory proteins were subsequently assessed in vitro before and after epigenetic (Bobcat339) modulation. Results: mRNA-seq identified 57 inflammatory genes up-regulated in RAS-STCs versus Normal-STCs (&gt;1.4 or &lt;0.7-fold, P&lt;0.05), of which 14% exhibited lower 5mC and 5% higher 5hmC levels in RAS-STCs versus Normal-STCs, respectively. Inflammatory gene and protein expression was higher in RAS-STCs compared with Normal-STCs but normalized after epigenetic modulation. Conclusions: These observations highlight a novel modulatory mechanism of this renal endogenous repair system and support development of epigenetic or anti-inflammatory therapies to preserve the reparative capacity of STCs in individuals with RAS.

Abstract Aims This study aims to identify whether adding peripheral microvascular dysfunction (PMED), a marker of atherosclerosis to established risk scores has an incremental prognostic value for major adverse cardiovascular events (MACE). Methods and results This is a retrospective study of patients who underwent measuring peripheral arterial tonometry from 2006 to 2020. The optimal cut-off value of the reactive hyperaemia index (RHI) that had maximal prognostic value associated with MACE was calculated. Peripheral microvascular endothelial dysfunction was defined as the RHI lower than the cut-off. Traditional cardiovascular risk factors such as age, sex, congestive heart failure, hypertension, diabetes, stroke, and vascular disease were determined to calculate the CHA2DS2-Vasc score. The outcome was MACE defined as myocardial infarction, heart failure hospitalization, cerebrovascular events, and all-cause mortality. A total of 1460 patients were enrolled (average age 51.4 ± 13.6, 64.1% female). The optimal cut-off value of the RHI was 1.83 in the overall population and in females and males was 1.61 and 1.8, respectively. The risk of MACE during 7 [interquartile range (IQR): 5,11] years of follow-up was 11.2%. Kaplan–Meier analysis showed that lower RHI is associated with worse MACE-free survival (P &lt; 0.001). Multivariate Cox proportional hazard analysis, controlling for classic cardiovascular risk factors or risk scores such as CHA2DS2-Vasc and Framingham risk score revealed that PMED is an independent predictor of MACE. Conclusion Peripheral microvascular dysfunction predicts cardiovascular events. Non-invasive assessment of peripheral endothelial function may be useful in early detection and improving the stratification of high-risk patients for cardiovascular events. Lay summary Key findings

Significance Statement Glomerular size differs by cortex depth. Larger nephrons are prognostic of progressive kidney disease, but it is unknown whether this risk differs by cortex depth or by glomeruli versus proximal or distal tubule size. We studied the average minor axis diameter in oval proximal and distal tubules separately and by cortex depth in patients who had radical nephrectomy to remove a tumor from 2019 to 2020. In adjusted analyses, larger glomerular volume in the middle and deep cortex predicted progressive kidney disease. Wider proximal tubular diameter did not predict progressive kidney disease independent of glomerular volume. Wider distal tubular diameter showed a gradient of strength of prediction of progressive kidney disease in the more superficial cortex than in the deep cortex. Background Larger nephrons are prognostic of progressive kidney disease, but whether this risk differs by nephron segments or by depth in the cortex is unclear. Methods We studied patients who underwent radical nephrectomy for a tumor between 2000 and 2019. Large wedge kidney sections were scanned into digital images. We estimated the diameters of proximal and distal tubules by the minor axis of oval tubular profiles and estimated glomerular volume with the Weibel–Gomez stereological model. Analyses were performed separately in the superficial, middle, and deep cortex. Cox proportional hazard models assessed the risk of progressive CKD (dialysis, kidney transplantation, sustained eGFR <10 ml/min per 1.73 m 2 , or a sustained 40% decline from the postnephrectomy baseline eGFR) with glomerular volume or tubule diameters. At each cortical depth, models were unadjusted, adjusted for glomerular volume or tubular diameter, and further adjusted for clinical characteristics (age, sex, body mass index, hypertension, diabetes, postnephrectomy baseline eGFR, and proteinuria). Results Among 1367 patients were 62 progressive CKD events during a median follow-up of 4.5 years. Glomerular volume predicted CKD outcomes at all depths, but only in the middle and deep cortex after adjusted analyses. Proximal tubular diameter also predicted progressive CKD at any depth but not after adjusted analyses. Distal tubular diameter showed a gradient of more strongly predicting progressive CKD in the superficial than deep cortex, even in adjusted analysis. Conclusions Larger glomeruli are independent predictors of progressive CKD in the deeper cortex, whereas in the superficial cortex, wider distal tubular diameters are an independent predictor of progressive CKD.

BACKGROUND: The financial burden linked to the diagnosis and treatment of patients with chest pain on the health care system is considerable. Angina and nonobstructive coronary artery disease (ANOCA) is common, associated with adverse cardiovascular events, and may lead to repeat testing or hospitalizations. Diagnostic certainty can be achieved in patients with ANOCA using coronary reactivity testing (CRT); however, its financial effect on the patient has not been studied. Our goal was to assess the effect of CRT on health care–related cost in patients with ANOCA. METHODS: Patients with ANOCA who underwent diagnostic coronary angiography (CAG) and CRT (CRT group) were matched to controls who had similar presentation but only underwent a CAG without CRT (CAG group). Standardized inflation-adjusted costs were collected and compared between the 2 groups on an annual basis for 2 years post the index date (CRT or CAG). RESULTS: Two hundred seven CRT and 207 CAG patients were included in the study with an average age of 52.3±11.5 years and 76% females. The total cost was significantly higher in the CAG group as compared with the CRT group ($37 804 [$26 933–$48 674] versus $13 679 [$9447–$17 910]; P <0.001). When costs are itemized and divided based on the Berenson-Eggers Type of Service categorization, the largest cost difference occurred in imaging (any type, including CAG; P <0.001), procedures (eg, percutaneous coronary intervention/coronary artery bypass grafting/thrombectomy) ( P =0.001), and test (eg, blood tests, EKG; P <0.001). CONCLUSIONS: In this retrospective observational study, assessment of CRT in patients with ANOCA was associated with significantly reduced annual total costs and health care utilization. Therefore, the study may support the integration of CRT into clinical practice.