American Journal of Physiology-Renal Physiology

Kidney disease affects over 850 million people and leads to degradation of the kidney cortical compartments and kidney failure. Interstitial fibrosis and tubular atrophy (IFTA) is a universal feature of chronic kidney disease, yet the sequence of tubulointerstitial matrix protein (matrisome) changes that underlie the development and progression of IFTA are not fully understood. In particular, post-translational modifications which may stabilize key matrisome components and result in irreversible and functionally detrimental interstitial fibrosis or tubular basement membrane thickening, have yet to be characterized. Post-translational modifications (e.g., acetylation) have been shown to contribute to kidney diseases, yet they have not been investigated in chronic kidney disease progression. Herein, we established feasibility of matrisome protein quantification in the tubulointerstitium, with concurrent matrisome protein post-translational modification quantification, in archived diagnostic human kidney biopsies, using laser capture microdissection, liquid chromatography tandem mass spectrometry (LC-MS/MS), and bioinformatics analysis. In this pilot study, we focused on two conditions associated with IFTA progression: diabetic nephropathy and kidney transplantation. We examined both healthy and chronically damaged tubulointerstitium in two human kidney biopsies, one representing each condition. We identified signature protein markers of tubulointerstitium (e.g., UMOD, SLC12A1) in abundance, confirming sample composition, and observed uniformity in protein abundance across replicates from the same biopsies. We found increases in certain collagen isoforms (COL1A1, COL6A3) and other ancillary proteins in areas of IFTA. We also successfully quantified post-translational modifications, confirming that hydroxylproline was the most abundant modification in healthy tubulointerstitium from the kidney transplant biopsy, and that hydroxyllysine was also highly abundant and could be confidently quantitated on two collagen isoforms (COL1A1, COL4A2). Lastly, we performed power analysis to determine the required sample size to achieve significant power and false discovery rate. Several proteins exhibited large Hedge effect sizes in the tubulointerstitium (ANPEP, PRODH2), indicating their potential usefulness as fibrosis signatures. Our study is a first step toward comprehensive characterization of tubulointerstitial matrisome changes, including post-translational modifications seen with IFTA progression. Subsequent studies will potentially reveal new molecular markers of key early events in chronic kidney disease progression, and new therapeutic targets for mitigating this process. Data are available via ProteomeXchange identifier PXD079025.

Introduction: The inability to measure renal sympathetic outflow clinically poses a significant problem for patient identification and intraprocedural feedback for renal denervation. Sympathetic vasomotion is a novel physiologic measure of rhythmic vascular control that reflects renal sympathetic nervous system activity, but previous measurements of sympathetic vasomotion relied on 15-minute data recordings, which may be impractical in many clinical settings. We therefore tested whether shorter data segments can provide reliable sympathetic vasomotion assessment to enhance decision making for renal denervation. Methods: Ten swine that had previously undergone unilateral surgical renal denervation underwent four rounds of catheter-based denervation of the contralateral kidney. Sympathetic vasomotion was quantified from the time-varying arterial pressure-resistive renal blood flow transfer function of recordings obtained before and after each round of ablation. Randomly placed 1- to 14-minute data segments were scored and compared with the corresponding 15-minute sympathetic vasomotion scores across 100 passes using correlation, intraclass correlation, error, and Bland-Altman agreement metrics, and by whether they reproduced the denervation decisions made from the full recordings. Results: Agreement with the 15-minute reference score improved steeply with recording length up to approximately four minutes and slowly thereafter, with the point of diminishing returns at four minutes for every group-level measure. Four-minute data segments showed an intraclass correlation coefficient of 0.94, reproduced the group-level statistical results across ablations, and preserved intraprocedural denervation decisions. Bland-Altman analysis revealed that individual-level precision continued to improve with recording length; the proportion of scores falling within a quarter of the average difference between innervated and denervated kidneys at baseline was 65 percent at four minutes and 96 percent at ten minutes. Discussion: Four-minute data segments provide a feasible minimum recording duration when the question is a group comparison or a paired, within-individual decision, such as intraprocedural confirmation that an innervated kidney has shifted toward a denervated state. When a single score must be interpreted on its own, as in preprocedural patient selection, ten-minute recordings provide greater precision. Further studies are needed to prospectively evaluate sympathetic vasomotion in clinical settings and determine whether this technology can support procedural feedback and inform patient selection.

Renal fibrosis represents an irreversible endpoint of chronic kidney disease. Although macrophage efferocytosis regulates the response to acute renal injury, the mechanisms by which early efferocytosis confers long-term protection against chronic fibrosis remain elusive. Here, we show that phagocyte-specific deletion of the inhibitory receptor CD300a (Cd300afl/flLyz2-Cre) mitigates obstructive renal injury and subsequent fibrosis. CD300a deficiency enhanced efferocytosis by tissue-resident macrophages during the early phase of injury, thereby suppressing the release of damage-associated molecular patterns (DAMPs) and TGF-{beta} expression. This early attenuation of the inflammatory microenvironment altered the composition of recruited neutrophils, shifting them from profibrotic SiglecF+ toward non-profibrotic SiglecF- subsets. Furthermore, loss of CD300a cell-autonomously primed neutrophils for enhanced matrix metalloproteinase-9 (MMP9) production upon injury, whereas neutrophil depletion abolished the protective effect of CD300a deficiency. Importantly, administration of a neutralizing anti-CD300a antibody to wild-type mice recapitulated these protective effects, promoting the shift toward SiglecF- neutrophils and significantly attenuating renal fibrosis. Together, our findings identify CD300a as a regulator of early macrophage efferocytosis and neutrophil-derived MMP9 production, and establish pharmacological CD300a blockade as a potential therapeutic strategy for progressive renal fibrosis.

Background: Dietary sodium intake is a modifiable determinant of blood pressure, and impaired renal sodium handling is implicated in the pathogenesis of hypertension. Whether dietary sodium intake modulates renal tubular sodium transporter abundance among normotensive adults at risk for hypertension is unknown. Methods: Forty-six normotensive adults with normal kidney function at risk of developing hypertension were prospectively enrolled to complete a very low sodium (LS) diet followed by a high sodium (HS) diet. Urinary extracellular vesicle (uEV) proteins were quantified from a 24-hour urine collection at the end of each diet. We compared normalized uEV protein concentrations between diets and tested whether diet modified uEV protein associations with serum aldosterone, urinary aldosterone, and serum cortisol-cortisone ratio under either diet using Wilcoxon rank sums and linear mixed effects models, respectively. Results: Participants achieved median [IQR] urine sodium 13.2 [7.7-19.9] mEq/24h on the LS diet and 244.0 [197.1-297.0] mEq/24h on the HS diet. LS intake induced greater renin activity, greater plasma and urinary aldosterone, and lower urinary cortisol, when compared to HS intake. In parallel, NCC, ENaC, SGLT2, OXSR1, and Pendrin uEV concentrations were significantly higher during LS intake than HS intake (p<0.05 for all), whereas TRPV5 and AQP2 uEV concentrations were lower during LS intake than HS intake (p<0.001 for both). Conclusions: Controlled dietary sodium modulation induced nephron-segment specific changes in uEV protein abundance. These findings demonstrate the ability of uEVs to serve as a non-invasive tool to probe in vivo renal sodium, water, and electrolyte handling.

End-stage kidney disease (ESKD) is a major driver of cardiovascular disease (CVD) and requires dialysis treatment leading to a unique metabolic phenotype with cyclic transitions between accumulation and clearance of uremic toxins. Although ESKD patients display the highest cardiovascular mortality among CKD patients, the effect of cyclic uremia on cardiovascular disease remains subject to discussion. Existing mouse models rely on induction of kidney injury and do not resolve the interconnected pathophysiological effects of metabolic, hypertensive and endocrine renal failure. They are therefore unable to distinguish uremia-driven effects from well-established drivers of cardiovascular disease such as hypertension. Here, we established a simple, reproducible, and sex-inclusive mouse model of uremia in absence of kidney injury or hypertension leveraging a bistable vesico-peritoneal shunt (VPS). Strikingly, we identify cyclic uremia as an independent driver of ESKD-induced CVD, that induces heart failure with preserved ejection fraction (HFpEF), vascular inflammation and immunosenescence. As such, the VPS represents the first preclinical model that enables investigation of cyclic uremia as a driver of cardiovascular disease.

Purpose: Videourodynamics (VUDS) are used to assess bladder dysfunction and risk of upper urinary tract deterioration in patients with spina bifida (SB), but interpretation is subject to interrater variability. Machine learning models have previously classified bladder dysfunction severity and predicted incident hydronephrosis using VUDS data. This study evaluated the performance of adapted versions of these models in an independent external cohort. Materials and Methods: VUDS data were collected from SB patients who underwent VUDS at a single institution between 2016 and 2025. Previously developed machine learning frameworks were adapted for implementation in the independent dataset. Three models were utilized: a deep learning convolutional neural network model using pressure-volume data, a deep learning imaging model using fluoroscopic imaging data, and an ensemble model that averaged the risk data from the pressure-volume and fluoroscopic data. The models were evaluated for prediction of incident hydronephrosis and classification of bladder dysfunction severity compared to expert pediatric urologist reviewers. Results: 70 patients were included in the hydronephrosis cohort, of whom 13 (18%) developed incident hydronephrosis. The ensemble model outperformed individual modalities, with a concordance index of 0.77 (pressure-volume only: 0.70, imaging only: 0.72) and an overall AUROC of 0.75 (pressure-volume: 0.67, imaging: 0.74). For bladder dysfunction classification in 95 VUDS studies, the ensemble model achieved 71% accuracy compared to 60% for pressure-volume data alone and 66% for imaging alone, with no substantial disagreements with expert reviewers. Conclusions: The machine learning models that were developed to predict incident hydronephrosis and classify the severity of bladder dysfunction using VUDS data were able to be adapted to an external, independent dataset and perform with similar discrimination and accuracy as previously reported.

Background: Chronic kidney disease (CKD) is a major risk factor for cardiovascular morbidity, promoting left ventricular hypertrophy (LVH) prior to overt heart failure. The mechanisms underlying early cardiac remodeling in CKD remain incompletely understood. The IL-23/Th17/IL-17A inflammatory axis has emerged as a key mediator of cardiovascular pathology; however, its role in CKD-associated cardiac remodeling remains unclear. This study aimed to elucidate the contribution of this pathway to the early cardiac changes in CKD. Methods: Using a 5/6 nephrectomy (5/6Nx) mouse model of CKD, we evaluated cardiac structure, mitochondrial function, and inflammatory signaling. Cardiomyocyte transcriptomes were analyzed by RamDA-seq to identify molecular alterations. Circulating cytokines were profiled longitudinally, and IL-17A neutralization experiments were performed to assess causality. In addition, serum IL-12p40 concentrations were measured in patients with CKD and correlated with their renal function and cardiac remodeling indices. Results: At 16 weeks post-5/6Nx, mice developed LVH with preserved systolic function, accompanied by increased oxidative stress and mitochondrial structural abnormalities, but without significant myocardial fibrosis. Transcriptomic analysis identified oxidative phosphorylation as the most significantly downregulated pathway in cardiomyocytes. Despite increased mitochondrial DNA copy number and elevated PGC-1 expression, ATP production and mitochondrial respiratory chain proteins were reduced, indicating mitochondrial dysfunction. Circulating IL-12p40 and IL-23 levels increased progressively, concurrent with expansion of splenic Th17 cells and elevated cardiac IL-17A expression. Recombinant IL-17A directly impaired mitochondrial respiration in cultured cardiomyocytes. In vivo, IL-17A neutralization attenuated LVH, reduced oxidative stress, and partially restored mitochondrial protein expression without improving renal function. In patients with CKD, serum IL-12p40 levels correlated inversely with estimated glomerular filtration rate and positively with left ventricular mass index, supporting clinical relevance. Conclusions: Activation of the systemic IL-23/Th17/IL-17A axis contributes to early CKD-associated cardiac remodeling by promoting oxidative stress and mitochondrial dysfunction. Circulating IL-12p40 may serve as a biomarker of cardiorenal remodeling, and the IL-23/IL-17A pathway represents a potential therapeutic target.

Abstract Background. Tuberous Sclerosis Complex (TSC) is caused by inactivating mutations in either the TSC1 or TSC2 genes, leading to activation of the mammalian target of rapamycin complex 1 (mTORC1) and unhindered cell growth and proliferation. The epithelium of TSC renal cysts in both mice and humans is composed of proliferating A-intercalated (A-IC) cells. The exact molecular mechanism of kidney cystogenesis in TSC remains speculative. Hypothesis.Superfluous cell proliferation driven by mTORC1 activation increases metabolic demand, causing oxidative stress and excess reactive oxygen species. If unchecked, this overwhelms antioxidant defenses and causes cell death. In TSC kidney cystogenesis, Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2), also known as NRF2, serves as the "master regulator" of antioxidant and anti-inflammatory responses, enabling cells to survive and proliferate by clearing the toxic environment and supplying nutrients and fuel. Results. RNA-seq and proteomics, along with western blot analysis, showed robust downregulation of Fumarate Hydrase 1 (FH1) and upregulation of NRF2, STAT3, and HIF1 in kidneys from TSC mice with moderate or heavy cyst burden. Confocal microscopy and immunohistochemical staining on kidney sections, and/or western blot studies on nuclear and cytoplasmic fractions, showed nuclear localization of NRF2, STAT3, and HIF1. In cyst-lining cells in TSC mouse models, FH1 downregulation was associated with inactivating succination of KEAP1 in immunoprecipitation experiments, promoting NRF2 nuclear localization in A-IC cells lining the cysts. NRF2 nuclear localization was associated with ectopic induction of the glutamine transporter SLC38A3 (SNAT3) on the basolateral membrane and activation of the NH3/NH4+ transporters RHCG and RHBG in A-IC cells lining the cysts. Twenty-four h urine NH3/NH4+ excretion rates increased significantly in Tsc1 KO vs. WT mice. The activation of NRF2, STAT3, and HIF1 can drive metabolic reprogramming and activate survival genes in proliferating cells. Together with SLC38A3 induction and upregulation of other glutamine and NH3/NH4+ transporters, these factors activate glutaminolysis and aerobic glycolysis, supplying nutrients to proliferating cystic epithelial cells and supporting cyst expansion in TSC. Consistent with this central role for glutaminolysis in kidney cystic epithelium and TSC cystogenesis, we find a significant reduction in kidney cyst burden in Tsc1 KO mice on a glutamine-free diet. Conclusions. NRF2 plays a critical role in antioxidant defense. Along with STAT3 and HIF1, NRF2 is a key player in metabolic reprogramming through glutaminolysis, which supplies nutrients to proliferating cystic epithelial cells and supports cyst expansion in TSC. These findings suggest that inhibiting or inactivating NRF2, alone or in combination with HIF1 or STAT3, may represent a potential treatment strategy for kidney lesions in TSC.

Abstract Endothelial cell (EC) injury is central to the pathogenesis of various renal disorders, highlighting the critical importance of vascular repair for restoring organ health. However, due to limited access to human tissue during active regeneration, the exact reparative processes remain largely unknown. This study aimed to investigate regeneration mechanisms specifically within the glomerular microvasculature by utilizing a selective endothelial cell injury (ECI) model in mice.Lineage tracing identified the origin of regenerating ECs. To uncover the transcriptional dynamics driving these reparative changes, we combined single-cell RNA sequencing and Xenium spatial transcriptomics (ST), mapping distinct glomerular EC (gEC) subpopulations across different phases of injury and recovery. Apold1 emerged as a key regulatory candidate within this process. To functionally validate this gene's specific role during the regenerative phase, Apold1-deficient (Apold1-/-) mice underwent the ECI model. After showing that gECs regenerate primarily from local, pre-existing renal ECs, our transcriptomic analysis identified 10 distinct gEC subpopulations and revealed a complex temporal pattern of regeneration. This process begins with the expression of genes associated with inflammation, followed sequentially by endothelial cell (EC) migration, the activation of immediate early genes, vascular remodeling and cellular proliferation. Within this dynamic process, the EC-specific gene Apold1 was identified as a key regulatory candidate. Apold1-/- mice exhibited impaired endothelial regeneration, characterized by increased endothelial activation and greater immune cell infiltration seven days post-ECI. To our knowledge, this is the first study to elucidate the spatiotemporal mechanisms of glomerular endothelial cell regeneration. By identifying local gECs as the primary source of repair, and stablishing the change in their expression pattern during regeneration, as well as identifying Apold1 as an intrinsic regulator, we have laid the groundwork for future research on renal microvascular disorders.

Obesity is a major risk factor for interrelated cardiorenal metabolic diseases including type 2 diabetes, heart failure and chronic kidney disease (1-4). Although obesity develops when energy intake chronically exceeds energy expenditure, individuals vary markedly in the amount of adiposity and metabolic dysfunction that develop in response to comparable caloric excess. Controlled overfeeding studies demonstrate several-fold variation in weight gain between individuals (5), while susceptibility to metabolic dysfunction also differs substantially between populations (6), suggesting considerable variation in how excess nutrients are partitioned. Energy balance is conventionally considered in terms of intake and expenditure, yet energy-containing nutrients can also be eliminated from the body. The kidney is uniquely positioned to regulate this third component of energy balance by determining whether filtered metabolites are reabsorbed or excreted. The metabolic benefits of SGLT2 inhibition demonstrate that reducing renal nutrient reabsorption can favourably alter systemic metabolic function (7,8), but whether naturally occurring variation in renal nutrient conservation contributes to metabolic disease susceptibility remains unknown. Here, screening eleven genetically divergent mouse strains identified BXD34 mice as strikingly resistant to diet-induced obesity, ectopic lipid accumulation and insulin resistance despite increased caloric intake. Mechanistically, this protection was associated with widespread urinary nutrient loss accompanied by coordinated suppression of renal solute transport, but came at the cost of profound susceptibility to acute kidney injury. Together, these findings identify renal nutrient conservation as an underappreciated determinant of whole-body metabolism and reveal a renometabolic trade-off in which protection from obesity is achieved at the expense of renal resilience.

Sickle cell disease (SCD) causes heterogeneous mineral imbalances including variable degrees of hypocalcemia. The kidney controls systemic calcium by reabsorbing calcium from the glomerular filtrate via paracellular transport and transcellular transport in the nephron tubules, yet it is unknown whether these processes are modulated by genetic or environmental factors or disrupted in SCD. Using SCD mouse models and single-cell multiomics, we identify the distal convoluted tubule (DCT) as the nephron segment most susceptible to calcium reabsorption dysfunction in SCD, mainly via reduction of calcium buffer protein calbindin 1 (CALB1). We show that CALB1 and its encoding mRNA are decreased in DCT cells in SCD, alongside decreased Klotho (KL)-dependent fibroblast growth factor (FGF) 23 signaling and intracellular calcium signaling. Dietary iron restriction reduces CALB1, KL, and calcium exporter SLC8A1 levels in SCD kidneys. Loss of CALB1 shifts DCT cells toward energy-inefficient glycolysis with the metabolite 2,3-diphosphoglycerate impairing KL-dependent FGF23 signaling to create a feed-forward loop suppressing calcium reabsorption. Analysis of gene expression and protein quantitative trait loci data from kidneys of genetically diverse mice revealed that Calb1 expression levels are highly heritable and co-regulated with Slc8a1, identifying a genetic axis that dictates differential capacities for calcium buffering and trafficking toward blood in the kidney. Together, these findings support a model in which genetic variation, dietary iron status, and FGF23 signaling converge on DCT calcium buffering to reduce renal calcium reabsorption in the SCD kidney. This points to personalized, genotype- and iron-dependent strategies for managing mineral metabolism in SCD patients.

Prostate cancer (PC) remains a significant health concern as the second leading cause of cancer-related death in American men. Androgen deprivation therapies represent the standard of care for patients with PC, regardless of disease stage. Gonadotropin-releasing hormone (GnRH) antagonists, such as Degarelix, are commonly used hormone therapies for PC and work by suppressing testosterone production. Previous studies indicate a prostate involution-regeneration cycle following surgical castration and testosterone addback. However, whether a similar response occurs following pharmacologic androgen deprivation with agents such as Degarelix remains poorly understood. In particular, the temporal dynamics of prostate involution and subsequent regeneration after chemical castration have not been systematically defined. Our study utilizes immunocompetent mouse models to evaluate the systemic and temporal effects of Degarelix treatment and determine its efficacy in suppressing PC tumor growth. We show that continuous Degarelix treatment significantly suppressed systemic testosterone levels in mice, whereas testosterone levels returned to physiological baseline following Degarelix withdrawal. Gross anatomical, H&E, and Trichrome analyses demonstrated that Degarelix induced castration resulted in prostate involution, characterized by reduced gland size and increased stromal remodeling. This phenotype was reversed after Degarelix withdrawal, highlighting the reversibility of pharmacological androgen deprivation. Immunofluorescence staining revealed changes in basal and luminal epithelial cell populations within the prostatic gland, indicating changes in prostate glandular architecture following Degarelix induced castration and subsequent regeneration. Next, we evaluated the impact of chemical castration on PC tumor growth. Although Degarelix initially suppressed tumor growth, this response was not sustained through the study endpoint, suggesting the emergence of acquired resistance to androgen deprivation. These findings establish this immunocompetent model as a physiologically relevant platform for dissecting the mechanisms that drive therapeutic resistance and tumor progression following androgen deprivation. Our findings highlight the effectiveness of chemical castration with Degarelix and establish a robust model to study both therapeutic responses and the emergence of castration resistance in PC.

During embryogenesis, the cortical and medullary compartments of the adrenal gland emerge from two distinct sources: intermediate mesoderm-derived steroidogenic progenitors that comprise the cortex, and migratory neural crest-derived catecholaminergic progenitors that give rise to the medulla. The distinct cellular origins and paracrine signaling environments that foster induction, differentiation, and/or migration of cortical and medullary layers present challenges to recapitulating their co-emergence and incorporation in a human pluripotent stem cell (hPSC) based system. To address this, we developed an approach for modeling the adreno-gonadal primordium (AGP) in compound organoids that are organized along a gradient of NR5A1 expression and which unexpectedly incorporate SOX10-expressing Schwann cell precursors that ultimately generate chromaffin cells. Deconstruction of paracrine signaling within AGP-like organoids (AGPLOs) identified a fibroblast growth factor 9 (FGF9)-driven mechanism that expands bridge cell-like chromaffin progenitors and correlates to paracrine signaling cues encountered by physiological correlates along their migration corridor to the cortex in vivo. Combination of NR5A1-expressing cells with chromaffin cell clusters generated corticomedullar-like assembloids (CMLAs) that generated steroids and catecholamines and were responsive to adrenocorticotropic hormone. This work establishes a platform for modeling the reciprocal signaling relationships that drive adrenal gland development and function in health and disease and provides an approach for generating hPSC-derived adrenal cells/tissues that can be applied therapeutically.

Background: Sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce cardiovascular mortality and chronic kidney disease (CKD) progression in cardiovascular-kidney-metabolic (CKM) syndrome, but this benefit derives primarily from reductions in heart failure and cardiovascular mortality rather than in atherothrombotic events. Sotagliflozin, which inhibits both intestinal SGLT1 and renal SGLT2, is the only drug in this class shown to lower rates of myocardial infarction and stroke in CKD. However, its mechanism remains unclear, and its effectiveness in non-diabetic CKD has not been studied. We hypothesized that dual SGLT1/2 inhibition reprograms systemic substrate utilization toward fatty acid oxidation, which may partially account for this protective effect. Methods: CKD was induced in male low-density lipoprotein receptor-deficient mice by 5/6 nephrectomy. Sham and CKD mice received a high-fat diet and drinking water containing sotagliflozin, empagliflozin, or vehicle for 16 weeks, at doses that produced equivalent fractional excretion of glucose. We quantified atherosclerosis en face and in the aortic sinus. Indirect calorimetry, echocardiography, hepatic RNA sequencing, plasma acylcarnitine profiling, and in vivo deuterium labeling of palmitate were performed. Results: At matched levels of glucosuria and plasma total cholesterol, sotagliflozin decreased body mass, atherosclerosis burden, triglycerides, and TNF- more than empagliflozin, and raised postprandial total GLP-1 approximately 3-fold, while both drugs similarly improved albuminuria and diastolic function. Sotagliflozin increased energy expenditure independent of body mass and lowered respiratory exchange ratio despite compensatory hyperphagia. Sotagliflozin reduced hepatic lipogenic genes and de novo lipogenic flux, while increasing {beta}-oxidation and ketogenesis; empagliflozin only activated the oxidative pathway. Conclusions: In experimental CKM syndrome, dual SGLT1/2 inhibition reduces atherosclerosis and metabolic syndrome more effectively than selective SGLT2 inhibition and is associated with reprogramming of hepatic lipid metabolism. These results suggest that sotagliflozin could be an attractive candidate for mitigating atherosclerotic risk in non-diabetic CKD.

Methylmalonic aciduria (MMA) is caused by deficiency of methylmalonyl-CoA mutase (MMUT), which catalyses the final step of propionate catabolism and fuels the tricarboxylic acid cycle for energy production. Previous studies reported disrupted mitochondrial homeostasis in MMA, including reduced mitochondrial membrane potential and increased oxidative stress, especially in energetically demanding and chronically affected tissues like brain and kidneys. However, how these changes impact mitochondrial energy production remains unclear. Here, we systematically investigated mitochondrial energy production using extracellular flux analysis in cellular models of MMA, including 293T cells, patient-derived fibroblasts, urine-derived epithelial kidney cells and induced pluripotent stem cells (iPSCs) as well as iPSC-derived neurons harbouring either complete loss (knockout, KO) or pathogenic missense variants in MMUT. We found no impact on mitochondrial energy production in MMUT-KO 293T cells and fibroblasts compared to controls. In contrast, fibroblasts and 293T cells expressing the pathogenic MMUT-p.N219Y variant showed decreased energy production. This corresponds to the altered mitochondrial membrane potential in 293T cells, but contrasts with their unchanged mitochondrial abundance. Depletion of glucose, glutamine and pyruvate from the media or provision of each individually as sole fuel source exacerbated the phenotype of MMUT-p.N219Y clones, but did not induce a phenotype in MMUT-KO 293T cells. Finally, we confirmed reduced mitochondrial energy production in patient-derived kidney cells but found no evidence in MMUT-p.N219Y iPSCs and their derived neurons. Overall, our work suggests that the impact of MMUT-deficiency on mitochondrial energy production is cell type- and variant-dependent. Further investigations should clarify molecular mechanisms and their clinical impact.

Background: Cardiovascular-kidney-metabolic (CKM) syndrome integrates cardiac, renal, and metabolic abnormalities that drive multi-organ injury. In pulmonary arterial hypertension (PAH), CKM manifests as systemic metabolic derangements, right ventricular dysfunction, and renal compromise. Renal dysfunction strongly predicts mortality in PAH, yet current therapies provide little renal benefit. Moreover, the mechanisms driving PAH nephropathy remain poorly understood, limiting our ability to effectively treat PAH-CKM. Methods: Single-nucleus RNA sequencing identified cell-type-specific transcriptional alterations in autopsy-derived kidneys from control (n=5) and PAH (n=4) patients. Mitochondrial, cytoplasmic, and phosphoproteomic analyses profiled proteomic alterations. AlphaFold 3/ChimeraX modeling defined the predicted structural consequences of altered protein phosphorylation. Histological analysis assessed renal fibrosis, glomerular structure, immune cell infiltration, and nephron segment density. Results: 3 of 4 PAH patients exhibited renal impairment, with a cohort mean estimated glomerular filtration rate of 61 plus or minus 36 mL/min/1.73m squared. snRNA-seq identified a distinct cellular architecture in PAH kidneys, marked by an increase in thick ascending limb, proximal tubule, and immune cell nuclei and depletion of collecting duct nuclei. Transcriptional profiling demonstrated proximal tubule and thick ascending limb cells both upregulated fatty acid oxidation, ferroptosis, and cuproptosis pathways. PAH lymphocytes displayed heightened T87 cell receptor signaling, natural killer cell-mediated cytotoxicity, and Th17 differentiation. Histological analyses demonstrated increased perivascular fibrosis, glomerular T-cell infiltration, and a reduction in glomerular basement membrane density in PAH kidneys. Mitochondrial and cytoplasmic proteomics revealed broad metabolic dysfunction characterized by impaired {beta}-oxidation, TCA cycle activity, amino acid metabolism, transsulfuration, and urea cycle pathways. Phosphoproteomics predicted increased GSK3{beta}, STK, and casein kinase activity in PAH kidneys. Finally, using the totality of our data, we nominated multiple druggable targets that could be evaluated to counteract PAH nephropathy. Conclusions: Integrated multi-omic profiling demonstrates PAH nephropathy is defined by glomerular structural remodeling, proximal nephron ferroptotic and cuproptotic signaling, amino acid metabolic dysregulation, and innate and adaptive leukocyte activation. Future studies intervening on these pathways could lead to the development of novel therapeutics to augment renal function in PAH.

Nephrotic syndrome, the leading cause of chronic kidney disease worldwide, is associated with injury and eventual loss of podocytes, the specialized epithelial cells that form the final layer of the kidney's filtration apparatus. The progression from injury to loss is largely irreversible. However, some forms of nephrotic syndrome undergo remission, suggesting that injured podocytes may retain a capacity for repair, the structural basis of which remains unknown. Podocytes respond to injury by the stereotypical morphological change known as foot process effacement (FPE), which can be visualized using transmission electron microscopy. Using modified ultrastructure expansion microscopy (U-ExM) protocol combined with super-resolution microscopy, we discovered that injured podocytes exhibit a three-stage progression of FPE maturation, with kinetics determined by the degree of actomyosin contractility. Through longitudinal study of adriamycin-induced nephropathy in mice, we identified three sequential stages of actomyosin reorganization within the sarcomere-like structures (SLSs) that define the injury response: focal infiltration of myosin IIA into synaptopodin-rich foot processes (Stage 1), disordered intermingling of synaptopodin/myosin IIA (Stage 2), and the formation of highly organized periodic contractile arrays oriented perpendicular to the direction of capillary blood flow (Stage 3). Longitudinal quantification revealed predictable temporal dynamics, with mature Stage 3 predominating by day 7-9 post-injury. To determine whether this sequence is conserved in human disease, we analyzed kidney biopsies from patients with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, and diabetic nephropathy. The same three stages were present across all four diseases, and their distribution tracked disease course: acute podocytopathies exhibited heterogeneous early-stage patterns, whereas chronic injuries converged on uniform, mature SLS organization. These findings demonstrate that podocyte injury is a graded, potentially reversible process rather than a binary switch. They identify structured actomyosin remodeling as a conserved wound response across mechanistically distinct kidney diseases and suggest that therapeutic strategies targeting specific stages of this progression could alter the trajectory of diverse glomerular diseases.

Certain glomerulopathies (GN) feature impaired protein homeostasis in the endoplasmic reticulum (ER) of podocytes. Protein misfolding causing ER stress activates the unfolded protein response (UPR), a protective signaling network. Mesencephalic astrocyte-derived neurotrophic factor (MANF) and ERdj3/DNAJB11 are ER chaperones that may be secreted from cells during the UPR. In rats with membranous nephropathy (MN) and focal segmental glomerulosclerosis (FSGS), secretion of MANF or ERdj3 from podocytes into urine identified glomerular ER stress/UPR activation. We address the biomarker potential of MANF and ERdj3 in human MN and FSGS. Glomerular gene expression of MANF and ERdj3 was increased in human MN and FSGS. Urinary MANF and ERdj3 were increased in proteinuric MN and FSGS compared to MN and FSGS in remission (low proteinuria), as well as other GNs, polycystic kidney disease, hypertension and healthy controls. In individuals with proteinuric MN or FSGS, amounts of urinary MANF and ERdj3 correlated weakly with proteinuria; correlation of MANF with ERdj3 was moderate. Males represented ~60% of persons in each group. eGFR in proteinuric MN and FSGS was >40 ml/min/1.73 m2. In MN, there were no significant differences in urinary MANF and ERdj3 between anti-PLA2R positive and negative individuals. In conclusion, proteinuric MN and FSGS show increased glomerular expression of MANF and ERdj3. High urinary levels of these ER chaperones correlate only weakly with proteinuria. In these GNs, urinary ER chaperones are thus non-invasive biomarkers of glomerular/podocyte ER stress. Such biomarkers may eventually be useful in identifying patients for therapies targeting ER protein misfolding.

Aldosterone is a key regulator of systemic sodium, potassium, and fluid homeostasis. Chronic elevation of aldosterone, as observed in primary aldosteronism, chronic heart and kidney diseases, promotes cardiac hypertrophy, inflammation, fibrosis, and heart failure, which are further enhanced in aging. Although these pathological effects are primarily attributed to the mineralocorticoid receptor (MR), emerging evidence suggests roles for nonclassical G protein-coupled receptor (GPCR) signaling. We identified lysophosphatidic acid receptor 2 (LPAR2) as a novel GPCR candidate that mediates aldosterone signaling in cardiomyocytes. Using AC16 human cardiomyocyte cells and reporter gene assays, we demonstrate that aldosterone activates a Gi/o-dependent GPCR pathway. Pharmacological inhibition of LPAR2 internalization confirms its specific reaction to aldosterone. Functional studies revealed that aldosterone-induced cardiomyocyte injury requires both MR and LPAR2, where specific antagonists to both receptors reduced the ability of aldosterone to induce collagen expression and cell death markers. These findings uncover a previously unrecognized non-classical mechanism of aldosterone action in the heart and highlight LPAR2 as a promising therapeutic target for aldosterone-driven cardiovascular disease.

Protein O-mannosylation in the endoplasmic reticulum (ER) regulates the maturation and function of extracellular proteins, yet the substrate spectrum and biological functions of individual O-mannosyltransferases remain incompletely understood. TMEM260 is an ER-resident O-mannosyltransferase that modifies immunoglobulin-like, plexins, transcription factors (IPT) domains. Here, we identify fibrocystin and fibrocystin-L, encoded by PKHD1 and PKHD1L1, respectively, as novel TMEM260-mediated O-mannosylated proteins. By combining screening of human proteomic datasets with mass spectrometry-based analysis of IPT domains expressed in TMEM260-deficient HEK293 cells, we demonstrate that multiple IPT domains of fibrocystin and fibrocystin-L are modified by TMEM260. Our findings expand the known substrate requirements of TMEM260 by revealing tolerance of substantial sequon variation and demonstrating serine as an alternative acceptor residue within IPT domains. In humans, loss-of-function variants in TMEM260 cause structural heart defects and renal anomalies (SHDRA) syndrome, whereas pathogenic variants in fibrocystin cause autosomal recessive polycystic kidney disease (ARPKD). Notably, patients with SHDRA and ARPKD share overlapping renal phenotypes, suggesting that impaired TMEM260 function may phenocopy aspects of fibrocystin-associated diseases. Given that fibrocystin is a substrate protein for TMEM260-mediated O-mannosylation, these findings raise the possibility that functional impairment of TMEM260 causes aberrant O-mannosylation of fibrocystin. Thereby fibrocystin function might be compromised leading to the renal phenotype observed in patients with TMEM260 mutations. To directly assess the functional relevance of this modification, we expressed recombinant fibrocystin fragments in TMEM260-deficient cells complemented with either wild-type TMEM260 or a catalytically inactive TMEM260 mutant. Further, we investigate the O-mannosylation of the threonine residue directly C-terminal to the ARPKD-associated patient mutation L1407R. Strikingly, substitution of leucine L1407 by the patient-associated arginine residue, completely abolished O-mannosylation of the adjacent threonine, demonstrating that this disease-associated mutation directly disrupts a TMEM260-dependent O-mannosylation site. Together, our findings establish fibrocystin and fibrocystin-L as TMEM260-dependent O-mannosylated proteins and reveal a potential molecular mechanism linking defective TMEM260-mediated O-mannosylation to the renal phenotype of SHDRA, which overlaps with PKHD1-associated ARPKD.