American Journal of Physiology-Renal Physiology

Biological sex is an important determinant of kidney disease susceptibility and outcomes. The epigenetic modifier KDM6A is an X chromosome-expressed lysine demethylase and molecular scaffold that escapes X chromosome inactivation. Here, we compared the effects of deletion of KDM6A from kidney tubule epithelial cells in female and male mice (KDM6ATubKO). Knockout of KDM6A from tubule cells aggravated kidney fibrosis caused by unilateral ureteral obstruction (UUO) in female mice, whereas male mice were unaffected by KDM6A absence. Unexpectedly, female (but not male) KDM6ATubKO mice developed spontaneous glucosuria that, when stressed by ligation of one ureter, presented as polyuria and a Fanconi renotubular syndrome-like picture affecting the unobstructed kidney. Absence of KDM6A from tubule epithelial cells of female mice caused mitochondrial circularization, tubule cell vacuolization with focal atrophy and lymphoid infiltration, and diminished sodium/glucose cotransporter 2 (SGLT2). Spatial transcriptomics and untargeted metabolomics revealed that knockout of KDM6A in female mice caused a shift in gene programs and metabolic pathways indicative of tubule cell metabolic dysfunction. In male mice, transcripts of the Y chromosome-expressed gametolog of Kdm6a, Uty were present in tubule epithelial cells at levels comparable to Kdm6a and they were upregulated with UUO. In summary, KDM6A is essential for normal tubule epithelial cell homeostasis in females but not in males. KDM6A and UTY are a dynamically regulated X-Y gene pair with at least partial compensatory overlap in function necessary for the preservation of kidney health and stress tolerance. Graphical abstract. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/740137v1_ufig1.gif" ALT="Figure 1000"> View larger version (29K): org.highwire.dtl.DTLVardef@7d26a4org.highwire.dtl.DTLVardef@740972org.highwire.dtl.DTLVardef@1851f7org.highwire.dtl.DTLVardef@ad6485_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chronic kidney disease (CKD) is a progressive disorder characterized by metabolic dysfunction, mitochondrial impairment, oxidative stress, and chronic inflammation, ultimately leading to irreversible renal damage. Despite advances in understanding CKD pathophysiology, effective therapies targeting these interconnected molecular processes remain limited. In this study, we performed a comprehensive data-independent acquisition (DIA)-based proteomic analysis to investigate the molecular alterations associated with CKD and to evaluate the therapeutic impact of DVA treatment. Using a CKD model with three treatment conditions (DVA, KY, and DVA+KY) alongside disease and healthy controls, we quantified global proteomic changes and applied statistical filtering (fold change [≥]2, p [≤]0.05) followed by K-means clustering (k=10). Distinct protein clusters revealed bidirectional modulation upon DVA treatment. Notably, Cluster 1 comprised proteins downregulated in CKD but significantly restored following DVA administration, while Cluster 2 included proteins elevated in CKD that were suppressed by DVA. Pathway enrichment and network analyses demonstrated that Cluster 1 proteins were predominantly associated with mitochondrial function, oxidative phosphorylation, and metabolic processes, whereas Cluster 2 proteins were enriched in immune signaling, oxidative stress, cytoskeletal remodeling, and proteostasis pathways. At the molecular level, DVA treatment restored key mitochondrial and metabolic regulators, including components of the electron transport chain (e.g., COX5A, NDUFS5, SDHB) and redox homeostasis proteins, indicating recovery of cellular bioenergetics. Concurrently, DVA suppressed inflammatory mediators (STAT2, IFI47, GBP2), oxidative stress-related proteins (CYBB, PRDX5), and cytoskeletal regulators linked to renal injury (ARHGEF12, FMNL2). Network and Reactome analyses further confirmed coordinated modulation of interconnected biological systems rather than isolated protein changes. Collectively, our findings demonstrate that DVA exerts a dual therapeutic effect by restoring essential mitochondrial and metabolic pathways while simultaneously suppressing inflammation, oxidative stress, and cytoskeletal dysregulation in CKD. This systems-level proteomic reprogramming highlights DVA as a promising candidate for CKD intervention and provides mechanistic insights into disease progression and therapeutic targeting.

Renal ischemia reperfusion injury (IRI) is a major driver of acute kidney injury and a critical determinant of long term renal outcomes. Although sex differences in susceptibility to renal injury are well recognized, the hormonal mechanisms underlying these disparities remain incompletely understood. Prolactin (PRL), a pleiotropic hormone, has been implicated in both protective and deleterious renal effects, while Cathepsin D (CTD), a lysosomal protease, cleaves PRL into Vasoinhibins that possess antiangiogenic and immunomodulatory properties. The impact of CTD deficiency on PRL's role in renal IRI has not been defined. Here, we investigated whether CTD regulated prolactin signaling modulates renal responses to IRI in a sex dependent manner. Wild type (WT) and heterozygous CTD mice of both sexes were subjected to unilateral renal IRI. PRL overexpression was achieved via lentiviral vector, and renal function, inflammatory cytokines, and angiogenic factors were assessed. In vitro Prolactin cleavage assays confirmed the enzymatic role of CTD. Proteomic analysis of renal cortex tissue was performed using label free LC MS/MS and pathway enrichment tools. CTD cleaved PRL into Vasoinhibins at physiological pH, an effect blocked by Pepstatin A. PRL effects varied by sex and genotype. In WT females, PRL reduced renal dysfunction and increased IL-6 and TGF beta; expression following IRI. CTD+/- females exhibited protection from injury, with PRL treatment reversing IL-6 induction. In males, PRL alone had modest effects, but CTD deficiency led to pronounced creatinine elevation, which PRL treatment normalized. Proteomic data revealed differential regulation of cytoskeletal and inflammatory proteins. Cathepsin D heterozygosity modifies PRL's biological effects on the injured kidney. The CTD, PRL axis regulates key inflammatory and angiogenic responses after IRI, revealing new mechanistic insights and potential therapeutic targets in acute kidney injury.

Renal cysts are categorized as non-pathogenic simple cysts and pathogenic malignant cysts based on their pathophysiological status. Cyst formation is divided by cyst initiation and cyst progression/promotion. Pathogenic cysts are thought to be developed through continuous initiation followed by progression until pathogenic status is achieved. Although many genetic and environmental factors are identified to cause pathogenic cyst formation, the mechanisms that discriminate cyst initiation and progression are poorly understood. Using genetic mutation models of ETV transcription factors, ETV1, ETV4, and ETV5, and a pharmacological inhibitor of hedgehog signaling, cyclopamine, we identified one of the mechanisms regulating cyst initiation and progression. Nephron specific deletion of ETV4 and ETV5 initiated cyst formation. However, cyst initiation did not continue as animals grow, and a limited number of the initial cysts underwent further growth. Additional deletion of ETV1 was required for continuous initiation in addition to promotion of cyst growth. Furthermore, administration of cyclopamine attenuated promotion of cyst progression but had little effect on cyst initiation. Therefore, we provide evidence that cyst initiation and progression is genetically and molecularly distinct and can be modulated. This information provides new insight into how to control renal cyst initiation and progression and can be used to suppress pathogenic cyst growth.

The urinary bladder functions to store and release urine, yet how the sensation of bladder fullness is conveyed and perceived to the central nervous system is not understood. During bladder filling, the detrusor smooth muscle (DSM) generates phasic contractions, resulting in pressure fluctuations within the bladder. These transient pressure events drive bursts of afferent nerve activity, yet the underlying mechanism leading to rhythmic contractions remains unclear. Here, we examined the role of Gq protein-coupled receptor (GqPCR) activity on DSM excitability and contractility. Using ex vivo pressurized urinary bladder preparations and sharp microelectrode experiments on bladder strips from mice, we evaluated whole bladder transient pressure events, whole bladder DSM Ca2+ activity, and membrane potential in bladder strips. We found that global inhibition of urinary bladder GqPCR activity with YM-254890 abates phasic contractility and transient pressure events through a reduction in DSM Ca2+ activity and propagation of Ca2+ waves. Further, we found inhibition of GqPCR significantly hyperpolarizes DSM, reducing action potentials and decreasing excitability, and activation of protein kinase C restores membrane potential to baseline levels. These findings highlight that GqPCR activity mediates DSM excitability and contractility in such a way as to result in phasic detrusor contractions and transient pressure events.

Wnt/{beta}-catenin signaling is a critical pathway that regulates nephron progenitor renewal versus differentiation as well as nephron patterning. In addition to its role as a transcription co-factor, {beta}-catenin also functions as a structural component of adherens junctions, where it interacts with cadherins to link cell-cell contacts to the cytoskeleton. However, the relationship between the nuclear and junctional localization of {beta}-catenin during vertebrate nephron development remains poorly understood. To define how endogenous {beta}-catenin localization changes during nephrogenesis, we optimized an accelerated-turnover {beta}-catenin chromobody for live imaging in Xenopus embryos. Using in vivo imaging of Xenopus pronephric development, we visualized endogenous {beta}-catenin within the nuclear, cytoplasmic, and junctional compartments. Across successive developmental stages, {beta}-catenin became progressively enriched at epithelial junctions during nephron maturation while remaining abundant within nuclear and cytoplasmic compartments. Quantitative analyses indicate that epithelial maturation is accompanied by coordinated expansion and partitioning of multiple intracellular {beta}-catenin pools rather than a simple redistribution from nuclear to junctional compartments.

BACKGROUNDChronic renin-angiotensin system (RAS) inhibition activates renin cells and induces afferent arteriolar hypertrophy, a maladaptive vascular response that may contribute to nephrosclerosis-like renal injury. Although genetic or cell ablation approaches have shown that renin cells are required for this remodeling, no pharmacological strategy to restrain hyperactivated renin cells while preserving RAS inhibition benefits has been established. Natriuretic peptides (NPs) counteract RAS; however, whether NP signaling modulates renin cell activation and afferent arteriolar remodeling remains unclear. METHODSWe examined direct effects of atrial natriuretic peptide (ANP) on As4.1 renin-producing cells using reverse transcription-quantitative PCR, ELISA, and RNA sequencing (RNA-seq). We established a mouse model of long-term RAS inhibition using valsartan, an angiotensin II receptor blocker (ARB), and compared it with sacubitril/valsartan, an angiotensin receptor-neprilysin inhibitor (ARNI). Valsartan was matched between the ARB and ARNI groups. Renin cell activation, afferent arteriolar remodeling, and renal injury were evaluated using biochemical assays, histology, immunostaining, single-nucleus RNA-seq, and region-specific photo-isolation chemistry RNA-seq of afferent arteriolar/juxtaglomerular regions. RESULTSANP suppressed Ren1 expression and renin secretion in As4.1 cells; this effect was attenuated by a natriuretic peptide receptor A antagonist. RNA-seq demonstrated that ANP induced receptor-dependent remodeling of renin cell gene programs. In mice, long-term ARB treatment induced renin cell hyperactivation, expansion of renin-positive juxtaglomerular regions, afferent arteriolar hypertrophy, renal dysfunction, tubular injury markers, and fibrosis. ARNI increased plasma ANP levels and attenuated these pathological changes, despite a comparable blood pressure reduction. Single-nucleus transcriptomics revealed attenuation of tubular injury-associated cellular states and altered renin cell-associated mesenchymal programs with ARNI. Region-specific transcriptomics further demonstrated distinct molecular states in afferent arteriolar/juxtaglomerular regions between ARB- and ARNI-treated kidneys. Integrated transcriptomic analysis suggested that NP signaling converges on vascular regulatory programs in hyperactivated renin-expressing cells. CONCLUSIONSNP signaling acts as a pharmacologically augmentable modulator of maladaptive renin cell activation. ARNI attenuates renin cell hyperactivation, afferent arteriolar hypertrophy, and renal injury during long-term RAS inhibition, suggesting that neprilysin inhibition may preserve RAS blockade benefits while limiting renin cell-driven renal vascular remodeling.

BackgroundAcute kidney injury (AKI) frequently progresses to chronic kidney disease (CKD), yet mechanisms governing this transition remain poorly understood. The endocannabinoid system (ECS), particularly cannabinoid-1 receptor (CB1R), regulates inflammation and metabolism in various organs, but its role in post-AKI maladaptive repair is less established. MethodsWe analyzed CB1R expression in kidney biopsies from pre- and post-transplant recipients and in murine AKI models (ischemia-reperfusion injury [IRI] and folic acid [FA]-induced AKI). Peripheral CB1R blockade was evaluated in FA-AKI model and in human primary kidney proximal tubule cells (hKPTCs). Spatial metabolomics, semi-targeted metabolomic profiling, and gene and protein expression characterized molecular mechanisms. ResultsCB1R expression was increased in kidneys undergoing maladaptive repair in both humans and mice, but remained unchanged during acute injury. In the FA-induced AKI model, the ECS showed stage-specific alterations, with temporal and spatial fluctuations in endocannabinoid levels and their enzymatic regulators. Peripheral CB1R blockade during the repair phase preserved kidney function, reduced injury, and maintained systemic glucose homeostasis. Metabolomic and molecular analyses revealed that CB1R blockade restored dysregulated arginine metabolism and reduced AKT/NF-{kappa}B-p65 pathway in post-AKI kidneys, linking CB1R activation to inflammatory signaling. In hKPTCs, 2-AG-induced activation of CB1R increased VCAM1 expression, a failed-repair marker, while its antagonism reduced TNF/2-AG-induced expression of pro-inflammatory adhesion molecules, chemokines, cytokines, and arginine metabolism enzymes. ConclusionsCB1R overactivation drives AKI-to-CKD progression by promoting inflammatory signaling and metabolic dysregulation. Peripheral CB1R blockade during the repair phase represents a novel therapeutic strategy to prevent maladaptive repair and CKD development after AKI. These findings establish CB1R as a phase-specific therapeutic target for post-AKI intervention. Translational StatementPeripheral CB1R antagonists offer a first-in-class therapeutic strategy to halt progression from acute kidney injury (AKI) to chronic kidney disease (CKD) by selectively targeting maladaptive tubular repair. By blocking CB1R signaling specifically in the kidney, these agents attenuate inflammation, metabolic dysregulation, and fibrogenic pathways that drive failed repair, while sparing central nervous system CB1R and thereby minimizing neuropsychiatric adverse effects. This phase-specific, peripherally restricted approach supports the development of peripheral CB1R antagonists as a viable translational therapy to improve long-term renal outcomes after AKI.

Spinal cord injury (SCI) disrupts innervation to the lower urinary tract, resulting in bladder dysfunction that predisposes to urinary infections and renal impairment. While inflammation is central to bladder pathology after SCI, the molecular events linking acute to chronic remodeling are poorly defined. We hypothesized that early treatment with pirfenidone, an anti-inflammatory and anti-fibrotic drug, would attenuate bladder pathology after SCI. Adult female C57BL/6J mice underwent contusive SCI or sham laminectomy, and bladders were collected at 2, 7, 16, and 45 days later. SCI induced bladder hypertrophy, edema, hemorrhage, neutrophil infiltration, cell proliferation and loss of voiding function in the first 48 hours. Transcriptomic profiling at this timepoint was characterized by activation of inflammatory and cytokine pathways including TNFalpha, IL-6, the complement cascade, and TGFbeta. Although bladder function partially recovered by day 7, inflammatory pathways persisted and extracellular matrix (ECM) remodeling programs emerged. By day 16, robust activation of ECM-remodeling pathways was evident in all bladders. Treatment with pirfenidone during the acute inflammatory phase (day 2-7) reduced bladder hypertrophy and suppressed expression of pro-fibrotic, inflammatory, and neuroplasticity-associated genes including Bdnf and Chrm2 that encodes muscarinic receptor 2 (M2). Mechanistically, pirfenidone attenuated TGFbeta signaling as shown by downregulation of phosphoSmad2 protein in whole bladders and decreased M2 receptor expression in the urothelium. These molecular changes correlated with improved function in pirfenidone-treated mice as shown by fewer voiding events with larger urine volumes up until 45 days after SCI. Early treatment with pirfenidone limits inflammation and fibrosis, normalizes neural signaling, and improves bladder function after SCI.

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: HNF4A is required for proximal tubule maturation during kidney development, but its role in maintaining proximal tubule identity in the adult kidney has not been defined. Following acute kidney injury, Hnf4a expression is rapidly suppressed in proximal tubule cells, coinciding with loss of mature epithelial features and activation of injury-associated transcriptional programs. It remains unknown whether HNF4A loss is sufficient to drive injury-associated transcriptional changes. Methods: We conditionally deleted Hnf4a in mature proximal tubules using Slc34a1Cre in mice and analyzed the consequences of Hnf4a loss in adult kidneys. To identify genes directly regulated by HNF4A, we performed proximal tubule-specific transcriptional profiling together with genome-wide mapping of HNF4A binding sites using CUT&RUN. Results: In Hnf4a mutant kidneys, HNF4A protein initially persisted in proximal tubules but was progressively lost from convoluted proximal tubule cells. Loss of Hnf4a resulted in downregulation of proximal tubule-specific transport and metabolic genes, accompanied by reactivation of developmental and injury-associated genes. CUT&RUN analysis revealed that HNF4A directly regulates genes predominantly involved in solute transport and metabolic processes. Conclusions: These findings identify HNF4A as a key regulator of proximal tubule identity and homeostasis in the adult mouse kidney. Genetic loss of Hnf4a in mature proximal tubules leads to loss of mature proximal tubule gene expression and activation of injury-associated transcriptional programs, implicating Hnf4a suppression as a potential contributor to maladaptive repair after kidney injury.

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.

In the United States, roughly 550,000 people receive routine hemodialysis for end-stage renal disease. This treatment requires an arteriovenous access, most commonly a brachiocephalic fistula. However, these accesses often fail due to stenosis in the cephalic arch (CA), a common complication whose underlying causes remain unclear (Bennet et al., 2015). To characterize the hemodynamic environment in the CA, we employed patient-specific millifluidic models that were perfused with blood-mimicking fluid containing fluorescently labelled beads to visualize flow behaviors. We perfused our models across physiologic (28-45 mL/min) and elevated (60-423 mL/min) flow rates and quantified wall shear stress (WSS) and streamline angle as a measure of flow disturbance. Our findings show that the bulk curvature and remodeled wall topography each create regions of persistently low WSS, consistent with prior clinical observations (Hammes et al., 2016). Moreover, remodeled wall topography promotes disturbed flow at elevated flow rates, a hemodynamic profile associated with various vascular pathologies (Chiu & Chien, 2011). Independently performed computational fluid dynamics (CFD) modeling complements these results, showing that remodeled wall topography promotes vortex formation at elevated flow rates, as assessed by Q-criterion. Collectively, our experimental and computational results provide strong evidence for geometry-driven disturbed flow in the CA at elevated flow rates. Notably, we observed disturbed flow at flow rates as low as 81 mL/min, far below the 600 mL/min required for hemodialysis. Disturbed flow thus offers a plausible mechanism that relates access flow rates to the vascular pathologies that precede access failure. Significance StatementHemodialysis requires an arteriovenous fistula (AVF) that must remodel and mature to withstand chronically elevated blood flow rates. However, how remodeled vessel geometry interplays with elevated flow to shape local hemodynamics remains poorly understood. Here, we used millifluidic models of the cephalic arch (CA) to show that vessel geometry and elevated flow rates promote regions of low wall shear stress and disturbed flow. Notably, geometry-driven disturbed flow is observed at flow rates above physiologic levels but well below 600 mL/min, the flow rate necessary for adequate dialysis. Because disturbed flow is injurious to the endothelium, our findings are the first to show that vascular damage begins before fistula maturation.

Introduction: The key role of maladaptive renal sympathetic activation in hypertension has led to the development of catheter-based renal denervation therapies. Unfortunately, the lack of practical physiological biomarkers impairs patient selection and prevents intraprocedural feedback for renal denervation. Methods: We tested whether renal arterial hemodynamic measures and the beat-to-beat variability of these measures reflect renal sympathetic innervation in two tightly controlled preclinical models of renal denervation. Bilateral renal hemodynamic data were obtained from ten conscious unilaterally denervated rabbits and ten anesthetized unilaterally denervated pigs that subsequently underwent stepwise catheter-based radiofrequency denervation of the contralateral kidney. Renal arterial mechanics were assessed by quantification of wave speed and input impedance modulus and phase shift. Variability was quantified as the within-recording standard deviation of each metric when measured on a beat-to-beat basis. Results: Wave speed trended down after surgical denervation in rabbits (P = 0.054) but not swine (P = 0.57); wave speed variability was unchanged in both models. Renal arterial input impedance modulus was not significantly affected by surgical denervation. Surgical renal denervation increased input impedance phase shift in rabbits (P = 0.0048) but not in swine (P = 0.85). The beat-to-beat variability of modulus did not differ significantly between innervated and surgically denervated kidneys in either species. Surgical renal denervation decreased phase shift variability in swine (P = 0.0048) but not rabbits. The difference in phase shift variability between kidneys in swine was eliminated after a single round of branch-vessel ablation (P = 0.42). Discussion: Renal arterial wave speed, input impedance, and their beat-to-beat variability did not consistently or dose-dependently reflect renal sympathetic innervation in these models. These findings argue against their use as direct physiological surrogates for renal sympathetic outflow.

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.

Modulation of renal nerves to improve blood pressure (BP) control has become a topic of intense investigation over the last 10-15 years. Given that renal innervation is composed of mixed nerve fibers containing both afferent (sensory) and efferent (sympathetic) fibers, subsequent preclinical studies have been investigating their respective roles in hypertension pathobiology in different genetic and salt-sensitive rat models. Here we set out to investigate how renal afferent and efferent nerves regulate hypertension development in the chronic mild stress model (CMS). We show that in male CMS rats, ablation of afferent renal nerves (ARDNx) and all renal nerves (TRDNx) resulted in similar BP (104 {+/-} 2 mmHg vs. 101 {+/-} 3 mmHg, respectively), both reduced compared to the SHAM group (118 {+/-} 1 mmHg, p = 0.003 and p < 0.001, respectively) arguing for a prominent role of afferent renal nerves in CMS hypertension. Additionally, we show a reduction of vasopressin (AVP) V1b but not V1a receptor abundance in ARDNx CMS males but not females, suggesting that afferent renal nerves are involved in increase in BP via V1b AVP receptor. We additionally show that despite normal BP, female CMS rats display increased renal sympathetic nerve activity (RSNA; 2.39 {+/-} 0.23 bursts/beat vs. 1.44 {+/-} 0.12 bursts/beat, p < 0.005) measured directly with implanted telemetry in conscious rats over one week and aortic stiffness, as evidenced by increased aortic pulse wave velocity (173.2 {+/-} 50.9 mm/s vs. - 10.7 {+/-} 54.6 mm/s in controls, p = 0.0393). NEW & NOTEWORTHYWe show that renal denervation mitigates the rise in blood pressure (BP) in a model that is not genetic nor diet-dependent, the chronic mild stress model (CMS). Specifically, we demonstrate the role of afferent, rather than efferent, renal nerves in mediating the rise in BP in male CMS rats. Finally, we report that renal sympathetic nerve activity, but not BP, is elevated in female CMS rats measured by telemetry over seven days in conscious rats.

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.

Introduction: Class II lupus nephritis (LN) is considered clinically benign, yet up to 50% of patients progress to more severe disease and no molecular characterization exists. We aimed to define the single-cell landscape of Class II LN and identify cellular and transcriptional features associated with kidney outcome. Methods: We performed single-cell RNA sequencing on kidney biopsies from fifteen Class II LN patients (eight de novo, seven regressed from prior proliferative or membranous disease) and six healthy controls, integrated with 155 LN and 30 healthy-control samples from the Accelerating Medicines Partnership in SLE spanning proliferative, membranous, and mixed LN. Findings were correlated with 52-week renal response and supported by urinary proteomics. Results: Despite histologically minimal changes, Class II LN exhibited marked immune cell expansion comparable to proliferative and membranous LN, including CCL3+ CCL4+ intermediate monocytes producing TNF, FOLR2+ SIGLEC1+ macrophages producing TGF{beta} and PDGF, and CD69+ CD8+ effector memory T cells producing IFN{gamma}. Fibroblasts and myofibroblasts were significantly expanded and displayed divergent transcriptional programs: pro-fibrotic and inflammatory myofibroblast modules were associated with worse 52-week outcomes, while interferon-responsive and matrix-remodeling fibroblast programs were associated with improvement. Pathogenic and protective fibroblast populations received overlapping immune-derived signals, suggesting that fibroblast transcriptional state, rather than the identity of incoming signals, shapes the stromal response. Baseline chronicity index captured this stromal heterogeneity and was the clinical parameter most associated with outcome. Urinary proteomics aligned Class II with proliferative rather than membranous disease. Conclusions: Class II LN harbors substantial molecular activity not captured by routine histology. Fibroblast transcriptional programs and baseline chronicity index are candidate correlates of kidney outcome that warrant validation in larger, prospectively followed cohorts.

BackgroundLupus nephritis remains a major cause of chronic kidney disease and kidney failure in systemic lupus erythematosus. Glucocorticoids are central to treatment but are limited by systemic toxicity. We evaluated whether a previously characterized collagen IV 3-targeted liposomal nanoparticle formulation carrying low-dose prednisolone could attenuate established lupus nephritis in MRL/lpr mice. MethodsFemale MRL/lpr mice with disease present at treatment initiation and C57BL/6J control mice received saline or collagen IV 3-targeted prednisolone-loaded nanoparticles (Col4-3-Pred-NPs). Renal outcomes were assessed by longitudinal proteinuria, glomerular filtration rate (GFR), survival, kidney histopathology, renal IgG and C3d deposition, dUTP/TUNEL-associated injury staining, and renal cytokine/chemokine profiling. Body weight, food and water intake, and blood glucose were monitored as measures of general condition and preliminary tolerability. ResultsCol4-3-Pred-NPs improved survival in MRL/lpr mice, reduced cumulative proteinuria burden, and attenuated terminal GFR decline compared with saline-treated MRL/lpr controls. Treatment reduced glomerular and tubulointerstitial injury, lowered composite EGTI histopathology scores, decreased terminal kidney enlargement, reduced glomerular IgG deposition and renal dUTP-positive injury signals, and reduced renal signals for IL-28A/B, IL-7, PD-ECGF, IL-11, CCL6/C10, and IL-15. C3d deposition was not significantly altered. Nanoparticle treatment was not associated with sustained treatment-related increases in blood glucose or body-weight loss during the measured study period. ConclusionsCollagen IV 3-targeted liposomal delivery of low-dose prednisolone attenuated established lupus nephritis in MRL/lpr mice and improved renal structural, functional, inflammatory, and survival outcomes. These findings support further evaluation of glomerulus-targeted nanotherapy as a potential strategy to improve the precision and therapeutic index of glucocorticoid treatment in lupus nephritis.

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.