American journal of physiology. Renal physiology最新文献

The stromal compartment of the developing kidney arises from forkhead box D1 (Foxd1)-expressing progenitors and gives rise to diverse cell types essential for nephrogenesis, including the renal stroma, capsule, mesangial cells, renin cells, pericytes, and vascular smooth muscle cells (VSMCs). However, the molecular mechanisms guiding their fate specification remain incompletely defined. Here, we identify the basic helix-loop-helix transcription factor Tcf21 as a critical determinant of stromal cell identity during kidney development. We performed single-cell RNA sequencing (scRNA-seq) on Foxd1-lineage cells isolated from embryonic day 14.5 (E14.5) Tcf21 conditional knockout (Tcf21-cKO) Foxd1^Cre/+;Rosa26^mTmG;Tcf21^f/f and control kidneys, revealing seven transcriptionally distinct stromal subpopulations. Loss of Tcf21 resulted in marked depletion of medullary/perivascular stroma, collecting duct-associated stroma, proliferating stroma, and nephrogenic zone-associated subpopulations, confirmed by immunostaining, which revealed severe constriction of medullary and collecting duct stromal spaces. In addition, we identified a novel cluster unique to Tcf21-cKO kidneys, characterized by high expression of endomucin (Emcn). These cells spanned pseudotime trajectories and were distributed broadly across the mutant kidney. These findings were corroborated by E14.5 single-cell ATAC sequencing (scATAC-seq), which confirmed altered chromatin accessibility in Tcf21-deficient stroma. To assess the persistence and downstream impact of these defects, we performed bulk and scRNA-seq at E18.5, revealing sustained expansion of Emcn⁺ cells with profibrotic and perivascular transcriptional programs. Histological analyses at 2 mo demonstrated lasting architectural disruption, interstitial fibrosis, and impaired renal function in Tcf21-cKO mice. Our results identify Tcf21 as a key regulator of stromal progenitor fate and establish a developmental origin for fibrotic remodeling and kidney dysfunction.NEW & NOTEWORTHY This study identifies Tcf21 as a key regulator of kidney stromal fate. Loss of Tcf21 disrupts the emergence of key stromal cell types and leads to the expansion of a dysregulated, Emcn-expressing stromal population. Integrating single-cell transcriptomics, chromatin accessibility, and histology, we show that this misdifferentiation contributes to fibrosis in adulthood. These findings suggest that TCF21-dependent stromal differentiation restrains maladaptive remodeling and links developmental fate decisions to later fibrotic disease.

Renin regulates blood pressure and fluid-electrolyte homeostasis via the renin-angiotensin-aldosterone system (RAAS), and renin cells function as renal baroreceptors that couple perfusion pressure to renin output. Krüppel-like factor 2 (Klf2), a canonical flow-responsive transcription factor, repeatedly emerged from our multiomics profiling linked to renin cell identity, but its role in renin cells remained unknown. We generated mice with renin-lineage-specific Klf2 deletion (Klf2cKO: Ren1^dCre/+;Klf2^fl/fl) and assessed renin expression and kidney morphology. Klf2cKO mice showed reduced plasma renin at 2 mo that persisted at older ages, decreased Ren1 mRNA, a lower juxtaglomerular renin area index by immunohistochemistry, and reduced carotid blood pressure, whereas the renal architecture and overall vasculature structure were largely conserved. Analysis of single-cell RNA-seq spanning Foxd1⁺ progenitors to mature renin-lineage cells revealed low Klf2 during embryogenesis and the neonatal period but enrichment in the mature postnatal juxtaglomerular cluster, consistent with a role in maintenance rather than early lineage specification. To test renin hypotensive stress and altered perfusion pressure, we challenged Klf2cKO mice with low-salt plus captopril and with surgical aortic coarctation (AoCo; right kidney high pressure, left kidney low pressure). In both models, plasma renin and cortical Ren1 mRNA remained lower than in controls, and AoCo yielded a significantly blunted left-to-right Ren1 ratio, indicating impaired pressure-responsive renin transcription. Together, the findings identify Klf2 as a transcriptional effector linking hemodynamic signals to renin transcription in mature juxtaglomerular cells. Identifying key transcriptional pathways in renin cells could reveal novel targets for modulating the RAAS and blood pressure.NEW & NOTEWORTHY Using renin lineage cell-specific Klf2 knockout mice, we identify Klf2 as a transcriptional effector linking hemodynamic pressure sensing to renin transcription in mature juxtaglomerular cells. Loss of Klf2 blunted the upregulation of renin in response to low-salt/captopril and the pressure-responsiveness to low and high perfusion.

Although injury in glomerular disease might only damage a subset of podocytes in any given glomerulus, the response of the healthy neighboring podocytes to the injured podocytes oftentimes determines the course of the disease. To investigate this relationship, we designed a dual-chamber open microfluidic coculture device to specifically examine paracrine signaling from podocytes undergoing targeted injury by either adriamycin, puromycin aminonucleoside, or a cytopathic antipodocyte antibody to healthy podocytes. Global transcriptomic analysis measured by RNA sequencing revealed shared and unique pathways between the three forms of targeted injury, with temporal differences in the transcriptomic responses to each form of injury. Transcriptional changes also showed that paracrine-induced injury to neighboring podocytes was similar to the targeted-injured podocytes and was specific for each podocyte injury model. In silico ligand-receptor analysis of ligands secreted by the insult-targeted podocytes and receptors expressed by the responsive, paracrine-injured counterparts identified 19 candidate mediator pairs that were shared among the three injury models. Several of these were enriched in patients with histological evidence of glomerular injury present in the Nephrotic Syndrome Study Network (NEPTUNE). One-factor-at-a-time candidate approaches validated the ability of these candidate pathways to mediate aspects of the podocyte injury models. Finally, an all-inclusive, comprehensive investigation of this signaling space using a systematic Design-of-Experiment analysis revealed that transforming growth factor-β1 (TGF-β1) signaling is a critical mediator of mitochondrial dysfunction during podocyte injury. Together, these findings define a new concept for future studies to understand the pathways involved in animal models and ultimately human studies.NEW & NOTEWORTHY From a clinical perspective, it is ideal if yet unknown common pathways could be therapeutically targeted in different forms of injury in diseases of podocytes, and if there were mitigation strategies to minimize further damage to yet unaffected podocytes. The results of the current studies showed that there are indeed common responses to different experimental forms of podocyte injury and identified common paracrine signaling from injured podocytes that adversely affects the neighboring healthy podocyte population.

The epithelial sodium channel (ENaC) is essential for sodium reabsorption and potassium homeostasis in the distal nephron, where its activity is controlled by mineralocorticoid signaling and downstream proteolytic processing of channel subunits. Although cleavage of the γ-ENaC subunit has been implicated in aldosterone-mediated sodium transport, the identity of mineralocorticoid receptor (MR)-regulated proteases responsible for this process remains uncertain. Here, we investigated the role of kallikrein-1 (encoded by Klk1), a serine protease expressed in the connecting tubule and cortical collecting duct (CNT/CCD), as a mediator of ENaC activation. Using CRISPR/Cas9, we generated a conditional Klk1-floxed allele and established mice with CNT/CCD-specific deletion of Klk1 by crossing with Calb1-Cre (CNT-Klk1^-/-). On a low-sodium, high-potassium diet, CNT-Klk1^-/-mice exhibited ∼85% less renal kallikrein-1 expression, yet maintained normal serum electrolytes, urinary potassium excretion, and aldosterone responses. Western blot analysis revealed significantly less cleavage of γ-ENaC and α-ENaC in CNT-Klk1^-/- kidneys, accompanied by more total NCC abundance. Despite impaired ENaC proteolysis, amiloride-sensitive sodium excretion was preserved, indicating intact ENaC function. These findings identify renal kallikrein-1 as a protease that contributes to ENaC subunit processing in vivo. However, the absence of overt sodium or potassium handling defects in CNT-Klk1^-/- mice suggests that kallikrein-1 deficiency is not sufficient to disrupt overall ENaC function, likely due to compensatory mechanisms from redundant proteolytic or nonproteolytic pathways. Together, our results refine the role of kallikrein-1 as a modulator, rather than a sole determinant, of ENaC activation and highlight the complexity of aldosterone-dependent sodium transport in the distal nephron.NEW & NOTEWORTHY Using a novel connecting tubule/cortical collecting duct specific kallikrein-1 knockout model, we show that γ- and α-ENaC cleavage is impaired by loss of renal kallikrein-1, without major disturbances in sodium or potassium handling. These findings highlight redundancy among ENaC regulatory pathways and suggest that proteolytic cleavage of ENaC, although useful as an indicator of ENaC-mediated transport under physiological conditions, may not, in and of itself, play a major role in ENaC function.

Urine extracellular vesicles (uEVs) originate from the genitourinary system, including the kidney's tubular epithelial cells. These cells control Na⁺ balance, for example, by increased aldosterone-induced Na⁺ reabsorption in response to a low-Na⁺ intake. We hypothesized that the uEV transcriptome reflects the physiological adaptation of tubular epithelial cells to variation in dietary Na⁺. Paired biobanked uEV samples from healthy young men after 5 days on a low (70 mmol/day) and high (250 mmol/day) Na⁺ diet were analyzed by RNA sequencing. From 20 samples, 17 produced high-quality data, yielding quantitative data for >13,000 genes. The Na⁺ diets only significantly affected the uEV abundance of 10 gene transcripts; 5 decreased, and 5 increased, including SLC12A3, encoding the Na⁺,Cl^- transporter NCC, in low-Na⁺ diet sample uEVs. We used transcriptomic deconvolution to estimate the uEVs' tissue and cell-type origins. The uEVs were mainly derived from the kidneys and bladder. Compared with the high-Na⁺ diet samples, the low-Na⁺ diet samples had a ∼30% higher kidney-derived uEV abundance. The estimated kidney-derived EV abundance was strongly correlated to plasma renin, plasma and urine aldosterone, and mean arterial blood pressure. At the kidney epithelial cell level, proximal tubule-derived EVs were most abundant. Although most of the cell-type-specific uEV abundances were not different between Na⁺ diets, β-intercalated cell-derived EVs were significantly less abundant in low-Na⁺ diet samples. Moreover, β-intercalated cell-uEV abundance estimates were negatively correlated with mean arterial pressure. In conclusion, uEV RNA analyses illuminate the pathways underlying physiological control of renal Na⁺ reabsorption in the human kidney.NEW & NOTEWORTHY The kidneys adapt to changes in Na⁺ intake by regulating tubular Na⁺ transport, and we investigated whether the RNA content of urinary EVs (uEV) reflects the physiological responses to dietary Na⁺ intake in humans. Dietary Na⁺ intake altered both transcript abundance and kidney-derived uEV levels, which correlated with renin, aldosterone, and blood pressure levels. Thus, uEV transcriptomics provide a noninvasive window for studying the molecular control of kidney Na⁺ handling in humans.

Septic acute kidney injury (AKI) is a life-threatening complication of systemic infection, characterized by rapid loss of renal function and high mortality. Despite its clinical significance, the underlying molecular mechanisms remain incompletely elucidated. In this study, we identify the nuclear factor-κB (NF-κB)/apoptotic protease-activating factor 1 (Apaf1)/caspase-9 signaling axis as a central regulator of tubular apoptosis and inflammation through suppression of autophagy. Using proximal tubule-specific Apaf1 knockout mice, we demonstrate that Apaf1 deficiency significantly mitigates lipopolysaccharide (LPS)-induced renal dysfunction, reduces histopathological injury, and decreases tubular apoptosis, as evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling staining and cleaved caspase-3 expression. Correspondingly, renal inflammatory cytokines, including Il6, Tnfa, and Mcp1, are markedly downregulated. In vitro, Apaf1 knockdown in LPS-treated BUMPT (the Boston University mouse proximal tubular cell line) cells similarly reduces apoptosis and inflammation, whereas Apaf1 overexpression exacerbates these pathological responses, confirming its pivotal role in tubular injury. Mechanistic studies reveal that Apaf1 mediates activation of caspase-9, which subsequently suppresses autophagic flux, as indicated by altered LC3 and p62 expression. Pharmacologic inhibition of caspase-9 using Z-LEHD-FMK restores autophagy, attenuates tubular apoptosis, and dampens inflammatory cytokine production in both cell culture and murine models, highlighting caspase-9 as a critical downstream effector. Furthermore, NF-κB functions as an upstream transcriptional activator of Apaf1, linking inflammatory signaling to autophagy suppression and tubular injury. Collectively, our findings delineate a sequential NF-κB/Apaf1/caspase-9/autophagy pathway that amplifies tubular inflammation and apoptosis in septic AKI. Targeting this axis may provide a novel therapeutic strategy to preserve tubular integrity, limit inflammation, and improve renal outcomes in patients with septic AKI.NEW & NOTEWORTHY Our findings delineate a sequential NF-κB/Apaf1/caspase-9/autophagy pathway that amplifies tubular inflammation and apoptosis in septic AKI. Targeting this axis may provide a novel therapeutic strategy to preserve tubular integrity, limit inflammation, and improve renal outcomes in patients with sepsis-associated AKI.

Diabetic nephropathy is a leading cause of end-stage kidney disease worldwide, yet its complex pathogenesis remains incompletely understood. This is partly due to limitations of existing preclinical models, which are often genotypic, monogenic, and fail to replicate the chronic progression and advanced renal damage observed in humans. We previously developed a nongenotypic rat model of type 2 diabetes model using obese male Sprague Dawley rats chronically treated with low-dose tacrolimus, which reproduced key metabolic features of human type 2 diabetes. In this study, we investigated the onset and progression of diabetic nephropathy in this model. Glomerular filtration rate was measured by iohexol-dried blood sample (DBS) plasma clearance. Twenty-four-hour urine collection was performed to assess albuminuria and proteinuria. At the endpoint, kidneys were collected for histological evaluation. Tacrolimus blood levels were monitored monthly. Diabetic animals initially exhibited glomerular hyperfiltration, followed by a decline in glomerular filtration rate at the final stage of the study, consistent with the trajectory observed in humans. This was accompanied by a trend toward increased proteinuria. Histological analysis revealed mesangial matrix expansion, a higher incidence of glomeruli with focal segmental glomerulosclerosis and significant glomerular hypertrophy. In addition, we observed increased kidney weight, tubular hypertrophy, intraglomerular and tubulointerstitial fibrosis, and elevated cortical expression of proinflammatory markers. This model reproduced both early and advanced pathological features of human diabetic nephropathy, representing a valuable tool for studying diabetic nephropathy pathophysiology in a chronic context and as a platform for evaluating potential therapeutic strategies.NEW & NOTEWORTHY We present a nongenotypic, obesity- and tacrolimus-induced rat model that recapitulates the chronic progression of human diabetic nephropathy. The model reproduces key early and advanced features, including hyperfiltration, glomerular filtration rate (GFR) decline, glomerular hypertrophy, mesangial expansion, nodular sclerotic lesions, and tubular-interstitial fibrosis. Its translational relevance and long-term progression provide a valuable platform for mechanistic studies and for evaluating potential therapeutic strategies.

Hypertension is a hallmark of cardiovascular abnormalities associated with Williams syndrome (WS), a rare genetic disorder involving microdeletion of genes on human chromosome 7, including the elastin gene (ELN). Heterozygous deletion of Eln (Eln^+/-) in mice recapitulates hypertension and arteriopathy associated with WS. Previously, differences in blood pressure elevation and sensitivity to dietary sodium were found to be less profound in female Eln^+/- mice. Here, we determined whether ovarian hormones play a role in sex-related difference in blood pressure elevation resulting from Eln haploinsufficiency. Female Eln^+/+ and Eln^+/- mice instrumented with radiotelemetry devices were subjected to sham surgery or ovariectomy (OVX). We found that OVX lowered diastolic but not systolic blood pressure (SBP) in Eln^+/- mice, resulting in increased pulse pressure. In Eln^+/- mice, diuresis induced by acute volume expansion was blunted, while anti-natriuresis was exaggerated. Furthermore, amiloride lowered SBP and increased urinary Na⁺ excretion, suggesting that Eln^+/--induced hypertension may be Na⁺-dependent. We conclude that increased Na⁺ and water retention by the kidney contribute to hypertension resulting from Eln haploinsufficiency. The underlying mechanism involves the alteration of ovarian hormone effects in the kidney and sustained signaling downstream of the V₂ receptor, leading to increased ENaC activity and water reabsorption.

To better understand the impact of accelerated aging on kidney function, we compared standard C57BL6 mice (C57BL6) with Senescence Accelerated Mouse-Prone 8 mice (SAMP8). Young male SAMP8 (3&6 months) showed glomerular hyperfiltration compared with C57BL6 (absolute and per body weight), followed by gradual GFR decline, lower blood pressure, and enhanced mortality over the first 15 months of life. This was associated with higher kidney, heart and liver but not brain weights. Female SAMP8 likewise showed a faster early rise in body weight, higher organ weights, and a somewhat higher mortality, but GFR and blood pressure appeared unaltered vs. C57BL6. Since GFR phenotype was stronger in male mice, they were subjected to bilateral renal artery clamping-induced kidney ischemia-reperfusion (IR). One day after IR, young SAMP8 (3 months) showed higher plasma creatinine and kidney VCAM1 expression and subsequent mortality but a blunted rise in kidney Kim-1 mRNA and urine KIM-1 vs. C57BL6. Kidney proteomics indicated suppressed pathways of phagocytosis and apoptosis but enhanced necroptosis in SAMP8 vs C57BL6. When ischemia time was lowered in SAMP8 to induce a similar initial rise in plasma creatinine and urine NGAL vs. C57BL6, plasma creatinine recovery over 24 days was similar between strains in young mice, and despite impaired plasma creatinine recovery in older SAMP8 (10 months) kidney injury or inflammation seemed not enhanced. In conclusion, male SAMP8 mice have a shortened life span, large kidneys, and at young age temporal hyperfiltration and enhanced sensitivity to IR-induced acute kidney injury associated with a blunted KIM-1 response.