American journal of physiology. Renal physiology最新文献

Interstitial fibrosis is a hallmark of chronic kidney disease, and extracellular matrix is secreted by kidney fibroblasts/pericytes that have differentiated into myofibroblasts. Class I histone deacetylases (HDACs) are highly expressed in the nucleus of kidney cells where they regulate transcription. Class I HDAC inhibitors prevent interstitial fibrosis in pre-clinical models of acute kidney injury (AKI). In the warm bilateral ischemia-reperfusion-injury (IRI) model, HDAC1 was the only class I HDAC with greater protein abundance following IRI including in interstitial cells. Thus, it was hypothesized that fibroblast/myofibroblast HDAC1 activation is profibrotic. Hdac1^flox/flox; hemizygous Col1a2-cre/ERT (iFibHDAC1KO) mice and Hdac1^flox/flox (control) were given tamoxifen to induce cre activity 3 weeks prior to warm bilateral IRI or sham surgeries (preventative strategy). The severity of AKI was similar at 24 h post-surgery among the IRI mice, but glomerular rate filtration recovered over the 4-week study. Despite this, only control male IRI mice developed progressive interstitial fibrosis and tubular injury, which was accompanied by increased kidney myofibroblasts. All the female mice were protected from developing fibrosis with this IRI model. Cultured kidney fibroblasts (NRK49F) overexpressing HDAC1 and/or differentiated to myofibroblasts with transforming growth factor-β1 had a significant shift in the cell cycle from G1 to S and G2 phases and increased proliferation. The HDAC1 overexpressing cultured fibroblasts had increased cell cycle/proliferation and pro-inflammatory transcriptomes. Indeed, control IRI male mice had significantly greater kidney CD3+ and F4/80+ immune cells 24 hours post injury compared to iFibHDAC1KO IRI mice. In conclusion, HDAC1 activation in the kidney fibroblast is profibrotic.

Urinary incontinence (UI) imposes a significant healthcare burden and reduces quality of life. Contributing factors such as aging, pregnancy/childbirth, stress, and injury are recognized, but incomplete understanding of underlying mechanisms limits new therapies. Hedgehog (Hh) signaling has been implicated in lower urinary tract development, but its specific role in female continence mechanisms has not been fully characterized. Here we investigate the functional and molecular consequences of reduced Hh signaling using Gli2^+/-;Gli3^Δ699/+ (Gli mutant) female mice. We assessed spontaneous voiding through void spot assays and uroflowmetry, then assessed contractility in bladder and urethral tissues ex vivo. Female Gli mutant mice display more small volume voids than wild-type mice. Gli mutant female bladder had reduced strength of contraction to electrical field and cholinergic stimuli, whereas the urethra had reduced sensitivity to serotonin-mediated contraction, but not to phenylephrine. Thus, unique changes to bladder and urethral contractility dynamics are present in Gli mutant mice and are dependent upon types of stimuli. Furthermore, expression of serotonin transporter (Sert) mRNA was increased in Gli mutant urethra compared with wild type. Uroplakin IIIa, typically localized to bladder urothelium, was ectopically expressed in distal urethral urothelium of adult but not embryonic (E) day 16 Gli mutant mice. These findings highlight a previously uncharacterized role of Hh signaling in maintaining female lower urinary tract function and urothelial patterning, and support further investigation of its contribution to continence.NEW & NOTEWORTHY This study identifies disrupted Hh signaling as a key determinant of female bladder and urethral contractility, providing new insights into molecular mechanisms maintaining continence. We observe impaired contractile responses to multiple stimuli, including urethral response to serotonin. In addition, we identify ectopic expression of uroplakin IIIa in urethra of Gli mutant mice, arising after prenatal development. By reducing-but not completely ablating-Hh signaling, we elucidate essential roles of this pathway in determining continence.

We investigated the interplay between the mineralocorticoid aldosterone and a mutation mimicking Liddle syndrome in the control of the processing of the epithelia Na⁺ channel (ENaC) in mouse kidneys. Rates of processing were assessed by the appearance of the cleaved form of the γENaC subunit. Cleaved γENaC increased with decreasing dietary Na intake and with administration of aldosterone. Measurements taken from isolated tubules indicated that enhanced processing was similar in connecting tubules and in late distal convoluted tubules. In a mouse model with a truncated βENaC subunit (Liddle mice), levels of cleaved γENaC were similar in wild-type (WT) and Liddle animals. The amounts of the full-length form of the subunit were lower in the Liddle mice on control and high-Na diets. Infusion of a low dose of aldosterone produced similar increases in cleaved γENaC in WT and Liddle mice, whereas with maximal doses, levels in Liddle animals were 35% higher than in WT. Acute Na repletion of Na-depleted mice decreased cleaved γENaC with a time constant of 5 h. Rates of decrease were similar in WT and Liddle genotypes. The Liddle's mutation produces modest changes in ENaC processing, and a major effect of the mutation is on the activation of processed channels.NEW & NOTEWORTHY Using a mouse model of Liddle syndrome we show that the effects of the mutation on ENaC activity do not correlate with effects on channel processing. We conclude that the hyperactivity of the channels likely results from increased activity of processed channels residing in the apical membrane.

Chronic kidney disease (CKD) is a risk factor for cardiovascular disease (CVD), partly due to phosphate-induced vascular calcification. Fetuin-A stabilizes calcium-phosphate complexes into calciprotein particles (CPPs), preventing precipitation, but CPPs can mature into crystalline particles that drive calcification, particularly in CKD. In this study, we investigated whether citrate, a calcium chelator, could mitigate CPP-induced vascular calcification in vitro. Vascular smooth muscle cells (VSMCs) were incubated with CPPs containing varying citrate concentrations. We quantified calcification using calcium assays and characterized CPPs using spectrophotometry, dynamic light scattering, cryogenic transmission electron microscopy (cryo-TEM), electron diffraction (ED), Raman spectroscopy, energy dispersive X-ray spectroscopy, and mass spectrometry (MS). The highest citrate concentration, reduced calcification by 88% versus standard CPPs (P < 0.0001). CPP maturation was delayed, and mean diameter was 9% lower (216 ± 2 nm vs. 236 ± 6 nm; P = 0.0022). Cryo-TEM showed a transition from primary to secondary CPPs with preserved morphology. Hydroxyapatite was detected by ED in the standard and high-citrate CPPs, with the latter showing a significant lattice shift. An increased mineral-to-protein ratio was observed by Raman spectroscopy and protein-to-calcium assays. EDX demonstrated unchanged Ca/P ratios, but differences were observed in Ca (P = 0.0003), P (P < 0.0001), Na (P < 0.0001), and Cl (P < 0.0001). Finally, proteomics revealed 18 proteins enriched in standard CPPs (fold-changes -1.2 to -3.4; FDR < 0.05), including lipid-related apolipoproteins APOM, APOA1, APOA2, APOC3, and APOE. These data indicate that citrate remodels CPPs toward a less calcifying phenotype, highlighting its potential as a therapeutic strategy against vascular calcification in CKD.NEW & NOTEWORTHY We show for the first time that CPPs can be directly modulated by incorporating citrate during their formation. Citrate-modified CPPs maintain their hydroxyapatite core but display altered crystall lattice structure, reduced size, and changes in protein composition with fewer apolipoproteins. Importantly, they induce 88% less calcification in VSMCs. These findings provide proof-of-principle that CPP remodeling may represent a novel therapeutic strategy to limit vascular calcification in CKD, warranting further investigation in vivo.

Background: Hypohydration reduces kidney function and increases acute kidney injury (AKI) risk. Aging increases hypohydration-induced kidney dysfunction in males, but female aging studies are lacking. Therefore, we compared the effects of mild hypohydration on kidney function and AKI biomarkers in young (YF) and older female adults (OF). Methods: In a random crossover design, seventeen YF (20-35 years old) and 9 OF (55-75 years old), who were apparently healthy, completed two hydration protocols with ≥1 week washout: 1) baseline hydration, and 2) stepwise water restriction over 3 days, concluding with 16 hours water deprivation. We assessed hydration, AKI biomarkers, and the renin-angiotensin-aldosterone system (RAAS) from blood and 24-hour urine samples. The effects of age group and condition were assessed using two-way mixed-effects analysis and reported as mean±SD. Results: Hypohydration increased urine specific gravity and osmolality (condition effect: p<0.001) with no other main effects. Neutrophil gelatinase-associated lipocalin excretion decreased with hypohydration (p=0.04), independent of age (p=0.62). Urine cystatin C excretion increased in YF (p<0.001) but not OF (p=0.69), with a significant interaction effect (p=0.017). Nephrin excretion and urinary IGFBP7*TIMP-2 increased after hypohydration (both p<0.001), independent of age (both p≥0.35). OF exhibited lower plasma renin activity than YF (p=0.046), with no other main or interaction effects for other RAAS markers. Conclusions: OF did not exhibit greater AKI biomarker responses to mild hypohydration, contrasting with male studies showing age-related kidney function decline. These results highlight the need for research to clarify potential sex-based differences in age-related decline in kidney function.

The farnesoid X receptor (FXR) plays a role in the regulation of renal transporters and ion channels. Our previous study reported that activation of FXR inhibited cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl^- secretion and retarded microcyst progression. The present study aims to investigate whether FXR regulates TMEM16A, a calcium-activated Cl^- channel, which plays a major role in renal cyst progression in polycystic kidney disease (PKD). In vitro experiments were conducted to investigate the roles of FXR in TMEM16A-mediated Cl^⁻ secretion and cyst progression using wild-type and pkd1-deleting collecting duct cells (mIMCD3^pkd1-/-). In vivo experiments were performed in cystic PCK rats. Treating the collecting duct cells with FXR agonists (GW4064 and altenusin) decreased TMEM16A-mediated Cl^-secretion, which required FXR activation. The inhibitory effect of FXR activation was correlated with the decreased TMEM16A protein level, without affecting mRNA expression. Decreasing TMEM16A expression was involved in the activation of lysosome-induced degradation processes. Altenusin and GW4064 retarded the enlargement of mIMCD3^pkd1-/- cells-derived cysts, which was attenuated by FXR inhibition. In cystic PCK rats, treatment with altenusin at doses of 7.5 and 15 mg/kgBW significantly reduced the cystic index, kidney weight, blood urea nitrogen, and serum creatinine levels compared with vehicle-treated rats. These effects were correlated with a decrease in TMEM16A expression in cystic kidneys. In addition, altenusin exhibited anti-inflammatory properties by attenuating inflammatory markers IL-6, MCP-1, and TNF-α. This study highlights the role of FXR in the regulation of TMEM16A and attenuating renal cyst progression, positioning FXR as a promising target for PKD treatment.

Epigenetic regulation through histone modifications plays a crucial role in driving cellular state transitions. Regulating gene transcription through bivalency, the co-occurrence of activating histone H3 lysine 4 trimethylation (H3K4me3) and repressive histone H3 lysine 27 trimethylation (H3K27me3) histone marks, drives cell fate in development; however, its role in kidney injury is not known. Here, we investigated bivalent gene activation in the adult male Mus musculus kidney following ischemia-reperfusion injury (IRI). We developed and validated a novel per-gene scoring method for identifying bivalent domains from CUT&RUN (Cleavage Under Targets and Release Using Nuclease) data. Our analysis revealed that bivalent genes in the mature kidney substantially overlap with known embryonic bivalent domains. Following IRI, a subset of bivalent genes became activated, defined by a loss of H3K27me3, enrichment of H3K4me3, and a corresponding increase in gene transcription. Activated bivalent genes were differentially expressed in kidney epithelial cells and strongly enriched for pathways involving inflammation and fibrosis. To uncover the regulatory mechanism associated with activated bivalent genes, we identified key transcription factors linking these genes which converged on the pioneer transcription factor, Spi1. We demonstrated that Spi1 targets are differentially expressed in both mouse and human kidney epithelial cells after injury and preferentially depleted of H3K27me3 and gain H3K4me3 enrichment after IRI, supporting its role in mediating the epigenetic switch. Our findings reveal a common epigenetic mechanism where transcription factors, acting on bivalent chromatin, contribute to inflammatory and fibrotic responses to kidney injury. This suggests that the progression from acute to chronic kidney injury is an active, transcriptionally driven failure of repair that is epigenetically mediated by histone modifications.NEW & NOTEWORTHY We performed the first identification of bivalent domains in the adult mouse kidney. We identified bivalent genes that, when activated after kidney injury, drive inflammation, proliferation, and fibrosis. Activation of bivalent genes is coordinated by transcription factors such as Spi1. Our research not only provides a valuable database of bivalent genes in the kidney but also demonstrates that activation of bivalent genes is crucial for the progression from acute to chronic kidney injury.

This study investigates the effects of environmental circadian disruption and high-fat diet (HFD) on cardiovascular and renal functions in Bmal1 knockout (KO) and wild-type (WT) rats. Under 12:12-h light:dark conditions, Bmal1 KO males and females on a normal fat diet (NFD) exhibit lower mean arterial pressure (MAP) compared with WT. These genotype differences were attenuated after subjecting rats to a weekly 6 h advance in the 12:12-h light:dark protocol to induce chronic circadian stress (CCS). CCS modestly elevated MAP in males, eliminating pre-existing genotypic differences, whereas in females, CCS had no significant effects on MAP and heart rate. Under HFD, genotype-based MAP differences are attenuated, and sex differences in heart rate are diminished. CCS further elevated MAP in male Bmal1 KO, accompanied by reduced blood pressure amplitude. Diurnal variations in sodium excretion are abolished post-CCS in both WT and Bmal1 KO males on HFD. In Bmal1 KO females, CCS combined with HFD disrupts sodium excretion rhythms, thus eliminating the protective effects seen on NFD. These findings highlight the complex interplay between circadian regulation, dietary fat, and environmental stress in modulating cardiovascular and renal physiology. This study further supports a "two-hit hypothesis," where CCS and HFD may synergistically disrupt sodium homeostasis and blood pressure circadian rhythms in both males and females.NEW & NOTEWORTHY We investigate the role of Bmal1, a core circadian clock gene, and diet in impairment of blood pressure and renal function during a chronic circadian stress protocol. This study finds that the endogenous molecular clock responds to circadian stress and high-fat diet in a sex-specific manner, warranting further investigation in the role of these systems in the regulation of blood pressure control and organ function.