American journal of physiology. Renal physiology最新文献

NaCl restriction upregulates pendrin, in part, through increased circulating aldosterone and the intercalated cell (IC) mineralocorticoid receptor (MR). Since 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enhances aldosterone binding to this receptor in other cells, we asked if pendrin abundance is reduced in NaCl-restricted 11β-HSD2 knockout (KO) rats. However, pendrin abundance was greater in 11β-HSD2 KOs than in controls, possibly from enhanced glucocorticoid MR activation. Moreover, the MR antagonist, spironolactone, reduced pendrin abundance in mice that do not produce aldosterone (aldosterone synthase KO). IC MR gene ablation also reduced pendrin protein abundance in corticosterone-treated, adrenalectomized mice. Therefore, the MR regulates pendrin independently of aldosterone. As such, we asked whether glucocorticoids, the other MR ligands, change pendrin abundance and/or subcellular distribution in adrenalectomized wild-type mice. We observed that corticosterone upregulated pendrin in a dose-dependent manner through both increased total protein abundance and subcellular redistribution. At higher doses, corticosterone increased pendrin abundance from greater pendrin-positive cell number within the late distal convoluted tubule 2 (DCT2) rather than increased pendrin abundance per cell. Finally, we asked whether pendrin contributes to the hypertension seen in rodent models of Cushing syndrome. Although corticosterone increased blood pressure in wild-type mice, it had no effect in pendrin KOs. In conclusion, glucocorticoids upregulate pendrin by increasing pendrin total protein abundance through an MR-dependent pathway and subcellular redistribution. Glucocorticoids increase pendrin abundance by increasing the number of pendrin-positive cells within the DCT2. In doing so, pendrin contributes to the hypertension seen in rodent models of Cushing syndrome.NEW & NOTEWORTHY Pendrin participates in the hypertension seen in Cushing syndrome.

The metabolic health of the kidney is directly correlated to the risk of progressive kidney disease. Our understanding of the metabolic processes that fuel the diverse functions of the kidney is limited by the kidney's structural and functional heterogeneity, especially in key metabolic organelles such as the mitochondria. As the kidney contains many different cell types, we sought to determine the intrarenal mitochondrial heterogeneity that contributes to cell-specific metabolism. To interrogate this, we used a recently developed mitochondrial tagging technique, MITO-Tag, to isolate kidney cell-type-specific mitochondria. Here, we investigated mitochondrial functional capacities and the metabolomes of the early and late proximal tubule (PT) and the distal convoluted tubule (DCT). The conditional MITO-Tag transgene was combined with Slc34a1-CreERT2, Ggt1-Cre, or Pvalb-Cre transgenes to generate mouse models capable of cell-specific isolation of hemagglutinin (HA)-tagged mitochondria from the early PT, late PT, or the DCT, respectively. Functional assays measuring mitochondrial respiratory and fatty acid oxidation (FAO) capacities and metabolomics were performed on anti-HA immunoprecipitated mitochondria from kidneys of ad libitum-fed and 24-h fasted male mice. The renal MITO-Tag models targeting the early PT, late PT, and DCT revealed differential mitochondrial respiratory and FAO capacities, which dynamically changed during fasting conditions. The renal MITO-Tag model captured differential mitochondrial metabolism and functional capacities across the early PT, late PT, and DCT at baseline and in response to fasting.NEW & NOTEWORTHY This study describes the generation and application of mouse models capable of interrogating kidney tubular epithelial cell-specific mitochondrial metabolism. Applying the MITO-Tag system in the kidney, we have, for the first time, defined the mitochondrial metabolic heterogeneity of renal cortical tubular epithelium and discovered differential mitochondrial functional capacities in response to an acute metabolic stress such as fasting.

Obesity and sex influence the salt-sensitivity of blood pressure, but it is unknown whether this also affects tissue electrolyte accumulation. We hypothesized that obese female rats exhibit greater salt-sensitivity of blood pressure with higher nonosmotic sodium accumulation in skin compared with obese male or lean female rats. To investigate this, male and female lean and obese ZSF1 rats received either a deoxycorticosterone acetate (DOCA) pellet plus a high-salt diet (6% NaCl) or a placebo pellet plus normal salt diet at 19 wk of age. Systolic blood pressure (SBP) and 24-h sodium excretion were measured biweekly from 12 to 26 wk. At 26 wk, sodium and potassium content in the skin was measured. SBP was higher in obese than in lean rats within the DOCA + high-salt groups. Lean female rats showed no SBP increase in response to the DOCA + high-salt diet. Analysis of pressure-natriuresis curves confirmed that obesity increased the salt-sensitivity of blood pressure. Furthermore, there was a significant interaction between sex and obesity on salt-sensitivity of blood pressure: lean female rats were completely salt-resistant, whereas obese female rats showed the greatest salt-sensitivity of blood pressure. Similar trends were observed in males, but the effects were less pronounced. Despite differences in salt-sensitivity of blood pressure, there were no differences in skin electrolytes. In conclusion, obesity enhances salt-sensitive hypertension more in female than in male rats, independent of skin electrolytes.NEW & NOTEWORTHY In the presence of obesity, salt-sensitivity of blood pressure was greater in female than in male ZSF1 rats, whereas in the absence of obesity, female rats were salt-resistant. Interestingly, these differences occurred without changes in sodium or potassium accumulation in the skin. These findings highlight that obesity leads to loss of female advantage in salt-sensitive hypertension and call into question the role of nonosmotic sodium storage in salt-sensitivity of blood pressure.

We examine the role of mTORc2 in mediating overnight-high-K⁺-(HK)-intake-induced stimulation of renal outer medullary K⁺ channels (ROMK) in late distal-convoluted-tubule (DCT2)/early connecting-tubule (early-CNT). Also, we explore whether mTORc2 simultaneously inhibits Kir4.1/Kir5.1 and stimulates ROMK/epithelial Na⁺ channel (ENaC) during overnight-HK. We performed patch-clamp experiments, immunoblotting, metabolic cage, and in vivo measurement of urinary K⁺ excretion in kidney tubule-specific RICTOR (rapamycin insensitive companion of mTOR) knockout mice (Ks-RICTOR-KO) and Rictor^flox/flox mice (wild type). Ks-RICTOR-KO mice had a lower urinary K⁺-excretion and higher plasma K⁺ concentration than Rictor^flox/flox mice. Moreover, overnight HK intake further increased plasma K⁺ level in Ks-RICTOR-KO mice but not in Rictor^flox/flox mice. Ks-RICTOR-KO mice had higher basolateral Kir4.1/Kir5.1 activity in the DCT than in Rictor^flox/flox mice. In contrast, tertiapin-Q (TPNQ)-sensitive K⁺ currents (ROMK) were lower in both DCT2/early-CNT and cortical-collecting-duct (CCD) of Ks-RICTOR-KO mice than in Rictor^flox/flox mice. Amiloride-sensitive Na⁺ currents (ENaC) were significantly lower in DCT2/CNT in Ks-RICTOR-KO mice than in wild type. Overnight HK intake decreased Kir4.1/Kir5.1 activity of DCT and increased amiloride-sensitive Na⁺ currents and TPNQ-sensitive K⁺ currents in DCT2/early-CNT and in the CCD in Rictor^flox/flox mice. However, these effects of overnight-HK were absent in Ks-RICTOR-KO mice. Finally, in vivo measurement of urinary K⁺ excretion showed that urinary K⁺ excretion in Ks-RICTOR-KO mice on overnight-HK was lower than in Rictor^flox/flox mice. In summary, mTORc2 plays a role in maintaining baseline activity of Kir4.1/Kir5.1, ROMK, and ENaC and it mediates the effect of overnight-HK on ENaC and ROMK in aldosterone-sensitive distal nephron (ASDN). We conclude that mTORc2 plays a key role in stimulating renal K⁺ excretion during high-K⁺-loading by inhibiting Kir4.1/Kir5.1 in the DCT and stimulating ENaC and ROMK in ASDN.NEW & NOTEWORTHY Our study has two novel findings. First, we demonstrate that mTORc2 plays a key role in mediating high-K⁺-intake-induced stimulation of ENaC/ROMK activity in late DCT. Second, we demonstrate that overnight-HK-induced stimulation of kidney K⁺ excretion is achieved by simultaneously stimulating ENaC and ROMK in the late DCT, CNT, and CCD, and inhibiting Kir4.1/Kir5.1 in the DCT. Thus, we have illustrated an integrated mechanism by which mTORc2 regulates kidney K⁺ excretion.

The Dahl salt-sensitive (SS) rat is an established model of hypertensive kidney injury, where the gut microbiota has been shown to causally contribute to disease progression. Gut bacteria-derived metabolites serve as mechanistic links between the microbiota and disease, with dietary fiber providing a critical source of protective metabolites such as short-chain fatty acids (SCFAs). The current study hypothesized that the substitution of nonfermentable fiber cellulose with the fermentable fiber inulin would attenuate hypertension and renal damage in SS rats via increased circulating SCFAs. Male and female SS rats were placed on the 0.4% NaCl (low-salt, LS) inulin diet for 1 wk before the switch to a 4.0% NaCl (high-salt, HS) inulin diet for 4 wk. Controls were maintained on diets containing cellulose. Rats consuming inulin had a reduction in mean arterial pressure compared with cellulose, though the antihypertensive effect was more robust in females. The inulin diet significantly protected both sexes from albuminuria, medullary protein cast formation, and renal immune cell infiltration, and was associated with specific changes to the fecal microbiota. Assessed by mass spectrometry, inulin consumption resulted in increased circulating propionate and butyrate, and the administration of these SCFAs revealed a protective effect of propionate against salt-sensitive hypertension and kidney damage in males, which coincided with an expansion of renal T regulatory cells. In conclusion, substitution of cellulose for the fermentable fiber inulin lowered blood pressure and significantly attenuated salt-induced renal damage in both sexes, which may be attributed to greater production of the protective, anti-inflammatory SCFA propionate.NEW & NOTEWORTHY The dietary switch to inulin, a fermentable fiber, reduced salt-sensitive hypertension and kidney injury in male and female Dahl SS rats, and caused gut microbiota composition shifts and increases in SCFA production (propionate and butyrate). Direct administration of propionate ameliorated salt-sensitivity in males, which coincided with renal T regulatory cell expansion. These findings provide the mechanistic basis for leveraging the microbiota and its metabolites through dietary interventions as a therapeutic for hypertension and kidney disease.

Atonal BHLH transcription factor 8 (ATOH8) is a basic helix-loop-helix (bHLH) transcription factor; however, its role in glomerular epithelial cells (podocytes) remains unclear. This study aimed to elucidate the function of ATOH8 in podocytes. First, ATOH8 expression in the mouse kidney was confirmed in podocytes by immunofluorescence staining and in situ hybridization. In cultured human podocytes, transforming growth factor-beta (TGF-β) treatment significantly reduced ATOH8 mRNA expression. To examine the functional consequences of ATOH8 downregulation, ATOH8 expression was knocked down with shRNA. Subsequent RNA sequencing analysis of ATOH8-knockdown podocytes revealed increased extracellular matrix gene expression and activation of TGF-β signaling. ATOH8-knockdown podocytes also showed SMAD2/3 nuclear translocation, increased SMAD transcriptional activity, as determined by a luciferase assay, and upregulated TGFB1 mRNA even without TGF-β stimulation, consistent with TGF-β signaling activation. In vivo, C57BL/6 Atoh8-deficient mice showed no renal abnormalities at baseline. However, in an adriamycin (ADR)-induced focal segmental glomerulosclerosis (FSGS) model, Atoh8-deficient mice developed significantly more severe glomerulosclerosis than wild-type mice, with higher renal cortical Tgfb1 and Col4a1 mRNA levels. Reduced ATOH8 expression was also observed in ADR-induced nephropathy in mice and rats and in various human glomerular diseases. These findings suggest that ATOH8 downregulation enhances TGF-β signaling and glomerulosclerosis progression, indicating a protective role for ATOH8 in maintaining podocyte integrity and preventing kidney injury.NEW & NOTEWORTHY This study identifies atonal transcription factor 8 (ATOH8) as a previously unexplored regulator of podocyte function. We demonstrate that ATOH8 knockdown activates TGF-β signaling and increases extracellular matrix gene expression. Notably, ATOH8 deficiency alone does not cause renal injury but exacerbates glomerulosclerosis in an adriamycin-induced nephropathy model, accompanied by increased Tgfb1 mRNA expression in the renal cortex. These findings indicate that ATOH8 plays a protective role in podocyte function and limits glomerulosclerosis during kidney injury.

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 while 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 type of stimuli. Further, expression of serotonin transporter (Sert) mRNA was increased in Gli mutant urethra compared to wild type. Uroplakin IIIa, typically localized to bladder urothelium, was ectopically expressed in 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.

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 (VSMC) were incubated with CPPs containing varying citrate concentrations. We quantified calcification using calcium assays and characterized CPPs using spectrophotometry, DLS, cryo-TEM, ED, Raman spectroscopy, EDX, and MS. At 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 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 towards a less calcifying phenotype, highlighting its potential as a therapeutic strategy against vascular calcification in CKD.

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, while 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 hours. 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.

Magnetic resonance imaging (MRI) is increasingly important in preclinical and clinical investigations of the kidney. However, there are few user-friendly, flexible, and standardized tools for evaluating MR images for quantitative imaging analysis. Here, we develop AutoGlom, an open-source, modular, and expandable imaging software tool that incorporates artificial intelligence (AI) for segmentation, analysis, and visualization of three-dimensional (3-D) MR images of the kidney. This initial version of AutoGlom focuses on morphological segmentation and quantification. We describe kidney segmentation from MR images, followed by the use of the graphical user interface of AutoGlom. Using AutoGlom, we measure glomerular number and volume from ex vivo cationic ferritin-enhanced MRI (CFE-MRI) in mice. We further demonstrate a 3-D-printed holder to allow for simultaneous imaging of up to 16 mouse kidneys at high resolution (50 μm) within several hours. The streamlined workflow facilitates rapid image analysis and accelerates optimization of cationic ferritin dosing and imaging parameters. These tools are a resource for the kidney community that may accelerate the identification of candidate imaging biomarkers from 3-D MRI of the kidney and have the potential to be extended to in vivo studies and other imaging modalities.NEW & NOTEWORTHY We present AutoGlom, an open-source software for quantitative kidney MRI analysis. AutoGlom integrates deep learning-based glomerular segmentation, parameter tuning, and visualization within a user-friendly interface. It enables high-throughput analysis using a 3-D-printed holder for simultaneous imaging of multiple kidneys and introduces a new image quality metric, glomerular contrast, to improve reliability. AutoGlom provides standardized, reproducible workflows for glomerular quantification, bridging preclinical and translational kidney imaging and enabling future physiological discoveries.