American Journal of Physiology-Renal Physiology

Juxtaglomerular (JG) cells are crucial regulators of blood pressure and fluid-electrolyte homeostasis. Under normal conditions, renin secretion by JG cells is sufficient to maintain homeostasis. However, under physiological stress such as narrowing of one of the renal arteries, heart failure, dehydration, or chronic administration of renin-angiotensin system (RAS) inhibitors, additional cells along the renal arterioles are transformed to the renin phenotype to meet the demands for renin and regain homeostasis. In cases of prolonged and persistent stimulation of renin cells, concentric arteriolar hypertrophy develops. The study of renin cell identity, plasticity and function often requires the isolation of this rare cell type. Here, we report on the generation of a mouse model to label renin-expressing cells with a bright fluorescent reporter under control of the Ren1c locus for the tracking and isolation of renin cells. Kidneys from adult heterozygous (Het) Ren1ctdTomato/+ mice showed tdTomato signal confined to the JG area under basal conditions, and extending along the afferent arterioles and in the intraglomerular mesangium upon treatment with captopril + low-salt diet to induce the endocrine transformation of renin cells. Unexpectedly, homozygous (Homo) Ren1ctdTomato/tdTomato mice exhibited increased tdTomato signal that extended along the afferent arterioles and into the mesangium even under normal physiological conditions, with progressive thickening of the kidney arterioles with age. Despite reduced renin immunostaining in the renal cortex, Ren1ctdTomato/tdTomato Homo mice exhibited significantly higher kidney Ren1 mRNA and circulating renin levels when compared to Het controls. Moreover, Homo mice showed significantly lower blood pressure measured under anesthesia and angiotensin I (Ang I) plasma levels, indicating compromised renin activity. In addition, Homo mice developed interstitial fibrosis and compromised kidney function. The concentric arteriolar hypertrophy phenotype observed in these mice is identical to that described when RAS is genetically or pharmacologically inhibited, including the presence of mutations in the renin gene. Unlike mice with global deletion of renin, these animals did not require neonatal saline injections to survive and did not develop other kidney abnormalities, indicating that the bicistronic approach rendered a renin hypomorphic mouse. Ren1ctdTomato mice constitute an excellent model for the bright and strong labeling of renin-expressing cells and for the study of the mechanisms involved in the development of concentric vascular hypertrophy under RAS inhibition. In addition, this model may provide a better understanding of factors controlling renin protein folding, stability, packaging, and release.

Kidney function is regulated by the circadian clock. Not only do glomerular filtration rate (GFR) and urinary excretion oscillate during the day, the expressions of several renal transporter proteins also exhibit circadian rhythms. Interestingly, the circadian regulation of these transporters may be sexually dimorphic. Thus, the goal of this study is to investigate the mechanisms by which kidney function of the mouse is modulated by sex and time of day. To accomplish this, we have developed the first computational models of epithelial water and solute transport along the mouse nephrons that represent the effects of sex and circadian clock on renal hemodynamics and transporter activity. We conduct simulations to study how the circadian control of renal transport genes affects overall kidney function, and how that process differs between male and female mice. Simulation results predict that tubular transport differs substantially among segments, with relative variations in water and Na+ reabsorption along the proximal tubules and thick ascending limb tracking that of GFR. In contrast, relative variations in distal segment transport are much larger, with Na+ reabsorption almost doubling during the active phase. Oscillations in Na+ transport drive K+ transport variations in the opposite direction. Model simulations of BMAL1 knockout mice predict a significant reduction in net Na+ reabsorption along the distal segments in both sexes, but more so in males than females. This can be attributed to the reduction of mean ENaC activity in males only, a sex-specific effect that may lead to a reduction in blood pressure in males.

Single-cell RNA sequencing (scRNAseq) is a crucial tool in kidney research. These technologies cluster cells according to transcriptome similarity, irrespective of the anatomical location and ordering within the nephron. Thus, a cluster transcriptome may obscure heterogeneity of the cell population within a nephron segment. Elevated dietary fructose leads to salt-sensitive hypertension, in part by fructose reabsorption in the proximal tubule (PT). However, organization of the four known fructose transporters in apical PTs (SGLT4, SGLT5, GLUT5 and NaGLT1) remains poorly understood. We hypothesized that cells within each subsegment of the proximal tubule exhibit complex, heterogenous fructose transporter expression patterns. To test this hypothesis we analyzed rat and kidney transcriptomes and proteomes from publicly available scRNAseq and tubule microdissection databases. We found that microdissected PT-S1 segments consist of 81{+/-}12% cells with scRNAseq-derived transcriptional characteristics of S1, whereas PT-S2 express a mixture of 18{+/-}9% S1, 58{+/-}8% S2, and 19{+/-}5% S3 transcripts, and PT-S3 consists of 75{+/-}9% S3 transcripts. The expression of all four fructose transporters was detectable in all three PT segments, but key fructose transporters SGLT5 and GLUT5 progressively increased from S1 to S3, and both were significantly upregulated in S3 vs. S1/S2 (Slc5a10: 1.9 log2FC, p<1x10-299; Scl2a5: 1.4 log2FC, p<4x10-105). A similar distribution was found in human kidneys. These data suggest that S3 is the primary site of fructose reabsorption in both humans and rats. Finally, because of the multiple scRNAseq transcriptional phenotypes found in each segment our findings also imply that anatomic labels applied to scRNAseq clusters may be misleading.

The ENaC gamma subunit is essential for homeostasis of Na+, K+, and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (Q4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5 in cultured unpolarized cells. Using biotinylation of cell-surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+.

Fructose high-salt (FHS) diets increase blood pressure (BP) in an angiotensin II (Ang II)-dependent manner. Ang II stimulates aldosterone release, which, by acting on the mineralocorticoid receptor (MR), regulates Na+ reabsorption by the aldosterone-sensitive distal nephron (ASDN). The MR can be transactivated by glucocorticoids, including those locally produced by 11{beta}-HSD1. The epithelial sodium channel (ENaC) is a key transporter regulated by MRs. We hypothesized that fructose-induced salt-sensitive hypertension depends in part on abnormal activation of MRs in the ASDN with consequent increases in ENaC expression. We found that aldosterone-upregulated genes in mice ASDN, significantly overlapped with 74 genes upregulated by FHS in the rat kidney cortex (13/74; p[&le;]1x10-8), and that these 74 genes are prominently expressed in rat ASDN cells. Additionally, the average z-score expression of mice-aldosterone-upregulated genes is highly correlated with FHS compared to glucose high-salt (GHS) in the rat kidney cortex (Pearson correlation; r=0.66; p[&le;]0.005). There were no significant differences in plasma aldosterone concentrations between the FHS and GHS. However, 11{beta}-HSD1 transcripts were upregulated by FHS (log2FC=0.26, p[&le;]0.02). FHS increased BP by 23{+/-}6 mmHg compared to GHS, and blocking MRs with eplerenone prevented this increase. Additionally, inhibiting ENaC with amiloride significantly reduced BP in FHS from 148{+/-}6 to 134{+/-}5 mmHg (p[&le;]0.019). Compared to GHS, FHS increased total and cleaved ENaC protein by 89{+/-}14 % (p[&le;]0.03) and 47{+/-}16 % (p[&le;]0.01) respectively. FHS did not change {beta}- or {gamma}-subunit expression. These results suggest that fructose-induced salt-sensitive hypertension depends, in part, on abnormal Na+ retention by ENaC, resulting from the activation of MRs by glucocorticoids.

BackgroundN-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood. Nonetheless, NMDAR inhibitors can increase serum K+ and reduce GFR, which suggests they have an important physiological role in the kidney. We hypothesized that NMDARs in the distal nephron induce afferent-arteriole vasodilation through the vasodilator mechanism connecting-tubule-glomerular feedback (CNTGF) that involves ENaC activation. Methods and resultsUsing a tubule-specific transcriptome database combined with molecular biology and microscopy techniques, we showed kidney expression of NMDAR subunits along the nephron and specifically in ENaC-positive cells. This receptor is expressed in both male and female mice, with higher abundance in females (p=0.02). Microperfusing NMDAR agonists into the connecting tubule induced afferent-arteriole vasodilation (EC50 10.7 vs. 24.5 mM; p<0.001) that was blunted or eliminated with the use of NMDAR blocker MK-801 or with the ENaC inhibitor Benzamil, indicating a dependence on CNTGF of the NMDAR-induced vasodilation. In vivo, we confirmed this CNTGF-associated vasodilation using kidney micropuncture (Stop-flow pressure 37.9{+/-}2.6 vs. 28.6{+/-}1.9 mmHg, NMDAR agonist vs vehicle; p<0.01). We explored NMDAR and ENaC channel interaction by using mpkCCD cells and split-open connecting tubules. We observed increased amiloride-sensitive current following NMDAR activation that was prevented by MK-801 (1.14 vs. 0.4 Amp; p=0.03). In split-open tubules, NMDAR activation increased ENaC activity (Npo Vehicle vs. NMDA; p=0.04). ConclusionNMDARs are expressed along the nephron, including ENaC-positive cells, with higher expression in females. Epithelial NMDAR mediates renal vasodilation through the connecting-tubule-glomerular feedback, by increasing ENaC activity.

Obesity is a risk factor for hypertension. Obesity-related hypertension has been associated with elevated plasma soluble prorenin receptor (sPRR) particularly in men. Additionally, renal PRR and sPRR protein expression is upregulated during obesity and diabetes. However, whether renal-derived human sPRR (HsPRR) may influence the intrarenal RAS status to regulate blood pressure and kidney function during obesity has not been investigated. Therefore, we studied the role of collecting duct (CD) derived-HsPRR on blood pressure and kidney function in male and female mice during obesity. Eight-week-old male and female CD-HsPRR mice were placed on a high fat diet for 25 weeks. HsPRR increased renal sPRR concentration but did not change its circulating levels in male and female littermates compared to CTL mice. GFR, water intake and urine flow were not influenced by the CD-HsPRR expression in either sex. Moreover, after 21 weeks of HFD, blood pressure was similar between groups, while only male CD-HsPRR mice showed an impaired pressor response to losartan. In the renal cortex, male CD-HsPRR mice showed increased renin and AT1R mRNA expression associated with increased AQP2, and ENaC subunits protein expression. These data indicate that renal-derived HsPRR induces local upregulation in renin, AT1R and sodium/water transporters in male mice without altering renal hemodynamics or blood pressure. In obese females, CD-HsPRR expression did not affect blood pressure or renal function, which suggests that females may be protected from obesity induced renal function impairment and hypertension.

Histology is the cornerstone of clinical pathology and an essential tool for many areas of medicine. Nevertheless, conventional histological methods, which rely on fixation, embedding, sectioning, and staining, distort cellular architecture, extract key molecules such as lipids, and introduce variability that severely limits reproducibility. Here, we present Normalized Raman Imaging (NoRI), a form of stimulated Raman scattering applied to organ pathology. NoRI enables quantitative, label-free measurements of proteins and lipids at high spatial resolution. NoRI overcomes heterogeneous light scattering from homogeneous tissues by computationally correcting each signal by the combined Raman signals of protein, lipid, and water. This enables quantitative biomass measurements while preserving tissue architecture, thereby facilitating advanced analysis by convolutional neural networks and feature discovery. Here we apply NoRI to the mouse kidney, showing that such imaging can be used to accurately classify tubule types (median F1 [harmonic mean of precision and recall against manual annotation]=0.93), anatomical regions (F1=0.91), and biological sex (F1=0.97) from regions as small as 132.5{micro}m{superscript 2}. Under these circumstances, NoRI has revealed novel sex-specific features, including higher cytoplasmic lipid (+6.9mg/mL; p=0.028), nuclear protein (+26.3mg/mL; p<0.001), and capillary protein concentrations (+3.1mg/mL; p<0.001) in female tubules, along with differences in intracellular lipid droplet morphology. In a time-course model of acute kidney injury (AKI), NoRI captured dynamic changes in protein and lipid organization, most pronounced at day 2 post-injury (F1=0.97), and quantified recovery of brush border structures and lipid droplets over 25 days. Lipid measurements were particularly critical for the high accuracy of feature classification and discovery in AKI (F1=1.0). These results establish NoRI as a reproducible, high-resolution, and fully quantitative framework for tissue analysis and feature discovery, far surpassing conventional histology. By preserving tissue architecture and accurately quantifying lipids and proteins, NoRI provides a unique platform to explore and identify unknown biological phenomena in complex tissues, and present as a powerful diagnostic tool for histopathology.

BackgroundLymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. MethodsWe utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy) that abrogates its kinase ability. Vegfr3Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using a combination of tissue clearing, light-sheet microscopy and computational analyses. ResultsIn the kidney, we found Vegfr3 is expressed not only in lymphatic vessels, but also various blood vessels. Vegfr3Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in blood pressure, renal function and histology. Similarly, there was no difference in the degree of renal injury after cisplatin, although Vegfr3Chy/+ mice developed more perivascular inflammation by histology. Control mice treated with cisplatin had a measurable increase in cortical lymphatic density despite no change in cortical lymphatic volume and length. ConclusionsWe demonstrate that Vegfr3 is required for development of renal lymphatics, but a reduction in lymphatic density does not alter renal function and induces only modest histological changes after injury. Our data suggests that an increase in lymphatic density after cisplatin injury may reflect the loss of cortical volume associated with chronic kidney disease rather than growth of lymphatic vessels. SIGNIFICANCE STATEMENTDefects in renal lymphatics occur in various kidney diseases, but their role in maintaining kidney structure and function is unknown. We combine tissue clearing, light-sheet microscopy and computational analysis to characterize lymphatics and find that mice with a heterozygous mutation in Vegfr3 (Vegfr3Chy/+) have severely reduced renal lymphatics. Strikingly, these mice have indistinguishable renal function and histology compared with controls. Even after cisplatin injury, there are no differences in renal function, although Vegfr3Chy/+ mice developed more perivascular inflammation. Our data present a novel method of lymphatic quantification and suggest that a normal complement of renal lymphatics is dispensable for renal structure and function.

The kidney regulates extracellular fluid and electrolyte homeostasis. Its function is modulated by the renin-angiotensin-aldosterone system (RAAS). Overactivation of the RAAS, such as in chronic Ang II infusion, promotes vasoconstriction, anti-natriuresis, and hypertension. Kidney function is also modulated by circadian clocks. Indeed, glomerular filtration rate, filtered electrolyte loads, urine volume, and urinary excretion all exhibit notable diurnal rhythms, which reflect, in part, the regulation of renal transporter proteins by circadian clock genes. Key renal transporters that are regulated by clock gene proteins include sodium-hydrogen exchanger 3 (NHE3), sodium-glucose cotransporter 1 (SGLT1), Na+-K+-2Cl- cotransporter (NKCC2), Na+-Cl- cotransporter (NCC), and epithelial sodium channel (ENaC), which are targeted by diuretics used to treat hypertension. The objective of the present study was to assess the effect of administration time on the natriuretic and diuretic effects of loop, thiazide, and K+-sparing diuretics, which are common treatments for hypertension, and how those effects differ between a normotensive and hypertensive kidney. Loop diuretics inhibit NKCC2 on the apical membrane of the thick ascending limb; notably, In Ang II-induced hypertension, NKCC2 is differentially regulated along the medullary versus cortical thick ascending limb. Thiazide diuretics inhibit NCC on the distal convoluted tubule, and K+-sparing diuretics inhibit ENaC on the connecting tubule and collecting duct. We simulated Na+ transporter inhibition using computational models of kidney function at different times of day. The simulation results predicted qualitatively similar diurnal oscillations in segmental transport in the normotensive and hypertensive kidneys, and highlighted significant time-of-day differences in the natriuresis, diuresis, and kaliuresis responses. NEW & NOTEWORTHYChronic infusion of angiotensin II promotes vasoconstriction, salt retention, and hypertension. Blood pressure and kidney function exhibit circadian rhythms. Given the diurnal variations in the expression levels of key renal electrolyte transporters, how do the natriuretic and diuretic effects of diuretics, a common treatment for hypertension that targets renal transporters, vary during the day? To answer this question, we simulated Na+ transporter inhibition using computational models of kidney function at different times of day.

BackgroundRenal sodium reabsorption occurs via both transcellular and paracellular pathways. Tight junction proteins play a key role in mediating paracellular transport. The collecting duct (CD) is critical for the fine-tuning of Na+ balance and is sensitive to changes in dietary salt intake. A low-sodium diet, which increases endogenous aldosterone secretion, stimulates transcellular sodium transport via epithelial Na+ channels (ENaC) and Na,K-ATPase. We hypothesized that a low-sodium diet also modulates paracellular Na+ permeability by regulating the expression or function of claudin-3, a major tight junction protein in the CD, in order to limit the back-leak of reabsorbed sodium and preserve sodium balance. MethodsWe used in vivo mouse models and cultured mouse CD principal cells (mCCDcl1) to assess aldosterones effects on tight junction proteins. In mCCDcl1 cells, aldosterone-induced changes in claudin-3 expression and localization were evaluated via Western blotting and immunofluorescence, and Ussing chamber assays were used to assess paracellular Na+ and Cl- permeability after modulating claudin-3 expression. Wild-type and claudin-3 knockout mice were fed low (0.01%) or normal (0.18%) sodium diets for seven days. In subsets of low sodium diet mice, spironolactone (a mineralocorticoid receptor antagonist) was administered. ResultsIn mice, a low-sodium diet upregulates renal claudin-3 expression. Concordantly, in vitro studies using mCCDcl1 cells showed that aldosterone treatment increased claudin-3 protein levels and promoted its localization to the lateral membrane. Functional analyses demonstrated that claudin-3 overexpression reduced paracellular permeability to both Na+ and Cl-, while claudin-3 silencing increased it. Claudin-3 knockout mice subjected to a low-sodium diet exhibited compensatory upregulation of the - and {gamma}-subunits of ENaC, alongside increased expression of claudin-4, claudin-8, and claudin-10. This highlights an adaptive response that maintains sodium homeostasis in the absence of claudin-3. Importantly, this compensatory mechanism persists even under spironolactone treatment, suggesting that the adaptation of claudin-3-deficient mice occurs independently of mineralocorticoid receptor activation. ConclusionsOur findings demonstrate that aldosterone enhances claudin-3 expression, reinforcing the paracellular barrier to Na+ and complementing its classical role in transcellular Na+ transport. Under low-sodium conditions, claudin-3-deficient mice adapt through complementary mechanisms aimed at increasing sodium reabsorption via ENaC activation and upregulation of claudin-4 and claudin-8, both barrier-forming claudins that restrict paracellular sodium leakage in the CD. This is associated with increased claudin-10 abundance in the thick ascending limb of Henle, a pore-forming claudin that facilitates paracellular sodium permeability. This study advances our understanding of the complex control of renal sodium handling, revealing adaptive mechanisms in response to low-salt diet and claudin-3 deficiency.

We reported that Shroom3 knockdown, via Fyn inhibition, induced albuminuria with foot process effacement (FPE) without glomerulosclerosis (FSGS) or podocytopenia. Interestingly, knockdown mice had reduced podocyte volumes. Human minimal change disease, where podocyte Fyn inactivation was reported, also showed lower glomerular volumes than FSGS. We hypothesized that lower glomerular volume prevented the progression to podocytopenia. To test this hypothesis, we utilized unilateral- and 5/6th nephrectomy models in Shroom3 knockdown mice. Knockdown mice exhibited lower glomerular volume, and less glomerular and podocyte hypertrophy after nephrectomy. FYN-knockdown podocytes had similar reductions in podocyte volume, implying Fyn was downstream of Shroom3. Using SHROOM3- or FYN-knockdown, we confirmed reduced podocyte protein content, along with significantly increased phosphorylated AMP-kinase, a negative regulator of anabolism. AMP-Kinase activation resulted from increased cytoplasmic redistribution of LKB1 in podocytes. Inhibition of AMP-Kinase abolished the reduction in glomerular volume and induced podocytopenia in mice with FPE, suggesting a protective role for AMP-Kinase activation. In agreement with this, treatment of glomerular injury models with AMP-Kinase activators restricted glomerular volume, podocytopenia and progression to FSGS. In summary, we demonstrate the important role of AMP-Kinase in glomerular volume regulation and podocyte survival. Our data suggest that AMP-Kinase activation adaptively regulates glomerular volume to prevent podocytopenia in the context of podocyte injury.

Dietary treatment is seminal for management of chronic kidney disease (CKD). The aim of our project was to assess the effects of potassium intake on the progression of CKD. We used 2 mouse CKD models to analyze the effects of potassium intake on CKD : the unilateral ureteral obstruction (UUO) and the POD-ATTAC models. POD-ATTAC mice display a podocyte-specific apoptosis after the administration of a chemical inducer. We also studied the effect of mineralocorticoid receptor (MR) using UUO in kidney tubule-specific MR knockout mice. In both UUO and POD-ATTAC mice, high potassium diet increased interstitial fibrosis. High potassium diet also increased the abundance of the extracellular matrix protein fibronectin and decreased the abundance of the epithelial marker Na+-K+ ATPase. Consistently, POD-ATTAC mice fed with high potassium diet displayed lower glomerular filtration rate. Spironolactone, a MR antagonist, decreased fibrosis induced by high potassium diet in POD-ATTAC mice. However, kidney tubule-specific MR knockout did not improve the fibrotic lesions induced by UUO under normal or high potassium diets. Macrophages from high potassium-fed POD-ATTAC mice displayed higher mRNA levels of the pro-inflammatory chemokine MCP1. This effect was decreased by spironolactone, suggesting a role of MR signaling in myeloid cells in the pro-fibrotic effect of potassium-rich diet. High potassium intake generates more fibrosis leading to decreased kidney function in experimental CKD. MR signaling plays a pivotal role in this potassium-induced fibrosis. The effect of reducing potassium intake on CKD progression should be assessed in future clinical trials. Translational statementDietetic approach is a cheap and effective therapy to slow down the development of chronic kidney diseases and kidney fibrosis. Potassium-rich diets are protective against renal and cardiovascular events in the general population, albeit some conflicting data were obtained in patients with chronic kidney disease. We showed that potassium-rich diet accelerates fibrosis development, by enhancing kidney inflammation in two mouse models of chronic kidney disease. These data suggest that potassium-rich diets should not be advised in patients with chronic kidney disease, unless future clinical trials demonstrate any beneficial effect in these patients.

Vasopressin regulates transcription of the aquaporin-2 gene (Aqp2) in collecting duct principal cells. To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP, a vasopressin analog, these cells exhibited low or undetectable GFP and Aqp2 expression in all cells. dDAVP stimulation (1nM dDAVP for 48hrs) markedly increased both GFP and Aqp2 expression together with reversal upon dDAVP removal. These observations demonstrate that GFP faithfully tracks Aqp2 expression. Interestingly, fewer than 50% of cells express GFP and Aqp2 after dDAVP or forskolin, indicating significant variability even though they were clonally-derived. We flow-sorted the GFP- cells (Aqp2-) and GFP+ cells (Aqp2+), regrew them, and restimulated them separately with dDAVP. Cells originating from GFP- cells gave rise to both GFP- cells and GFP+ cells, and GFP+ cells similarly regenerated both GFP- and GFP+ populations in the same proportion. Flow cytometry analysis of the DNA content showed variability in cell cycle phases, with most GFP+ cells in G0/G1, and more GFP cells in G2/S. RNA-seq analysis of the GFP- and GFP+ cells revealed increased abundance of cell-cycle related transcripts in the GFP- cells. We conclude that: 1) heterogeneity in Aqp2 expression is related to cell cycle state; and 2) the newly generated reporter cell line will likely serve as a useful tool to study Aqp2 transcriptional regulation. NEW & NOTEWORTHYTo investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. We demonstrate that the GFP reporter accurately and dynamically tracks the expression and regulation of endogenous Aqp2. We reveal that Aqp2 heterogeneity in mpkCCD cells is at least partly driven by differences in cell cycle phase.

BackgroundPhosphoproteomics studies in both cultured and native collecting duct (CD) cells showed that vasopressin strongly increases PKA-dependent phosphorylation of {beta}-catenin at Ser552. Relatively little is known about the role of Ser552 phosphorylation. MethodsTo address the role of {beta}-catenin Ser552 phosphorylation in the mature renal CD, we have inserted a Ser552Ala mutation in mice using CRISPR-Cas9. ResultsThe mutation did not affect the renal abundance of the vasopressin-regulated water channel aquaporin-2 (AQP2) or urinary osmolality. However, the structure of the CD system was altered. Specifically, the cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 {+/-} 0.66 in control mice to 3.33 {+/-} 0.82 in Ser552Ala mutant mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between control and Ser552Ala mutant mice (both [~]13,500 per kidney). RNA-seq in microdissected cortical CDs of the mice revealed a highly significant enrichment of genes involved in regulation of mitosis and the cell cycle, along with decreases in mRNAs coding for two CDK inhibitor proteins, Cdkn1b and Cdkn1c. At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. ConclusionsThe observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of {beta}-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation. We speculate that vasopressin may act to slow or halt branching morphogenesis perinatally and may affect the collecting duct elongation process that normally produces the unbranched region of the CD system in the cortex and outer medulla. Key pointsO_LIVasopressin regulates collecting duct (CD) transport by triggering phosphorylation of multiple proteins including {beta}-catenin at Ser552. C_LIO_LIMutating Ser552 to a non-phosphorylable amino acid in mice resulted in altered CD branching without loss of differentiation in adult CDs. C_LIO_LIThe findings point to a role for {beta}-catenin Ser552 phosphorylation in CD branching and sub-segmental CD elongation. C_LI

Common intronic enhancer SNPs in Shroom3 associate with CKD in GWAS, although there is paucity of detailed mechanism. Previously, we reported a role for Shroom3 in mediating crosstalk between TGF{beta}1- & Wnt/Ctnnb1 pathways promoting renal fibrosis (TIF). However, beneficial roles for Shroom3 in proteinuria have also been reported suggesting pleiotropic effects. Here we focused on identifying the specific profibrotic Shroom3 motif. Given known therapeutic roles for Rho-kinase inhibitors in experimental CKD, and the established interaction between Shroom3 and Rock via its ASD2 domain, we hypothesized that Shroom3-mediated ROCK activation played a crucial role in its profibrotic function in high expressors. To test this hypothesis, we developed transgenic mice and cell lines that inducibly overexpressed wild-type- (WT-Sh3) or ASD2-domain deletion- Shroom3 (ASD2{Delta}-Sh3). Prior scRNAseq data showed that during TIF, Shroom3 and Rock co-expression occurred in injured tubular cells and fibroblasts, highlighting cell-types where this mechanism could be involved. Using HEK293T cells, we first confirmed absent ROCK binding and inhibited TGF{beta}1-signaling with ASD2{Delta}-Sh3-overexpression vs WT-Sh3. In mIMCD cells, ASD2{Delta}-Sh3 overexpression, reduced Rock activation (phospho-MYPT1), pro-fibrotic and pro-inflammatory transcripts vs WT-Sh3. Fibroblast proliferation (3T3) was also reduced with ASD2{Delta}-Sh3. In vivo, we studied ureteric obstruction (UUO) and Aristolochic nephropathy (AAN) as TIF models. In AAN, inducible global-, or Pan-tubular specific-, WTSh3-overexpression showed increased azotemia, and TIF vs ASD2{Delta}-Sh3 mice. WT-Sh3 mice consistently showed significant enrichment of Rho-GTPase, TGF{beta}1- and Wnt/CtnnB1- signaling in kidney transcriptome, paralleling Shroom3-coexpressed genes in tubulo-interstitial transcriptomes from human CKD. In UUO, again WT-Sh3 mice recapitulated increased fibrosis vs ASD2{Delta}-Sh3. Importantly, ASD2{Delta}-Sh3 did not develop albuminuria vs WT-Sh3, while mutating a disparate Fyn-binding Shroom3 motif induced albuminuria in mice, suggesting motif-specific roles for Shroom3 in the kidney. Hence, our data show a critical role for the Rock-binding, ASD2-domain in mediating TIF in milieu of Shroom3 excess, with relevance to human CKD.

Chronic kidney disease (CKD) is a progressive disorder marked by a decline in kidney function. Obesity and sedentary behavior contribute to the development of CKD, though mechanisms by which this occurs are poorly understood. This knowledge gap is worsened by the lack of a reliable murine CKD model that does not rely on injury, toxin, or gene deletion to induce a reduction in kidney function. High-fat diet (HFD) feeding alone is insufficient to cause reduced kidney function until later in life. Here, we employed a small mouse cage (SMC), a recently developed mouse model of sedentariness, to study its effect on kidney function. Wildtype C57BL/6J male mice were housed in sham or SMC housing for six months with HFD in room (22{degrees}C) or thermoneutral (30{degrees}C) conditions. Despite hyperinsulinemia induced by the SMC+HFD intervention, kidneys from these mice displayed normal glomerular filtration rate (GFR). However, the kidneys showed early signs of kidney injury, including increases in Col1a1 and NGAL transcripts, as well as fibrosis by histology, primarily in the inner medullary/papilla region. High-resolution respirometry and fluorometry experiments showed no statistically significant changes in the capacities for respiration, ATP synthesis, or electron leak. These data confirm the technical challenge in modeling human CKD. They further support the notion that obesity and a sedentary lifestyle make the kidneys more vulnerable, but additional insults are likely required for the pathogenesis of CKD.

Polycystic kidney diseases (PKD) are genetic disorders characterised by the formation of fluid-filled cysts, which disrupt kidney architecture and function. Autosomal recessive PKD (ARPKD) is a rare form of PKD, caused by mutations in PKHD1, and clinically more severe than the more common autosomal dominant PKD (ADPKD). Prior studies have implicated the ciliary-located Hedgehog (Hh) pathway in ADPKD, with increased levels of Hh components in experimental ADPKD models, and reduced cystogenesis following pharmacological Hh inhibition. In contrast, the role of the Hh pathway in ARPKD is poorly understood. We hypothesised that Hh pathway activity would be elevated during ARPKD pathogenesis, and its modulation may inhibit cystogenesis, akin to prior findings in ADPKD. To test this, we utilised Cpk mice, a model which replicates the pathophysiology of ARPKD, and generated a human cellular ARPKD 3-dimensional cystogenesis model by mutating PKHD1 in human collecting duct cells through CRISPR-Cas9 technology. We found significantly elevated levels of the Hh transcriptional effector Gli3 in the Cpk mouse, a finding replicated in our human cellular ARPKD model. In the Cpk mouse, we also observed an increase in total GLI3 and GLI3 repressor protein levels. However, reduction of increased Gli3 levels via genetic deletion in the Cpk mouse did not affect cyst formation. Similarly, lowering GLI3 transcript to wildtype levels, did not influence cyst size in our human cellular ARPKD model. Collectively, these data show that elevated Gli3 does not modulate cyst progression in the context of ARPKD, highlighting the complexity of the Hh pathway in PKD. New and NoteworthyThe role of the Hedgehog pathway in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood. Here, we describe elevated levels of Gli3, the Hedgehog transcriptional effector, in murine and human ARPKD models. However, reversal of the increase in Gli3 did not significantly affect cystogenesis in a human cell model of ARPKD or disease progression in a mouse model which replicates ARPKD pathophysiology. Collectively, our data indicates that Gli3 does not modulate ARPKD progression.

Podocytes are highly specialized epithelial cells of the glomerular filtration barrier essential for maintaining proper kidney function. Their intricate cellular projections, called foot processes, wrap around the capillary endothelium, interdigitate, and connect to one another via slit diaphragm intercellular junctions to form a sieve-like barrier. Compromise to this unique podocyte architecture can lead to altered glomerular function and eventual kidney disease. We previously identified Immunoglobulin-like domain containing receptor 2 (Ildr2) as a component of the podocyte foot process through proteomic analyses. Ildr2 is a tricellular tight junction constituent and localizes to distinct puncta in podocytes that likely represent these specialized junctions. However, the significance of tricellular tight junctions and Ildr2 to podocyte integrity is unknown. To this end, we generated a conditional knockout mouse with podocyte-specific deletion of Ildr2 (Podocin-Cretg/+;Ildr2fl/flor Ildr2PodKO). Kidneys from Ildr2PodKO adult animals show disruptions to podocyte foot process architecture including effacement and the unique formation of electron dense strands connecting subsets of processes. Additionally, the glomerular basement membrane was significantly thicker in conditional knockouts and histological analyses revealed glomerular collagen and glycoprotein accumulation. Surprisingly, Ildr2PodKO animals displayed no significant albuminuria. When Ildr2PodKO mice were challenged with a stress to kidney function, immunohistochemistry revealed podocyte loss from a subset of glomeruli with concomitant detection of podocytes in urine, although function was still preserved. Collectively, our data provide novel insights into the function of Ildr2 and suggest tricellular junctions help preserve podocyte architecture but are likely not necessary to maintain proper filtration in no or low stress physiological states.