American Journal of Physiology-Renal Physiology

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder marked by numerous renal cysts that impair kidney function, with about half of affected individuals progressing to kidney failure by midlife. Patients exhibit reduced circulating apelin, a ligand of the apelin receptor, known to regulate cardiovascular function including hypertension. We tested whether diminished apelin signaling contributes to cystogenesis and if exogenous apelin receptor activation can improve disease outcomes. MethodsPlasma samples from age- and sex-matched healthy controls and ADPKD participants were analyzed for circulating apelin peptides. To assess direct cystic effects, primary ADPKD renal epithelial cells were grown as 3D collagen-embedded cysts and treated with apelin agonists. Male and female Pkd1RC/RC; Pkd2+/- (PKD) mice were treated for 27 days with apelin agonists, vehicle, or the standard of care drug, Mozavaptan. Kidney and heart weight ratios, BUN, renal cAMP, and kidney transcriptional profiles were evaluated. ResultsCirculating apelin peptides were significantly reduced in ADPKD patients despite normal kidney function (eGFR, BUN, and creatinine). In vitro, both apelin and the small molecule apelin receptor agonist Azelaprag inhibited cyst growth. Apelin and Mozavaptan reduced kidney weight, cystic index, blood urea nitrogen and renal cAMP in PKD mice, whereas Azelaprag did not. Apelin downregulated expression of genes associated with cyst progression, including Lcn2 (Ngal), Postn, and Havcr1 (Kim-1). Mozavaptan, but not apelin, induced diuresis and reduced urinary concentration. ConclusionApelin receptor activation by exogenous apelin inhibited cAMP synthesis and cyst growth and improved kidney function in an orthologous mouse model of ADPKD. We propose that the apelin receptor may be a potential therapeutic target in ADPKD.

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.

IntroductionThe podocyte slit diaphragm (SD) is a complex filtration unit localized to the blood and urine interface and governs the glomerular selectivity. However, the greater details of the SD composition and the mechanism of assembly of the SD protein as a macromolecular complex remain elusive. CD2-associated protein (CD2AP) serves as a central scaffold within the SD, and mutations in CD2AP are strongly associated with nephrotic syndrome (NS) and focal segmental glomerulosclerosis (FSGS). However, the mechanisms by which such mutations alter the architecture and higher-order organization of CD2AP are poorly understood. MethodsWe employed biophysical, structural, and proteomic approaches to investigate the impact of the disease-associated K301M mutation on CD2AP structure and its interaction with Podocin. Oligomerization was analyzed using size-exclusion chromatography, blue native PAGE, Dynamic light scattering, and small-angle X-ray scattering. Secondary and tertiary structural properties were assessed by far- and near-UV circular dichroism, thermal denaturation, and intrinsic fluorescence spectroscopy. CD2AP-podocin interactions were quantified using in vitro pulldown and surface plasmon resonance (SPR), and mutation-dependent changes in interaction networks were examined through interactome profiling. ResultsWild-type (WT) CD2AP assembled into flexible higher-order oligomers ([~]9-12-mers), whereas the K301M variant collapsed into lower-order species ([~]3-6-mers), indicating destabilization of the coiled-coil assembly interface. Spectroscopic analyses revealed subtle secondary-structure rearrangements, but profound tertiary packing defects, as well as reduced and markedly diminished thermal resilience in the mutant. SPR analysis demonstrated loss of binding between Podocin and mutant CD2AP, whereas WT CD2AP showed high-affinity interaction (KD = 211 nM) with Podocin. Complementary interactome profiling revealed widespread rewiring of protein-protein interactions in the case of mutant CD2AP, characterized by the loss of core partners and the emergence of aberrant associations. ConclusionThese findings define a mechanistic model in which the K301M mutation destabilizes CD2AP oligomerization, disrupts podocin recognition, and remodels interaction networks essential for SD stability. This work signifies the importance of CD2AP in SD assembly and the permselective filtration function of the kidney, and the impact of a single mutation in the pathogenesis of NS and FSGS. Translational Statement: Inherited nephrotic syndrome, characterized by heavy proteinuria, frequently arises from mutations in scaffolding proteins of the slit-diaphragm (SD). This study demonstrates that the nephrotic syndrome-associated K301M mutation in CD2-associated protein (CD2AP) compromises higher-order oligomerization, abolishes its binding to the binding partner (Podocin), and reshapes protein-protein interaction networks that are critical for SD assembly and stability. By establishing a direct link between mutation-induced collapse of CD2AP architecture and loss of podocyte scaffolding function, these findings provide mechanistic insight into the pathogenesis of CD2AP-associated proteinuric kidney disease. The results further identify oligomeric assembly interfaces as potential targets for therapeutic strategies aimed at preserving the integrity of the SD and glomerular filtration function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/698362v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1f49f1dorg.highwire.dtl.DTLVardef@fa2e06org.highwire.dtl.DTLVardef@e399c0org.highwire.dtl.DTLVardef@831c17_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

BackgroundLymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease. As injury related lymphangiogenesis is driven by signaling from the receptor VEGFR-3 in response to the cognate growth factor VEGF-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remains poorly defined. MethodsWe generated a new mouse model expressing Vegf-C under regulation of the nephron progenitor Six2Cre driver strain (Six2Vegf-C). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and micro computed tomography 3-dimensional imaging. ResultsSix2Vegf-C mice had reduced body weight and kidney function compared to littermate controls. Six2Vegf-C kidneys demonstrated large peripelvic fluid filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. 3D imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+/PDPN+/VEGFR3+ lymphatic capillaries extending alongside EMCN+ peritubular capillaries. There was no change in EMCN+ peritubular capillary density. ConclusionsKidney lymphangiogenesis was robustly induced in the Six2Vegf-C mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR-3. The model resulted in a severe cystic kidney phenotype that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGF-C signaling during kidney development and provides new insight into a mimicker of human cystic kidney disease.

BackgroundNephrotic syndrome (NS) is characterized by massive sodium chloride retention. Along the kidney tubule, sodium and chloride reabsorption are coupled via a combination of transcellular and paracellular transport pathways. The mechanism of sodium retention in NS has been extensively studied, but the associated chloride transport pathway has not been elucidated. MethodsTo investigate the pathway of chloride retention in NS, we assessed the expression levels of both paracellular and transcellular components of chloride transport in the CD of POD-ATTAC mice and PAN rats, two rodent models of NS. We also used cultured mouse cortical collecting duct cells to see how overexpression or silencing of claudin-4 affect paracellular permeability. Finally, human renal biopsies were used to confirm our in vivo results. ResultsIn control animals, claudin-4 was expressed at low levels in collecting duct (CD). In POD-ATTAC mice and PAN rats, claudin-4 expression was strongly increased in CD beta-intercalated cells (B-IC) and to a lesser extent in CD principal cells and was also induced in connecting tubules. Similarly, we found that claudin-4 was expressed at low levels in normal human kidneys and was dramatically increased in CD cells of nephrotic human kidneys (focal and segmental glomerulosclerosis). In parallel, the expression of pendrin, which exchanges chloride for bicarbonates in B-IC, was decreased in nephrotic compared to control animals. However, the increase in claudin-4 expression observed in NS is likely independent of pendrin abundance. Increased claudin-4 abundance is coupled with increased ENaC-dependent sodium transport. Overexpression or silencing of claudin-4 in mCCDcl1 cells confirmed the preferential permeability of claudin-4 to chloride over sodium. ConclusionsThese results suggest that during NS, transcellular Cl-/HCO - transport decreases while paracellular chloride transport via claudin-4 may increase along the collecting system. Paracellular chloride permeability may constitute a chloride shunt that favors Na+ reabsorption and opposes K+ secretion along the CD in NS. Significance StatementNephrotic syndrome is a common disease characterized by massive proteinuria, hypoalbuminemia and edema due to renal sodium-chloride retention. We demonstrate for the first time an induction of claudin-4 expression indicating a partial shift from transcellular to paracellular chloride transport in the renal collecting system of nephrotic rodents. We confirmed the increased expression of claudin-4 in kidney biopsies of nephrotic patients, highlighting the translational significance of these results. Whether the paracellular pathway may represent a novel target to treat edema in nephrotic syndrome remains to be elucidated.

Intercalated cells (ICs) of the renal collecting duct are traditionally recognized for their role in acid-base homeostasis, but growing evidence suggests they also participate in innate immune defense. Although ICs have been implicated in renal antimicrobial function, their specific role in coordinating immune responses during urinary tract infection (UTI) remains unclear. Using Ae1 R607H knock-in mice, a distal renal tubular acidosis (dRTA) model with A-intercalated cell (A-IC) dysfunction, we examined the renal response to uropathogenic Escherichia coli (UPEC). Mice with A-IC dysfunction exhibited higher bacterial loads 24 h post-infection and increased renal expression of antimicrobial peptides lipocalin-2 (Lcn2), galectin-3 (Lgals3), and cathelicidin-related antimicrobial peptide (Camp). Pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-1{beta} (IL-1{beta}) were elevated at both transcript and protein levels, whereas tumor necrosis factor- (TNF-) increased only at the protein level. Interleukin-10 (IL-10) showed a modest rise in mRNA. Chemokines C-X-C motif chemokine ligand 2 (Cxcl2) and C-C motif chemokine ligand 2 (Ccl2) were also upregulated, accompanied by excessive neutrophil infiltration and a marked shift in renal myeloid-cell composition. A-IC dysfunction therefore disrupts epithelial-immune homeostasis, resulting in exaggerated inflammation and impaired immune resolution. These findings identify A-ICs as essential epithelial immunomodulators that integrate antimicrobial defense, cytokine regulation, and immune-cell recruitment during UTI.

Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we used a multi-omic approach in a mouse unilateral nephrectomy model to identify signaling processes associated with compensatory hypertrophy of the renal proximal tubule. Morphometry applied to microdissected proximal tubules showed that growth of the proximal tubule involves a marked, rapid increase in cell volume rather than cell number. Measurements of DNA accessibility (ATAC-seq), transcriptome (RNA-seq) and proteome (quantitative protein mass spectrometry) independently identified patterns of change that are indicative of activation of the lipid-regulated transcription factor, PPAR. Activation of PPAR by fenofibrate administration increased proximal tubule cell size, while genetic deletion of PPAR in mice decreased it. The results indicate that PPAR is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.

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.

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.

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.

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.

Dipeptidyl peptidase 4 (DPP4) is a transmembrane serine exopeptidase abundantly expressed in the kidneys, predominantly in the proximal tubule (PT); however, its non-enzymatic functions in this nephron segment remain poorly understood. While DPP4 physically associates with the Na+/H+ exchanger isoform 3 (NHE3) and its inhibitors exert natriuretic effects, the DPP4 role in blood pressure (BP) regulation remains controversial. This study investigated the effects of PT-specific Dpp4 deletion (Dpp4{Delta}PT) and global Dpp4 deletion (Dpp4-/-) on systolic blood pressure (SBP), natriuresis, and NHE3 regulation under baseline and angiotensin II (Ang II)-stimulated conditions in both male and female mice. Global and PT-specific Dpp4 deletion increased diuretic and natriuretic responses to acute saline loading, correlating with enhanced phosphorylation of NHE3 at serine 552 (pS552-NHE3). However, baseline SBP remained unchanged. Ang II stimulation increased DPP4 activity in control mice, with a greater effect in males than in females, reflecting sex-dependent regulation of renal DPP4. In Dpp4{Delta}PT mice, residual kidney DPP4 was unresponsive to Ang II, indicating that PT DPP4, rather than DPP4 in other nephron segments, is regulated by Ang II. Ang II administration increased SBP in all groups; however, the pressor response was significantly attenuated in both Dpp4{Delta}PT and Dpp4-/- mice, coinciding with sustained elevated levels of pS552-NHE3. Collectively, these findings demonstrate that PT DPP4 modulates NHE3 activity through mechanisms that prevent the accumulation of pS552-NHE3, exerting an anti-natriuretic effect. In the absence of DPP4, these mechanisms are disrupted, reducing Ang II sensitivity and maintaining high pS552-NHE3 levels, underscoring the role of DPP4 in PT signaling and function.

Chronic kidney disease (CKD) affects over 850 million people worldwide and is characterized by progressive renal fibrosis driven by activated interstitial fibroblasts. Signaling by extracellular nucleotides and P2 receptors plays an important role in renal pathophysiology, yet its contribution to fibroblast activation and fibrosis remains poorly understood. Here, we investigated the expression and function of Gq/11-coupled P2Y receptors in renal interstitial fibroblasts and their involvement in experimental kidney fibrosis. Using highly selective RNA in situ hybridization, we detected P2Y1 (P2ry1) and P2Y6 (P2ry6) receptor expression in interstitial fibroblasts. Notably, P2Y6 expression was markedly upregulated in several experimental mouse models of renal fibrosis. Functional assays in primary cultured renal fibroblasts confirmed Gq/11-coupled P2Y receptor activity, as evidenced by transient intracellular Ca2+ elevations upon nucleotide stimulation. Primary cultured renal fibroblasts exhibited enhanced migration in response to extracellular uridine diphosphate (UDP). To assess the contribution of interstitial P2Y6 receptors to fibrosis progression, we employed an adenine-induced nephropathy model with or without the selective P2Y6 antagonist MRS2578. Pharmacological inhibition of P2Y6 significantly reduced the mRNA expression of the myofibroblast marker -smooth muscle actin and collagen I. Collectively, these findings suggest that upregulated P2Y6 receptor signaling promotes the transition of resident interstitial cells into myofibroblasts during renal fibrosis, likely by modulating fibroblast migration. Inhibition of P2Y6 signaling could represent a new strategy for reducing excessive renal fibrosis. TRANSLATIONAL STATEMENTThis study reveals the role of the P2Y6 receptor (P2ry6) in fibrotic processes in the kidney. P2Y6, a Gq/11 protein-coupled UDP-sensitive receptor, is expressed in renal interstitial PDGFR-{beta}-positive cells and macrophages. Its pharmacological inhibition significantly reduces fibrosis in the mouse adenine nephropathy model. Blocking P2Y6 therefore represents a promising therapeutic strategy for kidney diseases characterized by excessive scarring.

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.

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.

BackgroundThe distal convoluted tubule (DCT) and connecting tubule (CNT) are critical for fine-tuning electrolyte reabsorption and maintaining renal homeostasis. While the Hippo pathway effector Yap is known to regulate kidney development, its specific role within the distal nephron segments remains unknown. MethodsTo investigate Yap function in the distal nephron segments, we generated a Cre-inducible Yap gain-of-function allele (Col1a1-Yap5SA) and a distal nephron-specific Slc12a3Cre to drive an active form of Yap in the distal nephron segments. We performed phenotypic assessments along with molecular and transcriptomic analyses to examine changes in epithelial organization and nephron segmentation. ResultsLineage tracing showed that Slc12a3Cre targets both DCT and CNT, suggesting that CNT cells arise from Slc12a3+ DCT cells. Constitutive Yap activation in these segments caused increased proliferation and ectopic cell migration into adjacent nephron segments. This was accompanied by disrupted expression of DCT/CNT marker genes, loss of apicobasal polarity, and compromised junctional architecture, indicating epithelial-to-mesenchymal transition. Notably, mutant kidneys also exhibited downregulation of proximal tubule markers, indicating a non-cell-autonomous effect. ConclusionsOur findings demonstrate that sustained Yap activation in the distal nephron segments disrupts epithelial identity and structure while also exerting non-cell-autonomous effects on nephron patterning, particularly in the proximal tubule. These results underscore the importance of tightly regulated Hippo-Yap signaling in maintaining epithelial integrity and proper nephron segmentation. Key PointsO_LIA novel Slc12a3Cre targets both distal and connecting tubules in the mouse kidney, revealing the developmental origin of connecting tubules. C_LIO_LIConstitutive Yap activation in distal nephron segments disrupts their segmental identity, leading to epithelial-to-mesenchymal transition. C_LIO_LIConstitutive Yap activation in distal nephron segments suppresses expression of proximal tubule-specific genes. C_LI

BackgroundPolycystic kidney disease (PKD) is a genetic disorder characterized by progressive cyst formation, epithelial disorganization, and impaired fluid transport, ultimately leading to renal failure. Disruption of cytoskeletal dynamics and epithelial polarity is central to PKD pathogenesis. Malpighian tubules (MTs) of Drosophila melanogaster serve as a conserved renal analog, and caspase-3/Drice-deficient flies exhibit a robust PKD-like tubular phenotype, providing a powerful in vivo model to investigate therapeutic interventions. PurposeThis study evaluates the therapeutic potential of the Brahmi Ghrita (BG) in ameliorating PKD-like defects in Drosophila Drice mutants and elucidates the underlying cellular and molecular mechanisms. MethodsDrice mutant flies were reared with dietary BG supplementation, and developmental viability (pupation and eclosion) was assessed. Tubule morphology was analyzed by measuring cyst formation and tubule dimensions. Stellate cell (SC) number, shape, and nuclear size were quantified. Cytoskeletal organization and epithelial polarity were examined using F-actin and polarity markers. Molecular analyses included assessment of Rho1 signaling, Gelsolin, and Rho kinase (Rok) localization. Tubule physiology was evaluated by uric acid crystal deposition and Na{square}/K{square}-ATPase expression. ResultsBG supplementation significantly improved pupation and eclosion rates in Drice mutants and markedly reduced cystic dilation by restoring tubule width without altering developmental length. BG selectively increased stellate cell number and normalized aberrant morphology, while principal cell number remained unchanged. Cytoskeletal disorganization and polarity defects were rescued, accompanied by normalization of elevated Rho1 levels and restoration of the actin-severing protein Gelsolin. BG enhanced Na{square}/K{square}-ATPase expression and reduced uric acid accumulation, consistent with improved epithelial transport function. Additionally, BG promoted nuclear enrichment of Rok, indicating altered Rho-associated signaling dynamics. ConclusionBrahmi Ghrita confers nephroprotective effects in a genetic PKD model by coordinately restoring cytoskeletal integrity, epithelial polarity, and ion transport machinery. Rather than broadly suppressing Rho signaling, BG appears to rebalance the Rho1-Gelsolin axis and re-establish transport competency, culminating in structural and functional rescue. These findings provide mechanistic evidence supporting BG as a multi-target modulator of epithelial homeostasis in PKD-relevant contexts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/705756v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d909a9org.highwire.dtl.DTLVardef@10938bborg.highwire.dtl.DTLVardef@6ba065org.highwire.dtl.DTLVardef@1ef7857_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

A spatial gene expression pattern between the cortex (CTX) and inner medulla (IM) of the kidney has been observed, but the underlying mechanisms are unclear. Understanding these mechanisms is essential for elucidating renal function. Using the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) we analyzed the open chromatin structures and the involvement of epigenetic mechanisms in mediating gene expression differences between the renal CTX and IM. We also examined the role of the nuclear factor of activated T cells 5 (NFAT5), a key regulator of hypertonicity. ATAC-seq analysis was performed on CTX and IM samples from both wild-type (WT) and NFAT5 knockout (KO) mice. This work demonstrates for the first time that these differences in gene expression between renal CTX and IM are associated with an epigenetic mechanism driven by chromatin accessibility, which is partially modulated by the nuclear factor of activated T-cells 5 (NFAT5) in mice. Furthermore, spatial localization and NFAT5-promoted chromatin accessibility correlate with differential gene expression and altered promoter binding motif enrichment in CTX and IM. This study provides new insights into the spatial and NFAT5-mediated regulation of chromatin accessibility and gene expression in CTX and IM. This work advances our understanding of kidney physiology by uncovering previously unknown epigenetic factors influencing gene expression and provides a new perspective on renal adaptive mechanisms. TRANSLATIONAL STATEMENTThe study reveals new insights into the spatial and epigenetic regulation of gene expression in the renal cortex (CTX) and inner medulla (IM) in the mouse kidney. We used the Assay for Transposase-Accessible Chromatin with High-Throughput Sequence Analysis (ATAC-seq) to identify a key role of NFAT5 in modulating chromatin accessibility and to uncover previously unknown epigenetic factors. This research enhances our understanding of renal physiology and has important implications for clinical care by providing insights into potential adaptive mechanisms in the kidney. These findings suggest future investigations targeting epigenetic signaling pathways for therapeutic intervention in renal diseases.

Diabetic nephropathy remains the leading cause of end-stage renal disease. The ZSF-1 rat model combines features known as the metabolic syndrome, such as type 2 diabetes mellitus, hypertension and obesity, developing progressive kidney disease. In this study, we investigated the nephroprotective mechanisms of the SGLT2 inhibitor empagliflozin, focusing on mitochondrial function. Obese ZSF-1 rats were randomized at 24 weeks of age to receive either placebo or empagliflozin for eight weeks, while lean ZSF-1 rats served as healthy controls. Kidney function, assessed by glomerular filtration rate (GFR), was significantly reduced in obese rats and was not improved by empagliflozin treatment. However, obese animals exhibited increased tubular injury, tubular cast formation, and elevated total and tubular proteinuria, all of which were attenuated by empagliflozin. Mitochondrial function was evaluated in freshly isolated cortical kidney mitochondria by measuring oxygen consumption. Obese ZSF-1 rats showed impaired respiratory capacity and reduced protein expression of oxidative phosphorylation (OXPHOS) complexes II, III, IV, and V, indicating mitochondrial dysfunction. Empagliflozin treatment improved mitochondrial function by enhancing complex I- and IV-linked respiration and restoring the expression of OXPHOS complexes II, III, and IV. In addition, empagliflozin treatment was linked to improved mitochondrial dynamics and modulation of autophagic activity, suggesting enhanced mitochondrial quality control. Overall, these findings demonstrate that empagliflozin exerts nephroprotective effects primarily at the tubular level in obese ZSF-1 rats. The beneficial effects appear to be mediated through improved mitochondrial function, enhanced mitochondrial integrity, and reduced tubular injury.