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.

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.

Bladder outlet obstruction leads to pathological remodeling and emergence of lower urinary tract symptoms. Although relief of obstruction is associated with symptomatic improvement, it is not universally successful, reflecting persistent alterations in the bladder. Reliable surrogate biomarkers of obstruction are lacking, particularly early in the disease course before irreversible damage to the bladder may have occurred. In this study, re-analysis of publicly available transcriptomic datasets from diverse rodent models of obstruction identified tissue transcripts including Cthrc1, Grem1, Ltbp2 and Msn that were induced in response to injury. Candidate markers were validated experimentally in an independent model of neurogenic obstruction demonstrating time-dependent changes. Candidate markers were also attenuated with either surgical removal of obstruction or treatment with anticholinergic medication or inosine. Integrated analysis of tissue transcriptomics data and tissue and urine proteomics data from a model of neurogenic obstruction revealed significant concordance between markers observed in tissue and urine. Urinary proteomics analysis identified a statistically significant increase in MSN in patients with neurogenic bladder compared to unaffected controls. These findings identify tissue and urine biomarkers of both non-neurogenic and neurogenic obstruction that may reflect early changes in obstructive uropathy that could be monitored in a non-invasive manner.

Acute kidney injury (AKI) is a serious and common clinical syndrome that currently has no effective treatment. Emerging evidence links coagulation pathways to kidney injury, particularly through coagulation proteases. Protease-activated receptors (PARs) are a family of G-protein coupled receptors (GPCRs) that are activated by proteolytic cleavage of their N termini, exposing a tethered ligand that initiates receptor signaling. PARs have been shown to play a major role in inflammation, vascular regulation, and tissue injury. PARs play key roles in inflammation, vascular regulation, and tissue injury. Previous work from the Hamm laboratory demonstrated that PAR4 contributes to AKI progression, as PAR4 knockout mice were protected in both unilateral ureteral obstruction and ischemia-reperfusion-based models of kidney disease. In this study, we investigated the potential of a PAR4 antagonist, VU6073819, at mitigating AKI progression in an ischemia-reperfusion injury (IRI) mouse model. PAR4 antagonism not only alleviated kidney injury and inflammatory response, but it significantly improved the survival. These findings identify PAR4 as a promising therapeutic target for AKI.

Expression of the Csf1r gene in cells of the mononuclear phagocyte lineage is regulated by a conserved enhancer, the fms-intronic regulatory element (FIRE). In mice with a germ-line deletion of FIRE (Fireko) CSF1R expression is undetectable in bone marrow progenitors and classical monocytes. Fireko mice lack subpopulations of macrophages in the brain and periphery but develop normally. Here we show that loss of CSF1R expression in Fireko mice is partly overcome by CSF2 in vitro and inflammatory recruitment in vitro. Analysis of heterozygous mutant mice and deletion of the conserved AP1 motif in FIRE provide evidence that continuous receptor synthesis determines CSF1 responsiveness. The absence of macrophages in kidney and heart of Fireko mice was not associated with detectable loss of physiological function. In a model of renal injury macrophage recruitment and histopathology were similar in WT and Fireko mice. Tissue resident macrophages that were depleted in Fireko mice, including microglia, were replaced by donor-derived cells following intraperitoneal adoptive transfer of wild-type bone marrow at weaning. The Fireko mouse provides a novel platform to dissect the functions of tissue resident macrophages in development, homeostasis and pathology. Summary StatementThis study describes a unique model of selective tissue resident macrophage deficiency arising from dysregulated expression of the mouse Csf1r gene.

Microvascular inflammation and endothelial injury, triggered by interferon-gamma (IFN{gamma}), are hallmarks of antibody-mediated rejection (ABMR), the leading cause of premature kidney allograft loss. Glomerular extracellular matrix (ECM) remodeling and endothelial caveolae formation are important aspects of chronic ABMR. We found galectin-1, an immunomodulatory protein that interacts with the ECM, to be increased in the glomeruli of patients with ABMR, while its gene (LGALS1) expression was decreased by IFN{gamma} stimulation in glomerular endothelial cells. Mechanisms underlying endothelial dysfunction in ABMR, its links to ECM remodeling, and the role of immunomodulatory proteins such as galectin-1 remain incompletely understood. Here we studied the effects of galectin-1 modulation in glomerular microvascular endothelial cells (GMECs) in vitro. We demonstrated that galectin-1 was mainly expressed by glomerular endothelial cells in ABMR kidneys. To model key aspects of endothelial injury in ABMR, we knocked down LGALS1 in GMECs, followed by stimulation with IFN{gamma} and performed label-free quantitative proteomic and phosphoproteomic profiling of GMECs. Proteomic analysis identified 5446 proteins (FDR<0.01), of which 236, 827, and 267 were differentially expressed in response to LGALS1 knockdown, IFN{gamma} treatment, and their interaction, respectively (FDR<0.05). Both LGALS1 knockdown and the interaction between treatments significantly altered expression of adhesion proteins (FDR<0.01), particularly integrin subunit {beta}5, which was validated. Phosphoproteomic profiling identified 2727 phosphopeptides (FDR<0.01), with 28 that were differentially expressed across LGALS1 knockdown, IFN{gamma} treatment, and their interaction (P<0.01). Phosphorylation of CAVN1 and co-localization with its partner CAV1, critical for caveolar formation, were decreased in GMECs upon LGALS1 knockdown, IFN{gamma} stimulation, or both. In a microfluidic model of the glomerular microvasculature, addition of recombinant galectin-1 increased both endothelial permeability and secretion of proinflammatory cytokines, in LGALS1-silenced GMECs. Thus, endothelial signaling pathways regulated by inflammatory cues and galectin-1 contribute to endothelial injury and caveolae formation, highlighting galectin-1 as a potential therapeutic target in ABMR. SynopsisGalectin-1 is expressed by kidney glomerular endothelium. This study reveals that modifying galectin-1 in endothelial cells, in the presence of IFN{gamma} perturbs cytoskeletal, adhesion and caveolar proteins resulting in altered endothelial permeability. O_LILGALS1 knockdown increased ECM proteins and decreased interferon-induced proteins. C_LIO_LILGALS1 knockdown and IFN{gamma} treatment perturbed cell adhesion proteins such as ITGB5. C_LIO_LICAVN1 phosphorylation and colocalization with CAV1 decreased upon LGALS1 knockdown. C_LIO_LIExtracellular galectin-1 increased microvascular permeability in response to IFN{gamma}. C_LI

Kidneys play an essential role in balancing fluid and electrolyte levels. Two mouse strains, C57Bl/6 and 129S2/SV, are routinely used to study renal physiology in laboratory settings, and prior observations suggest that significant differences in salt and water handling exist between them. This study aims to further establish the sources of these observed differences at both expressional and functional levels, in male and female mice. At baseline, male 129S2/SV mice displayed decreased Na+ and increased K+ plasma concentrations compared to C57Bl/6 males, while no statistical differences were observed between female mice. Interestingly, 129S2/SV male mice had lower glomerular density than C57Bl/6 males. Immunoblotting shows that 129S2/SV mice of both sexes had increased expression of NHE3 and NKCC2 compared to their C57Bl/6 counterparts. Both total and phosphorylated NCC were more abundant in female mice as compared to males, indicating sexual dimorphism. Furthermore, 129S2/SV females had higher expression of total and phosphorylated NCC compared to C57Bl/6 females. In contrast, the expression of SGLT2, ENaC subunits, and Na+/K+-ATPase were comparable between C57Bl/6 and 129S2/SV mice of both sexes. When challenged with diuretics intended to block NKCC2, NCC or ENaC, 129S2/SV male mice responded with a smaller diuresis and natriuresis than their C57Bl/6 counterparts. Taken together, our data suggest that differential expression of key Na+ transporters along the nephron contributes to differences in Na+/K+ homeostasis between these two mouse strains. NEW & NOTEWORTHYWe assessed the influence of genetic background on the expression of key Na+ transporters along the nephron in two commonly used inbred mouse strains, C57Bl/6 and 129S2/SV. We found that the kidney expression of NHE3, NKCC2, and NCC are strain dependent. Additionally, murine strain significantly contributes to the diuretic responses induced by hydrochlorothiazide, amiloride, and furosemide.

BackgroundDysregulation of phosphate homeostasis contributes to reduced longevity and vascular complications in chronic kidney disease and aging. This study investigates the role of TMEM174, a proximal tubule-specific protein, in regulating the phosphate co-transporter NPT2A and its subsequent impact on lifespan and vascular health. MethodsTMEM174 knockout (KO) mice (C57BL6/J and DBA/2J) were fed diets with varying phosphate concentrations (0.6% vs. 1.2%). In OKP cells, TIRF and FRET microscopy, alongside immunoprecipitation, were used to identify the TMEM174 protein regions essential for NPT2A binding and endocytosis. ResultsTMEM174 KO mice exhibited significantly shorter lifespans than wild-type controls. High phosphate diets exacerbated vascular calcification, stiffness, and mortality, while low phosphate diets rescued these phenotypes. In vitro, TMEM174 siRNA blocked PTH-induced NPT2A endocytosis, increasing its apical membrane retention. FRET and biochemical assays revealed that the C-terminal region of TMEM174 is essential for its association with NPT2A. While intact TMEM174 and N-terminal mutants (TMEM174{Delta}N) facilitated NPT2A degradation, C-terminal deletions (TMEM174{Delta}C) failed to associate with or degrade NPT2A. ConclusionsTMEM174 is a critical regulator of phosphate homeostasis and longevity. The C-terminal region of TMEM174 is specifically required for NPT2A endocytosis and degradation, identifying it as a potential therapeutic target for managing phosphate-related vascular complications.

AimSecretin was recently found to play a pivotal role in the renal adaptation to acute base excess. Here, secretin increases pendrin-dependent HCO3- secretion from the beta-intercalated cells in the cortical collecting ducts. Whether secretin and its receptor play a role during prolonged base-loading remains unknown. MethodsUrine and blood acid-base analyses were carried out in secretin receptor (SCTR) KO and WT mice at baseline and after 1 and up to 8 days of base-loading with NaHCO3-enriched drinking water. Changes in pendrin protein abundance and function were assessed by immunoblotting and isolated tubule perfusion experiments. Plasma secretin levels and renal SCTR expression were assessed after 24 hours of acid/base-loading by radioimmunoassay and qPCR, respectively. ResultsSCTR KO mice responded with diminished urine alkalization and a lesser reduction of urinary acid excretion when base-loaded for 48 hours. Concordantly, SCTR KO mice presented with increased blood base retention compared with WTs. Base-loaded SCTR WT and KO mice showed comparable total pendrin protein abundance. Despite this, pendrin function was markedly lower in SCTR KO mice. Base-loaded mice had higher plasma secretin and renal SCTR levels compared with acid-loaded mice. Higher arterial HCO3- associated with higher renal SCTR mRNA expression. ConclusionPlasma secretin and renal SCTR levels are modulated by systemic acid-base status. Loss of the SCTR diminishes renal base excretion capacity and exacerbates systemic base accumulation during prolonged base-loading. These findings further support a central role of secretin and its receptor in the regulation of both acute and prolonged base excess.

NKCC2, localized to the apical membrane of thick ascending limb epithelial cells, is essential for renal salt handling and systemic electrolyte homeostasis. NKCC2 undergoes extensive ubiquitylation, with the E3 protein ligase Nedd4-2 implicated as a key regulator. However, progress has been limited by challenges expressing NKCC2 in mammalian cell lines, hindering mechanistic studies of NKCC2 ubiquitylation. Therefore, the aims of this study were to develop a mammalian cell model enabling mechanistic investigations of NKCC2 ubiquitylation, including the role of Nedd4-2 and the functional consequences of site-specific modification. A tetracycline-inducible MDCKI cell line was generated expressing human NKCC2 and used to assess Nedd4-2-dependent and site-specific ubiquitylation of NKCC2 using biochemical, imaging, and functional assays. The MDCKI cell line demonstrated stable, inducible expression of full-length human NKCC2. In this cell line, mutating the ubiquitylation site at K871 increased membrane abundance and uptake activity, without altering internalization rates. Nedd4-2 co-immunoprecipitated with NKCC2, and Nedd4-2 deletion increased total, but not membrane NKCC2 abundance. In summary, ubiquitylation on NKCC2 at K871 plays a key role in controlling NKCC2 membrane localization and thus function. Although Nedd4-2 can modulate NKCC2 abundance, it is not involved in NKCC2 trafficking. We conclude that the generated cell line provides a robust model for mechanistic studies of NKCC2 and will aid studies examining posttranslational regulation of NKCC2.

Salt-sensitive hypertension, a condition in which the blood pressure (BP) increases with an increase in salt intake, is influenced by behavioral, genetic, and environmental factors. Salt sensitivity is associated with variants of the G protein-coupled receptor kinase 4{gamma} (GRK4{gamma}) and the renal sodium bicarbonate cotransporter 2 (NBCe2), encoded by the solute carrier family 4 member 5 (SLC4A5). The R>65L variant (rs2960306) of human GRK4 (hGRK4{gamma} 65L) contributes to salt sensitivity through a signaling pathway and gene-gene interaction with SLC4A5. Global expression of GRK4{gamma} 65L in transgenic mice results in salt-sensitive hypertension, due in part to an increase in endogenous GRK4 and angiotensin type 1 receptor (AT1R) expression. Grk4 knockout (Grk4-/-) mice have decreased blood pressure and are salt-resistant. The expression of hGRK4{gamma} 65L only in the kidney of Grk4-/- mice increases BP in response to a high salt diet. The renal expression of SLC4A5 is increased in hGRK4{gamma} 65L transgenic mice, relative to mice expressing wild-type (WT) human GRK4 (hGRK4 65L), without endogenous mGrk4. Human renal proximal tubule cells (hRPTCs) endogenously expressing GRK4 WT and SLC4A5 WT, SLC4A5 variants, GRK4 65L, and both GRK4 65L and SLC4A5 variants were studied. SLC4A5 expression is increased in hRPTCs expressing GRK4 65L and in cells expressing both GRK4 65L and SLC4A5 variants compared with GRK4 WT and SLC4A5 WT. Luminal and basolateral sodium transport in hRPTCs is increased in the presence of both hGRK4 65L and SLC4A5 variants. GRK4 interacts with nuclear histone deacetylases (HDACs). Mice expressing hGRK4 65L only in the kidney have decreased expression but increased phosphorylation of HDAC1. HDAC1 expression is decreased and HDAC1 but not HDAC2 phosphorylation is increased in hRPTCs expressing both hGRK4 65L and SLC4A5 variants. The presence of hGRK4{gamma} 65L decreased HDAC1 expression but increased AT1R expression in the kidneys of mice on high salt diet. Our results show that GRK4{gamma} 65L causes salt-sensitive hypertension by increasing renal SLC4A5 and AT1R expressions by inhibiting the HDAC1 pathway.

ObjectivesLupus nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE), leading to progressive renal fibrosis and functional decline. Understanding the interplay between immune cells and stromal cells is needed to develop effective therapeutic strategies. Here, we investigated the landscape of macrophage-fibroblast interactions in human LN and validated these findings in mouse models. MethodsWe characterized distinct fibroblast subsets and their interactions with renal macrophages using single-cell RNA sequencing (scRNAseq) of 156 human LN biopsies and 30 healthy controls from the AMP-SLE cohort, and spatial transcriptomics of biopsies from 6 LN patients. In vitro co-culture studies using mouse models were performed to further define functional consequences of these interactions. ResultsWe identified two myofibroblast subsets: a pro-inflammatory subset (Myofib1) enriched in the tubulointerstitium, and a fibrotic/remodeling subset (Myofib2) in glomeruli, both correlating with the histologic chronicity index. Spatial transcriptomics revealed different colocalization patterns, with Myofib1 interacting with activated resident macrophage (RM) subsets and Myofib2 with glomerular infiltrating disease-associated macrophages. In vitro co-culture studies demonstrated that nephritic RMs promote a pro-inflammatory, remodeling fibroblast phenotype that impairs wound healing and drives a Myofib1-like gene program, whereas disease-associated macrophages generated profibrotic fibroblasts with dysregulated reparative capacity. Cell-cell communication analyses identified key ligand-receptor interactions mediating this crosstalk, including Spp1/integrins, Sema4/PlexinB, and NAMPT/INSR. ConclusionsOur data reveal a spatially and functionally heterogeneous landscape of macrophage-fibroblast crosstalk in LN. These findings advance our understanding of renal fibrogenesis in LN, highlighting specific fibro-inflammatory circuits that may represent therapeutic targets to prevent chronic renal damage.

BackgroundDeoxycorticosterone acetate (DOCA)-salt induces greater increases in blood pressure (BP) and a more pro-inflammatory T cell profile in males compared to females. T cells contribute to DOCA-salt hypertension, however, the mechanisms driving T cell activation remain unclear. The NLRP3 inflammasome has been implicated in DOCA hypertension in male mice. Little is known regarding NLRP3 in females. The goal of the current study was to test the hypothesis that NLRP3 contributes to greater increases in BP and renal inflammation with DOCA in males vs. females. MethodsRenal NLRP3 protein levels were measured in normotensive and hypertensive male and female subjects and in male and female Sprague Dawley uni-nephrectomized (UNX) control and DOCA-salt rats. Additional 11-wk-old Sprague Dawley rats were UNX and randomized to: 1) DOCA + vehicle or 2) DOCA + the NLRP3 inhibitor MCC950 (10 mg/kg/day in saline) from 11-14 wks of age. At 14-wks-of-age rats were euthanized, terminal plasma samples and remaining kidneys were collected for flow cytometric analysis of T cells. ResultsRenal NLRP3 levels were significantly greater in hypertensive males and females vs. normotensive controls. DOCA increased BP in both sexes, with greater elevations in males. MCC950 attenuated DOCA-induced increases in BP in male, but not female rats. MCC950 decreased circulating and renal CD4 and Th17 cells in both sexes, although the effect was greater in males. ConclusionDespite both males and females exhibiting an increase in NLRP3 in hypertension, NLRP3 contributes to BP elevations only in DOCA-salt males.

ObjectivesBenign Prostatic Hyperplasia (BPH) is the non-cancerous enlargement of the prostate accompanied by lower urinary tract symptoms, affecting 50% of men by the age of 501,2. Advanced highly symptomatic BPH exhibits large epithelial glandular nodules with microglandular/atypical adenomatous hyperplasia, but how these features form is unknown3. Our lab has reported that the common parasite Toxoplasma gondii can infect the prostate and induce glandular nodule formation in mice3. The objective of this study is to determine if T. gondii exposure in humans correlates to BPH and nodule formation and if it induces urinary dysfunction concurrent in the mouse model. MethodsWe assessed Toxoplasma exposure by serum ELISA in patients with BPH and non-BPH donor controls, and compared seropositivity rates between the groups. We further assessed the histopathology of these patients for the presence of inflammation and epithelial glandular nodule formation and compared Toxoplasma positive and negative samples. We determined voiding function in Toxoplasma-infected mice between 14 and 60 days of infection with void spot with Void Whizzard software. ResultsMen diagnosed with BPH are more likely to be seropositive for Toxoplasma than age-matched undiagnosed donor controls. In addition, BPH patients that are seropositive for Toxoplasma are more likely to exhibit glandular nodule formation with microglandular / adenomous hyperplasia than seronegative BPH patients. In animal studies, Toxoplasma infection results in abnormal void patterns concurrent with microglandular hyperplasia and nodule formation. ConclusionsThese results suggest that Toxoplasma may be contributing to BPH pathology and lower urinary tract dysfunction in both humans and mice, opening new insights into the development of this important disease. The results also serve to further characterize this model of prostatic hyperplasia and define it as a potential urinary dysfunction model.

IntroductionImmune checkpoint inhibitors (ICIs) enhance antitumor responses by blocking inhibitory receptors, including PD-1 and CTLA-4. Overactivation can trigger systemic toxicity akin to autoimmune diseases, including kidney manifestations. We sought to 1) profile immune signaling and 2) interrogate potential mechanisms of ICI-related kidney injury in a Human Immune System (HIS) tumor-bearing mouse model treated with nivolumab and ipilimumab. MethodsImmunodeficient BRGS (BALB/c-Rag2nullIl2r{gamma}nullSirpNOD) neonates were engrafted with human CD34+ cells to generate HIS-BRGS mice. Human MDA-MB-231 tumor cells were implanted subcutaneously; once tumors reached [~]150 mm3, mice received weekly intraperitoneal vehicle (PBS) or ICI (nivolumab 20 mg/kg + ipilimumab 10 mg/kg) for 4 weeks (Veh BRGS n=4; ICI BRGS n=6; Veh HIS-BRGS n=7; ICI HIS-BRGS n=7). Kidneys were evaluated by histopathology (H&E, TEM), flow cytometry for human immune phenotypes, multiplex ELISA (80 human proteins; 10 injury biomarkers), bulk RNA sequencing, and targeted qPCR. Pearson correlations identified predictors of histopathological injury. ResultsRenal vasculitis and interstitial nephritis were observed only in ICI-treated HIS-BRGS mice. These kidneys showed a shift toward CD4+ T-cell enrichment with an increased TNF- production capacity compared to CD8+ counterparts. Toxicity was accompanied by increased renal concentrations of human cytokines, chemokines, and soluble receptors. ICI treatment significantly elevated serine proteases (Granzyme A/B) and NGF-{beta}, while decreasing IL-4. Interstitial nephritis correlated with renal PD-1 and MIF. Renal vasculitis correlated with kidney PD-1, CCL1, MIF, Granzyme A, IL-15, and BAFF. Traditional injury biomarkers (KIM-1, NGAL) remained unchanged; however, a trending decrease in EGF was observed. ConclusionsOur study suggests that shifts in human T-cell populations and specific immune proteins could serve as promising biomarkers and mechanistic targets for ICI nephrotoxicity. The tumor-bearing HIS-BRGS mouse model reproducibly recapitulates the histopathological and immunological features of human ICI-induced nephrotoxicity and represents a validated preclinical platform for testing novel therapeutic interventions to preserve kidney function during cancer immunotherapy. Translational StatementImmune checkpoint inhibitor (ICI)-associated nephrotoxicity occurs in up to 25% of treated patients, yet the immunological mechanisms driving renal injury remain poorly characterized due to the scarcity of human biopsy material and the absence of robust preclinical models that recapitulate human immune responses. This study demonstrates that tumor-bearing humanized immune system (HIS) mice treated with combined nivolumab and ipilimumab reproducibly develop renal vasculitis and interstitial nephritis mediated by a human CD4+ T cell-dominant infiltrate, mirroring the clinicopathological features reported in patients with ICI-associated acute kidney injury. By integrating histopathology, flow cytometry, multiplex proteomics, and transcriptomics, we identify a coordinated immune network, including IL-15, CCL1, MIF, GZMA, and BAFF, that correlates with the severity of renal pathology and represents tractable mechanistic targets and candidate biomarkers. These findings provide a validated preclinical platform for dissecting irAE mechanisms and testing novel therapeutic strategies to preserve kidney function during cancer immunotherapy.

BackgroundPolycystic kidney disease (PKD) is the leading genetic cause of renal failure, resulting in the accumulation of fluid filled cysts and gross enlargement of the kidney. Mutations in PKD1 or PKD2, which encode ciliary polycystin proteins, are the most common cause of PKD. These proteins function in a cilia-dependent cyst activation (CDCA) pathway-one that requires cilia for its pro-cystic function-yet the molecular driver(s) of this pathway are unknown. ARL13B is a regulatory GTPase enriched in cilia and links to renal cystogenesis. ARL13B possesses guanine nucleotide exchange factor (GEF) activity for ARL3, another ARL with links to cilia. MethodsWe used two distinct Arl13b mouse alleles to investigate whether ARL13B is a component of the CDCA pathway: Arl13bV358Aencodes for enzymatically normal ARL13B that is undetectable in cilia, and Arl13bR79Qencodes for cilia-localized ARL13B lacking a residue critical for its ARL3 GEF activity. We used these alleles in a Pkd1-deficient adult mouse model and investigated renal morphology (H&E and cystic index analysis), physiology (blood urea nitrogen measurements), renal fibrosis (picrosirius staining and -smooth muscle actin levels), renal injury (SOX9 immunofluorescent staining and quantification), and Wnt signaling ({beta}-catenin and cyclin D1 protein levels). ResultsWe found that loss of ciliary ARL13B or mutation of a single residue critical for its ARL3 GEF activity suppressed Pkd1-dependent cysts. We observed reductions in kidney size, cystic index, and blood urea nitrogen. We also observed suppression of renal fibrosis, renal injury, and {beta}-catenin and cyclin D1 protein levels. ConclusionsOur results identified a subcellular location and mechanism driving Pkd1-dependent renal cystogenesis. We demonstrated that expression of a critical residue for ARL13Bs GEF activity specifically in cilia is a key mechanism of the CDCA pathway driving renal cystogenesis. Key PointsO_LILoss of ciliary ARL13B suppressed renal cystogenesis in an adult mouse model of polycystic kidney disease (PKD) without ablating cilia C_LIO_LILoss of ciliary ARL13B or mutation of the residue critical for its GEF activity did not affect renal morphology or physiology in a PKD mouse model C_LIO_LIMutation of a residue critical for ARL13B activation of ARL3 suppressed cystic phenotypes in Pkd1-dependent cysts C_LI

Ciliogenesis associated kinase 1 (CILK1) deficiency in human and mice results in kidney developmental defects including cystogenesis. However, the biology of CILK1 in autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disease, remains to be investigated. Here, we show that CILK1 is overexpressed in dedifferentiated cells of renal tissue from ADPKD human patients in comparison to normal control tissue samples. We demonstrate that CILK1 overexpression results in protein accumulation in a non-phosphorylated inactive form. Using mouse polycystic kidney disease models, we reveal that inactive CILK1 accumulation is progressive over the course of disease progression. We show that genetic inactivation of the Polycystic Kidney Disease 1 (PKD1) gene is sufficient to trigger CILK1 accumulation. Altogether, these findings demonstrate that CILK1 regulation is altered in ADPKD and it represents a hallmark of disease progression.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare condition causing nephrotic syndrome, neuropathy, and other manifestations. SPLIS is caused by mutations in SGPL1, which encodes sphingosine-1-phosphate lyase (SPL), a pyridoxal 5-phosphate (PLP)-dependent enzyme needed to degrade the bioactive sphingolipid sphingosine-1-phosphate (S1P). Supplementation with the PLP precursor pyridoxine benefits some individuals with PLP-dependent enzymopathies. We sought to establish whether pyridoxine has therapeutic activity in SPLIS. Neurological improvement, plasma S1P normalization, and increased SPL activity in patient-derived fibroblasts were observed after pyridoxine supplementation in a patient with R222Q-variant SPLIS. Additionally, PLP dose-dependently augmented recombinant R222Q-variant SPL activity. To further explore pyridoxines effects, gene editing was employed to create an R222Q-variant SPLIS mouse model. SPLR222Q mice fed pyridoxine-enriched chow lacked obvious phenotypes. However, SPL inactivation, S1P accumulation, wasting, anemia, proteinuria, and glomerulosclerosis developed in SPLR222Q but not WT mice fed chow with reduced pyridoxine. Ultrastructural analysis and super-resolution microscopy showed podocyte loss and foot process effacement. Transcriptional profiling revealed a pattern of cytokine upregulation and extracellular matrix remodeling. Inhibiting S1P production prevented nephrosis in SPLR222Q mice fed chow lacking pyridoxine. Our findings establish a novel SPLIS mouse model that recapitulates R222Q-variant SPLIS, demonstrates its responsiveness to pyridoxine, and implicates S1P in its pathophysiology.

Acute antibody-mediated rejection (aABMR) is an important cause of clinical kidney graft injury and failure. Transcripts associated with NK cell activation in graft biopsies are diagnostic of aABMR, but mechanisms underlying NK cell activation during ABMR remain poorly understood. In contrast to the long-term (> 60 days) survival of complete MHC-mismatched kidney allografts in wild type C57BL/6 mice, B6.CCR5-/- recipients develop high titers of donor-specific antibody (DSA) with allograft rejection between days 18 to 28 post-transplant. This has allowed investigation of mechanisms underlying NK cell activation within kidney allografts during aABMR. DSA titers first became detectable in B6.CCR5-/- (H-2b) recipients of A/J (H-2a) kidney allografts at day 8 and peaked on day 15 post-transplant and was accompanied by a parallel increase in mRNA levels of Rae-1e, a ligand for the NK cell activation receptor NKG2D. A/J kidneys in B6.CCR5-/-NKG2D-/- recipients and A/J.Rae-1e-/- kidneys in B6.CCR5-/- recipients survived >60 days, despite high serum DSA levels. Flow cytometric analysis of allograft infiltrating cells in B6.CCR5-/- recipients on day 15 post-transplant revealed inflammatory monocyte and NK cell infiltration and NK cell activation to proliferate and express CD107a, a marker of cytotoxic function. These features of aABMR were absent or markedly reduced by recipient NKG2D- or donor graft Rae-1e-deficiency. These findings suggest that interference with expression of allograft Rae-1e or recipient NK cell NKG2D abrogates aABMR despite persistently high DSA levels and that aABMR requires coordination between infiltrating NK cell and inflammatory monocyte activation within the kidney allograft.

BackgroundMaternal protein restriction results in a 28% reduction in nephrogenic cells and nephron units in rodent offspring by the 17th day of gestation compared to adequate protein intake. AimsThe present study investigates the association between growth factor expression and some developmental pathways that contribute to nephron reduction during embryonic and fetal development. Experimental DesignPregnant C57BL/6-Tg and C57BL/6J mice were assigned to either normal protein intake (NP-17%) or low protein intake (LP-6%) groups. Body weight of male offspring and kidney growth factor expression were assessed on gestation days (GD) 14 and 18. ResultsOn GD 14, LP pups exhibited a 4% higher body mass (0.1035 g) compared to NP pups (0.0995 g, p = 0.005). By GD 18, LP pups demonstrated a 4% decrease in body mass (0.939 g, p = 0.03) and a 10% increase in the number of cells per metanephric cap area. Three genes (Csf2, Il1b, Il2) were downregulated, while seven genes (Bmp2, Csf3, Fgf8, Gdnf, Bmp7, Fgf3, Ntf3) were upregulated. By GD 14, phagophores and autophagosomes in the ureteric bud increased by 197%, with further increases observed by GD 18. Bcl-2 expression increased significantly in ureteric bud cells, and mTOR activity was elevated by GD 18. ConclusionEarly gestational protein restriction modifies renal growth factor gene expression, influencing cell proliferation and autophagy, and may contribute to reduced nephron numbers by the 18th day of gestation. HIGHLIGHTSO_LIThis study examines the effects of a low-protein diet during pregnancy in mice and demonstrates a significant reduction in embryo-fetal body weight between gestational days 14 and 18. C_LIO_LIProtein restriction induces a distinct cellular pattern in the mesonephros, with a 21% increase in CAP cells at gestational day 14 (GD14), followed by a decrease by gestational day 18 (GD18) compared to offspring from mothers on a normal protein diet. C_LIO_LIAdditionally, increased expression levels of key growth factors essential for kidney development were observed at GD 14, comparing LP with NP intake during pregnancy. C_LIO_LISeven genes were upregulated (Gdnf, Bmp2, Bmp7, Tgf, Fgf8, Fgf3, Csf3, Ntf3), while three genes were downregulated (Csf2, Il1b, Il2). C_LIO_LIOverall, these findings indicate that gene regulation, autophagy, and mTOR signaling mechanisms significantly influence nephron numbers in response to gestational protein restriction beyond the 18th day of gestation. C_LI