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.

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.

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.

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.

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

Poor outcomes in proteinuric kidney diseases are challenging to successfully manage therapeutically due to the heterogeneity of underlying disease pathogenesis and associated risk for progression. The role of cytoskeleton-associated proteins, including the scaffolding protein Anillin (ANLN), are of specific interest in kidney disease given the importance of actin dynamics in the kidneys specialized epithelial cell types. In this study, we identify the prevalence of genetic variants in ANLN, the gene encoding ANLN, in a cohort of deeply phenotyped individuals with non-diabetic proteinuric kidney disease. Thirty-one individuals (of 864 genotyped) harbor heterozygously expressed variants in ANLN; 7 unrelated individuals shared the same variant (I1109V) in the C-terminal pleckstrin homology (PH) domain, a region necessary for interaction with the plasma membrane. Kidney organoids generated from I1109V induced pluripotent stem cells from 1 of these individuals showed increased epithelial cell mitogen-activated protein kinase 8 network activity and apoptosis, which was enhanced by tumor necrosis factor alpha (TNF-) and phenocopied by actin polymerization inhibition. TNF--treated I1109V organoids also exhibited tubular lumen expansion. Knockdown and re-expression of the analogous ANLN variant in Xenopus laevis embryonic epithelia resulted in defects in cell-cell junction dynamics including wavy cell membranes exhibiting increased transverse movements as well as abnormal junctional F-actin remodeling in response to mechanical stress and leaky barrier function. Taken together, these results indicate that enhanced tubular epithelial cell death, perturbed cell-cell contacts and barrier function defects are associated with a novel ANLN variant discovered in individuals with non-diabetic proteinuric kidney disease. ONE SENTENCE SUMMARYEnhanced tubular epithelial cell death and perturbed cell-cell junction integrity and barrier function are associated with a novel Anillin coding variant discovered in a cohort of individuals with proteinuric kidney disease.

Background & AimsDiabetes mellitus has been associated with both intestinal barrier dysfunction and peripheral neuropathy leading to increased risk of infection. The mucus layer forms a physical barrier against pathogens and is a critical component of the intestinal barrier that may be impaired in diabetes. This study aimed to assess how diabetes impacts goblet cells (GCs), mucus layer integrity, and innervation in the colon. MethodsFluorescence microscopy was used to investigate GCs, the mucus layer, and innervation in the colon of db/db mice. Custom open-access image analysis pipelines were developed to quantify GC numbers, location and content, mucus thickness, bacterial colonization, and innervation density in intestinal tissue sections. We also treated mice with the clinically used glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide to assess its capacity to reverse pathological changes to GCs and the mucus layer in a model of established type 2 diabetes (T2DM). ResultsThe mucus layer was significantly thinner in the colon of db/db mice with established diabetes and bacteria more readily colonized the epithelium and crypts. Intercrypt GC numbers were significantly reduced in db/db mice. However, there were significantly more GCs per crypt, and crypts were elongated in the db/db colon. Innervation was reduced in the mucosa and external muscle of the colon, consistent with diabetic neuropathic changes. Liraglutide treatment increased the size of GCs but had no effect on GC numbers, mucus thickness, or innervation in this model of established T2DM. ConclusionsMucus barrier dysfunction and GC hyperplasia is evident in the db/db colon. Increased microbial penetrability through the mucus layer suggests potential implications for the increased risk of gastrointestinal infection in diabetes.

Autosomal dominant polycystic liver disease (ADPLD) commonly results from a pathogenic variant in one of 6 genes (GANAB, ALG8, LRP5, PRKCSH, SEC61B, SEC63). Pathogenic variants in these genes are also associated with kidney cysts, which rarely cause kidney failure, but the genes are included in cystic kidney panels. This study determined the population frequency of predicted pathogenic variants in the ADPLD genes in the general population. Variants for each gene were downloaded from gnomAD and annotated with ANNOVAR. The population frequencies were calculated from the number of people with "predicted pathogenic" variants in gnomAD v.2.1.1:loss-of-function structural and copy number; null; and rare, computationally-damaging missense changes that affected a conserved residue. Frequencies were also estimated from the number of gnomADv.4.1 variants assessed as Pathogenic or Likely pathogenic in ClinVar. Predicted pathogenic variants affected one in 95 people using our strategy and gnomAD v.2.1.1, and one in 151 with ClinVar assessments of gnomAD v.4.1 variants. LRP5 and ALG8 which are associated with a milder clinical phenotype, were the commonest affected genes with both strategies. Predicted pathogenic variants in ADPLD appear more frequent in admixed American (one in 100), Finnish (one in 107) and African/African American (one in 130) people (p all <0.0001 compared with Europeans (one in 197).Predicted pathogenic variants for ADPLD may be even more common because of additional unidentified causative genes. However not all ADPLD variants result in liver cysts, nor indeed cystic kidneys, because of incomplete penetrance and variable expressivity.

BackgroundWithin the kidney, (pro) renin receptor (PRR) is abundantly expressed in the collecting duct (CD) and modulate physiological and pathophysiological processes. We have recently reported that activation of CD PRR mediates obstructive renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO). The current study addresses the underlying mechanisms by examining the profibrotic pathway mediated by soluble PRR (sPRR)-dependent alternative macrophage activation. MethodsWe performed UUO or sham surgery on mice with CD-specific deletion of PRR (CD PRR KO) and floxed controls. After 7 days, we assessed fibrosis-related parameters, inflammatory cytokines, M1/M2 macrophage markers, other gene expression markers of kidney injury, and the concentration of plasma sPRR. We also administered vehicle or site-1 protease (S1P) inhibitor PF-429242 (PF) to C57BL/6 mice with UUO to determine the role of sPRR. Experiments were performed in vitro to examine the mechanism of sPRR-His-mediated macrophage M2 polarization and activation of potential target genes in bone-marrow-derived macrophages (BMDMs). ResultsCompared with the floxed control, CD PRR KO decreased macrophage accumulation, M2 polarization, and Yap/Taz expression while improving renal fibrosis and suppressing plasma sPRR levels following UUO. In BMDMs, sPRR-His treatment promoted macrophage M2 polarization, fibrosis, and Yap/Taz expression, which was dependent on angiotensin type 1 receptor (AT1R). ConclusionCD PRR-derived sPRR acts via ATR to promote macrophage M2 polarization and stimulates the AT1R/Yap/Taz axis, which leads to renal fibrosis during UUO.

Acute kidney injury (AKI) is a sudden episode of kidney failure linked to a wide range of health conditions. High mortality in AKI highlights the need to identify new therapeutic approaches. Homeostasis in multicellular organisms is exquisitely regulated by phagocytosis of apoptotic cells, also known as efferocytosis. Apoptotic cells are frequently observed at sites of inflammation, including in AKI. Engulfment and cell motility protein-1 (ELMO1) is a regulator of the actin cytoskeleton that promotes apoptotic cell removal by phagocytes during efferocytosis. Mutations in the human ELMO1 gene are linked with diabetic nephropathy and, in animal models of this disease, high ELMO1 levels promote renal dysfunction. However, the role of ELMO1 in AKI was not known. Here, we describe the links between ELMO1 and kidney pathology and test global and tissue-specific ELMO1-deficient mice in models of AKI. While global loss of Elmo1 expression did not impact the immediate loss of renal function after ischemia-reperfusion elicited AKI, ELMO1 deficiency resulted in increased tissue injury in AKI caused by cisplatin injection. Cisplatin induced robust renal cell apoptosis that was significantly elevated in mice with the global loss of ELMO1, but not in mice with the macrophage-specific Elmo1 deletion. Using primary cell culture and immunofluorescence approaches, we highlight the role of ELMO1 in efferocytosis by several renal cell types, suggesting possible additive effects during nephrotoxic injury.

Autophagy is a critical homeostatic mechanism in podocytes, maintaining cellular integrity under stress and proteostatic challenges. Dysregulation of autophagy has been implicated in different glomerular diseases such as diabetes and membranous glomerulonephropathy (MGN), yet the underlying molecular drivers remain incompletely understood. We identified microRNA-378a (miR-378a), previously found upregulated in MGN, as a functional enhancer of autophagic flux in human podocytes and tubular epithelial cells. While miR-378a did not directly alter transcription of canonical autophagy genes (ATG2A, ATG5, ATG7, ATG12), it increased autophagic flux through suppression of mTOR phosphorylation at Ser2448. Given that NPNT is a miR-378a target and a key glomerular basement membrane component, we investigated its role in autophagy regulation. NPNT knockdown reduced ATG2A, ATG7, and BCN1 expression, but paradoxically increased autophagic flux, independent of mTOR, accompanied by enhanced ERK1/2 phosphorylation. These findings reveal a dual-layered regulatory network in which miR-378a promotes autophagy via mTOR inhibition, whereas NPNT modulates autophagy probably through MAPK-dependent signaling. Our results highlight the complex interplay between miRs, extracellular matrix components, and intracellular signaling pathways in podocyte autophagy. Dysregulation of these pathways in kidney disease may reflect both adaptive and maladaptive responses, providing mechanistic insights and potential therapeutic targets to preserve glomerular filtration barrier integrity in immune-mediated kidney disease.

BackgroundKidney cell lines are widely used to model kidney physiology and disease; however, their gene expression profiles may differ from primary cells due to immortalization, culture conditions, or experimental treatments. Determining whether a cell line resembles its native cell type is critical for interpreting in vitro findings. We developed a transcriptome-based approach that matches bulk RNA-seq data from kidney cell lines, primary cells, or tissues to reference cell types derived from single-cell RNA-seq (scRNA-seq) datasets. MethodsReference transcriptomic profiles were generated from two human and two murine kidney scRNA-seq datasets by pseudobulk aggregation. Bulk RNA-seq data from microdissected kidney tissue, non-kidney negative controls, and kidney cell lines were matched to these references using three statistical similarity measures (Spearman correlation, Euclidean distance, Poisson distance) and three machine learning classifiers (Random Forest, XGBoost, TabPFN). Each was assessed with global gene expression, curated kidney marker gene lists, and the most variable genes. Matching accuracy was evaluated through a three-step validation strategy: within-dataset matching, cross-reference comparison, and validation against primary kidney tissue and negative controls. ResultsGene expression rank-based Spearman correlation and TabPFN, a foundation model for tabular data, emerged as the most accurate and specific approaches, particularly with curated kidney marker gene lists. Both methods correctly identified microdissected kidney tubule segments and were robust against non-kidney negative controls. Applied to commonly used kidney cell lines, OK cells retained proximal tubule identity, particularly under shear stress, while other proximal tubule lines (HK-2, HKC-8, HKC-11) showed inconsistent matching. Collecting duct-derived mIMCD-3 maintained stable similarity across passages, culture conditions, and genetic modifications. ConclusionWe provide two complementary implementations: CellMatchR, an accessible web-based tool using Spearman correlation for routine use, and comprehensive scripts for TabPFN-based matching (link will be added after peer reviewed publication). Together, these resources enable researchers to make informed decisions about kidney cell culture model selection, interpretation, and stability. Translational StatementKidney cell lines are fundamental tools in nephrology research, yet their transcriptomic similarity to native cell types is rarely validated systematically. We demonstrate that combining bulk RNA-seq data with single-cell reference datasets enables robust assessment of cell line identity using gene expression-rank-based correlation and machine learning approaches. By providing a comprehensive evaluation of matching methods, curated kidney marker gene lists, and reference datasets, our study serves as both a practical resource and a methodological framework for the kidney research community, facilitating informed selection of cell culture models, quality control of experimental conditions, developing new experimental cell culture models, and more reliable translation of in vitro findings to kidney physiology and disease.

Genome-wide association studies (GWAS) of ischemic heart disease (IHD) remain underrepresented in Central Asian populations. We conducted a pilot GWAS of IHD with co-occurring arterial hypertension in a Kazakh cohort to identify candidate loci for future replication. A case-control GWAS was performed in 451 individuals (236 cases and 215 controls). Genotyping was conducted using the Illumina Infinium Global Screening Array-24 v3.0. Association testing was performed using a logistic regression under an additive genetic model adjusted for age, sex and the first ten principal components (PC1 - PC10). Multiple testing correction was applied using the Bonferroni adjustment. As an additional analysis, knowledge-guided GWAS (KGWAS) followed by MAGMA gene-based testing was used to prioritize candidate genes. After quality control, 345 371 variants were tested. Two loci surpassed the Bonferroni-corrected genome-wide significance threshold: rs28898595 at the UGT1A locus (effect allele C; OR = 0.33, 95% CI = 0.23 - 0.49; p = 3.01x10-8) and rs28709059 in the intron region of the ACTR3C gene (effect allele C; OR = 0.4, 95% CI = 0.29 - 0.55; p = 4.08x10-8). Several additional loci showed suggestive evidence of association. In gene-level analysis, the CSMD1 gene demonstrated a significant association signal in MAGMA consistent with the European (p = 1.16x10-11) and East Asian (p = 9.07x10-11) LD reference panels. This pilot study identifies genome-wide significant loci (UGT1A, ACTR3C genes) and supports CSMD1 gene as a prioritized candidate gene for the complex phenotype of IHD associated with co-occurring arterial hypertension in the Kazakh cohort. These findings are preliminary and require replication in larger Central Asian cohorts and further functional validation.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection worldwide and a leading cause of inflammatory reproductive tract disease and infertility in women. Much of the tissue damage associated with genital chlamydial infection arises from host inflammatory responses rather than direct bacterial cytotoxicity. Epithelial cells lining the female reproductive tract represent the primary host cells infected during chlamydial infection and play key roles in initiating innate immune responses. Among the cytokines produced by infected epithelial cells, type-I interferons have emerged as important regulators of host defense and inflammatory signaling; however, the specific contribution of interferon-{beta} (IFN-{beta}) to epithelial transcriptional responses during chlamydial infection remains incompletely defined. In the present study, we investigated the role of IFN-{beta} in coordinating epithelial immune signaling networks during infection with Chlamydia muridarum. Using wild-type murine oviduct epithelial cells (OE-WT) and IFN-{beta}-deficient epithelial cells (OE-IFN{beta}-KO), we performed pathway-focused RT{superscript 2} Profiler PCR array analyses examining transcriptional responses across four biological pathways: (1) innate and adaptive immune responses, (2) type-I interferon signaling, (3) inflammatory and autoimmune responses, and (4) fibrosis-associated pathways. Infection of OE-WT cells resulted in coordinated induction of cytokines, chemokines, and interferon-stimulated genes associated with antimicrobial defense and immune cell recruitment. In contrast, IFN-{beta} deficiency resulted in widespread dysregulation of these transcriptional programs, including reduced induction of interferon-responsive chemokines such as CCL5 and CXCL10, altered inflammatory cytokine expression, and transcriptional signatures consistent with enhanced tissue remodeling responses. Notably, IFN-{beta} deficiency resulted in increased TNF expression accompanied by reduced IL-6 induction, suggesting disruption of balanced inflammatory signaling networks. Pathway analyses further revealed dysregulated expression of fibrosis-associated genes including Serpine1, Ctgf, and Eng in IFN-{beta}-deficient epithelial cells, indicating potential mechanisms linking interferon signaling to tissue remodeling during infection. Collectively, these findings identify IFN-{beta} as a central regulator of epithelial immune networks during chlamydial infection and suggest that disruption of IFN-{beta} signaling may promote inflammatory and fibrotic pathology within the female reproductive tract. Author SummarySexually transmitted infections caused by Chlamydia trachomatis are a major cause of infertility worldwide. Although antibiotic treatment can eliminate the bacteria, damage to the reproductive tract often results from the bodys own immune response to infection. The epithelial cells lining the reproductive tract are the first cells infected and play an important role in initiating immune responses. In this study, we investigated how a specific immune signaling molecule, interferon-{beta} (IFN-{beta}), regulates the gene expression programs activated in epithelial cells during chlamydial infection. Using pathway-focused gene expression arrays, we found that IFN-{beta} coordinates multiple immune pathways, including interferon signaling, inflammatory cytokine networks, and genes associated with tissue remodeling. When IFN-{beta} was absent, many of these pathways became dysregulated, resulting in altered inflammatory signaling and gene expression patterns linked to fibrosis. These findings suggest that IFN-{beta} functions as a key regulator that helps balance protective immune responses with inflammatory processes that can damage reproductive tissues during infection.

BackgroundPersistent neurohormonal activation is a key driver of maladaptive remodeling and disease progression in heart failure (HF). Sodium-glucose cotransporter 2 inhibitors (SGLT2is) confer robust renoprotective effects in HF; however, the extent to which these benefits involve modulation of renal neurohormonal activity remains unclear. We hypothesized that SGLT2i-mediated renoprotection in HF is associated with attenuation of excessive renal neurohormonal activation. MethodsMale rats with myocardial infarction-induced HF and sham controls were fed standard chow or chow containing empagliflozin (EMPA, 300 mg/kg) for four weeks, followed by assessment of renal inflammatory and neurohormonal markers. Parallel in vitro studies in THP-1 macrophages and HK-2 proximal tubule cells evaluated the direct effects of EMPA on norepinephrine (NE)-dependent tubular inflammatory signaling. ResultsHF rats displayed higher renal cortical renin gene expression and angiotensin II concentrations, which remained unaffected by EMPA. Conversely, EMPA normalized the elevated urinary NE excretion and renal cortical NE content observed in HF rats. Given the inflammatory role of sympathetic hyperactivity, we assessed renal macrophage polarization. EMPA-treated HF rats showed reduced expression of pro-inflammatory markers (Tnf, Ccr2, Nos2, Il-6) and increased expression of markers associated with a reparative macrophage profile (Arg1, Mrc1, CD163), supported by higher CD206 macrophages in kidney sections. While EMPA did not directly alter THP-1 macrophage activation in vitro, it significantly reduced NE-induced SGLT2 expression and interleukin-6 (IL-6) release by HK-2 human proximal tubule epithelial cells. ConclusionThese findings support a model in which SGLT2 inhibitors confer renoprotection in HF by suppressing renal sympathetic hyperactivity, independently of the intrarenal renin-angiotensin system, thereby disrupting a maladaptive renal neuro-epithelial-immune axis and promoting a reparative macrophage phenotype. CLINICAL PERSPECTIVE Whats new?O_LIThis study identifies a renal neuro-epithelial-immune axis underlying empagliflozin-mediated renoprotection in heart failure. C_LIO_LIEmpagliflozin reduces renal cortical and urinary norepinephrine levels in heart failure without altering intrarenal renin-angiotensin system activity, revealing a distinct neurohumoral target of SGLT2 inhibition. C_LIO_LIThis sympatholytic effect is associated with a shift in renal macrophages toward a reparative (M2) phenotype, without changes in total macrophage abundance. C_LIO_LIEmpagliflozin blocks norepinephrine-induced SGLT2 upregulation, limiting proximal tubular glucose reabsorption and IL-6 production, and linking sympathetic signaling to renal inflammation. C_LI What are the clinical implications?O_LIOur findings provide a mechanistic basis for the additive cardiorenal benefits of SGLT2 inhibitors in heart failure, beyond conventional RAS-directed therapies. C_LIO_LITargeting renal sympathetic-driven inflammation may help preserve kidney function and attenuate the progression of cardiorenal syndrome. C_LIO_LISuppression of a renal neuroinflammatory pathway may help explain the early and sustained benefits of SGLT2 inhibitors across heart failure phenotypes, including nondiabetic patients. C_LI

ABSTRACT/SUMMARYVascular remodeling within the developing fetus and placenta is essential for supporting the growth and function of emerging tissues and organs. Pericytes (PCs) play a central role in stabilizing and maturing microvascular networks by extending along endothelial cells (ECs) and reinforcing vessel integrity. In the placenta, as in other organs, PC-EC communication is mediated in part by platelet-derived growth factor-BB (PDGF-BB) signaling, which governs PC differentiation, proliferation, migration, and survival, ultimately enabling their recruitment and retention along capillaries. In this study, we identified progressive PC investment along feto-placental capillaries in both murine and human tissues across gestation, supported by morphological and molecular evidence. Placental PCs displayed phenotypic heterogeneity comparable to that observed in the brain and heart, suggesting conserved diversity across organ systems. In addition to characterizing PC dynamics, we examined the expression of recently identified soluble PDGF Receptor-{beta} (sPDGFR{beta}) isoforms. These variants were detected at the protein and transcript levels in mouse and human placentas, as well as in a murine trophoblast-embryonic stem cell (TESC) differentiation model that recapitulates aspects of early placental vascular development. Within this model, sPDGFR{beta} expression was independent of ADAM10 activity and exogenous growth factors during early vessel formation but was markedly upregulated during hypoxia. To assess how elevated sPDGFR{beta} might influence PDGF-BB signaling, we exposed TESCl-derived vascular networks to excess PDGF-BB with or without a sPDGFR{beta} mimetic. PDGF-BB alone reduced full-length PDGFR{beta} levels while increasing receptor phosphorylation, consistent with known ligand-induced regulatory mechanisms. Inclusion of the sPDGFR{beta} mimetic shifted these responses toward baseline, suggesting a potential modulatory or feedback role for soluble receptor variants. Together, these findings demonstrate that PCs are progressively recruited to placental capillaries and exhibit diverse phenotypes during development, and that soluble PDGFR{beta} isoforms may modulate PDGF-BB signaling in a manner sensitive to oxygen tension. Understanding these mechanisms provides insight into the regulation of placental vascular maturation and may inform strategies to improve human health by targeting disorders rooted in impaired placental development.

Introduction: Podocyte injury is central to the pathogenesis of most glomerulonephritides (GN) and causes segmental glomerulosclerotic lesions that predict progression in IgA Nephropathy (IgAN). Recent advances in high-resolution microscopy and AI-assisted image analysis have enabled detailed quantification of podocyte foot process (FP) morphology. However, whether nanoscale podocyte morphometrics can predict disease progression or treatment response in GN has not been investigated. Aim: To evaluate whether nanoscale podocyte morphometric parameters predict clinical characteristics, disease progression, and treatment response in GN, with a focus on IgAN. Method: Podocyte morphometrics were analyzed in kidney biopsies from patients with GN using high-resolution microscopy and the deep learning-based tool Automatic Morphometric Analysis of Podocytes (AMAP). Four morphometric parameters were quantified: slit diaphragm length (SDL), FP area, FP circularity and FP perimeter. These parameters were correlated with clinical characteristics, conventional electron microscopy (EM) findings and longitudinal follow-up data. Results: The study included 37 patients with GN from Danderyd University Hospital (Stockholm, Sweden), with IgAN representing the largest diagnostic subgroup (n = 19). The median follow-up for the cohort was 3.0 years. SDL correlated significantly with urine albumin-to-creatinine ratio (uACR; p = 0.021), whereas conventional EM measurements did not (p = 0.22). Within the IgAN subgroup, lower SDL was associated with a steeper decline in eGFR, higher FP area with increased long-term proteinuria, and higher FP circularity with improvement in uACR during the first year. The association between lower SDL and eGFR decline remained as a trend in IgAN patients not treated with corticosteroids (p = 0.068) but was absent in the treatment group (p = 0.59). Conclusion: In this proof-of-concept study, nanoscale podocyte morphometrics demonstrated greater sensitivity than conventional EM in quantifying podocyte injury and predicting progression in IgAN. These findings suggest that high-resolution morphometrics may improve risk stratification in IgAN but require validation in larger, independent cohorts before clinical implementation.

ObjectiveTo establish a fetal lamb model of complex gastroschisis and characterize the impact on the intestines over time. Summary Background DataGastroschisis is a congenital abdominal wall defect and in its complex form is associated with serious morbidity. Robust large-animal models may help understanding are lacking. MethodsAt gestational day 75, gastroschisis was induced by creating a 1-cm abdominal wall defect reinforced by a silicone ring. Fetuses were assessed either at term or at mid-gestation (13-21 days post-induction). The primary outcome was complex gastroschisis occurrence, defined by bowel stenosis, atresia, volvulus, perforation or necrosis; otherwise classified as simple. At mid-gestation, occurrence was compared between early (13-16 days) and late (17-21 days) intervals. Secondary outcomes included prenatal ultrasound findings, in vivo bowel motility and morphology, ex-vivo bowel contractility, amniotic fluid composition, and histology across complex, simple, and normal groups. ResultsGastroschisis was induced in 32 fetuses. At term (n=14), all survivors (7/14; 50%) had complex gastroschisis, with impaired bowel motility, altered enteric neural contractile responses and smooth muscle remodeling. At mid-gestation (n=18), complex gastroschisis occurred more frequently in the late than in the early group (71% vs. 11%; p=0.035). Mid-gestation gastroschisis fetuses showed greater intra-abdominal bowel dilatation on ultrasound and higher amniotic fluid digestive enzyme levels compared with non-operated littermates, with the greatest dilation observed in complex gastroschisis. ConclusionsThis model consistently reproduces complex gastroschisis in term survivors. After induction, complex gastroschisis occurrence increases with disease duration and is accompanied by structural and functional bowel changes.

Smooth muscle (SM) contraction is well known to be regulated by the reversible phosphorylation of the myosin regulatory light chain. However, SM force generation and relaxation are often uncoupled from myosin phosphorylation levels (e.g. the latch-state), indicating that additional regulatory mechanisms must be at play. The precise effects of the actin binding protein caldesmon (CaD) on SM force production and relaxation remain ambiguous, largely due to contradictory findings in experiments performed at the tissue level. To date, there are no studies that have measured the effects of CaD on force and relaxation at the molecular level. Here, we use a laser-trap assay to measure the force produced by SM myosin molecules in the presence and absence of CaD. Measurements were performed before and during myosin dephosphorylation, thus simulating SM contraction and relaxation in-vitro. We demonstrate that CaD inhibits force generation, most likely through competitive inhibition of actomyosin binding while simultaneously introducing a resistive load via tethering of actin and myosin. We also establish CaD as a potentiator of relaxation, increasing force decay rate during myosin dephosphorylation. Finally, we show that CaD directly modulates the dependence of myosin-actin mechanics on myosin phosphorylation levels. These findings refine our understanding of SM regulation, highlighting CaD not merely as a passive structural stabilizer, but as a critical regulatory component of force development and relaxation. Ultimately, understanding these mechanical functions offers new perspectives on pathophysiologies involving SM, such as asthma, hypertension, and gastrointestinal disorders, potentially guiding targeted therapeutic strategies. SIGNIFICANCE STATEMENTSmooth muscle (SM) is responsible for controlling the internal diameter of blood vessels and viscera. Understanding the precise regulation of SM relaxation by actin-binding proteins remains a fundamental lacuna in physiology. Using a molecular mechanics chamber to manipulate the biochemical milieu during active measurements, we demonstrate, for the first time at the molecular level, that caldesmon (CaD) acts as a mechanical modulator that inhibits force generation and accelerates relaxation of SM myosin ensembles. Our results provide a molecular basis for resolving previous contradictory findings reported in tissue-level experiments. Ultimately, understanding the role of contractile and regulatory proteins of SM will provide the basis for understanding SM disorders, such as hypertension and asthma, and guide the development of targeted therapeutic strategies.