American journal of physiology. Renal physiology最新文献

Urinary incontinence (UI) imposes a significant healthcare burden and reduces quality of life. Contributing factors such as aging, pregnancy/childbirth, stress, and injury are recognized, but incomplete understanding of underlying mechanisms limits new therapies. Hedgehog (Hh) signaling has been implicated in lower urinary tract development, but its specific role in female continence mechanisms has not been fully characterized. Here we investigate the functional and molecular consequences of reduced Hh signaling using Gli2^+/-;Gli3^Δ699/+, (Gli mutant) female mice. We assessed spontaneous voiding through void spot assays and uroflowmetry, then assessed contractility in bladder and urethral tissues ex vivo. Female Gli mutant mice display more small volume voids than wild type mice. Gli mutant female bladder had reduced strength of contraction to electrical field and cholinergic stimuli while the urethra had reduced sensitivity to serotonin mediated contraction, but not to phenylephrine. Thus, unique changes to bladder and urethral contractility dynamics are present in Gli mutant mice and are dependent upon type of stimuli. Further, expression of serotonin transporter (Sert) mRNA was increased in Gli mutant urethra compared to wild type. Uroplakin IIIa, typically localized to bladder urothelium, was ectopically expressed in urethral urothelium of adult but not embryonic (E) day 16 Gli mutant mice. These findings highlight a previously uncharacterized role of Hh signaling in maintaining female lower urinary tract function and urothelial patterning, and support further investigation of its contribution to continence.

Chronic kidney disease (CKD) is a risk factor for cardiovascular disease (CVD), partly due to phosphate-induced vascular calcification. Fetuin-A stabilizes calcium-phosphate complexes into calciprotein particles (CPPs), preventing precipitation, but CPPs can mature into crystalline particles that drive calcification, particularly in CKD. In this study, we investigated whether citrate, a calcium chelator, could mitigate CPP-induced vascular calcification in vitro. Vascular Smooth Muscle Cells (VSMC) were incubated with CPPs containing varying citrate concentrations. We quantified calcification using calcium assays and characterized CPPs using spectrophotometry, DLS, cryo-TEM, ED, Raman spectroscopy, EDX, and MS. At the highest citrate concentration, reduced calcification by 88% versus standard CPPs (p < 0.0001). CPP maturation was delayed, and mean diameter was 9% lower (216 ± 2 nm vs. 236 ± 6 nm; p = 0.0022). Cryo-TEM showed a transition from primary to secondary CPPs with preserved morphology. Hydroxyapatite was detected by ED in the standard and high-citrate CPPs, with the latter showing a significant lattice shift. An increased mineral-to-protein ratio was observed by Raman spectroscopy and protein-to-calcium assays. EDX demonstrated unchanged Ca/P ratios, but differences in Ca (p = 0.0003), P (p < 0.0001), Na (p < 0.0001), and Cl (p < 0.0001). Finally, proteomics revealed 18 proteins enriched in standard CPPs (fold-changes -1.2 to -3.4; FDR < 0.05), including lipid-related apolipoproteins APOM, APOA1, APOA2, APOC3, and APOE. These data indicate that citrate remodels CPPs towards a less calcifying phenotype, highlighting its potential as a therapeutic strategy against vascular calcification in CKD.

We investigated the interplay between the mineralocorticoid aldosterone and a mutation mimicking Liddle syndrome in the control of the processing of the epithelia Na⁺ channel (ENaC) in mouse kidneys. Rates of processing were assessed by the appearance of the cleaved form of the γENaC subunit. Cleaved γENaC increased with decreasing dietary Na intake and with administration of aldosterone. Measurements taken from isolated tubules indicated that enhanced processing was similar in connecting tubules and in late distal convoluted tubules. In a mouse model with a truncated βENaC subunit (Liddle mice), levels of cleaved γENaC were similar in wild-type (WT) and Liddle animals. The amounts of the full-length form of the subunit were lower in the Liddle mice on control and high-Na diets. Infusion of a low dose of aldosterone produced similar increases in cleaved γENaC in WT and Liddle mice, while with maximal doses, levels in Liddle animals were 35% higher than in WT. Acute Na repletion of Na-depleted mice decreased cleaved γENaC with a time constant of 5 hours. Rates of decrease were similar in WT and Liddle genotypes. The Liddle's mutation produces modest changes in ENaC processing, and a major effect of the mutation is on the activation of processed channels.

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

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors improve outcomes in diabetic nephropathy (DN) and cardiovascular disease. To elucidate the underlying protective mechanisms, we explored the hypothesis that the lectin complement pathway, specifically through collectin kidney 1 (CL-K1), plays a critical role in early DN. We assessed: 1) CL-K1 abundance in the kidneys of diabetic mice, 2) the impact of SGLT-2 inhibition on CL-K1 and complement activation, and 3) the effect of CL-K1 deficiency on albuminuria and epithelial injury. Streptozotocin (STZ) was used to induce diabetes in male wild-type (WT) and CL-K1 knockout (KO) mice in three substudies. The studies evaluated time-dependent effects (14 days and 35 days), the effect of dapagliflozin, and the effect of COLEC11 gene deletion. Urine, plasma, and organ samples were analyzed for CL-K1 mRNA and protein levels by quantitative polymerase chain reaction (qPCR), Western blotting, and in situ hybridization. STZ-treated mice displayed elevated plasma glucose, increased kidney weight, urinary excretion of albumin, and kidney injury molecule-1. Kidney and plasma CL-K1 protein levels increased significantly and progressively in STZ-treated mice, whereas hepatic CL-K1 remained unchanged. Plasma mannose-binding lectin-C (MBL-C) and mannose-binding lectin (MBL)-associated serine proteases (MASP-1), as well as kidney CL-K1 and MBL-C mRNAs increased following STZ. Dapagliflozin reduced fasting blood glucose (P < 0.01), kidney (P < 0.05), and hepatic (P < 0.05) CL-K1 protein abundance, and plasma MBL-C levels (P < 0.01), without affecting mRNA levels. CL-K1 KO STZ mice exhibited a transient significant reduction in the albumin-creatinine ratio after 2 wk compared with WT STZ (P < 0.0001). Dapagliflozin reduced diabetes-related lectin pathway molecule levels in liver and kidney, potentially protecting the kidney through inhibition of this pathway.NEW & NOTEWORTHY This study investigated how SGLT-2 inhibitors (SGLT-2is) could improve kidney outcomes in early stages of diabetic nephropathy (DN) by examining changes in and contribution of collectins. We found that levels of collectin [CL-K1 and mannose-binding lectin (MBL)] increase in the kidneys of diabetic mice as injury progresses, and SGLT-2is reduce these levels. Notably, CL-K1 deletion offered temporary protection against filtration barrier injury. These findings suggest that collectins are involved in the early stages of DN, and SGLT-2is might protect the kidneys by influencing collectin activity and reducing inflammation.

Angiotensin type 2 receptor (AT₂R) activation promotes natriuresis, thereby contributing to sodium balance and blood pressure regulation. In this study, we explored a novel intermediate in AT₂R signaling, protein phosphatase 2A (PP2A) regulatory subunit B55α. Probing for PP2A subunit-AT₂R interactions in vivo using proximity ligation assays on kidney sections prepared from rats after renal interstitial (RI) infusion of vehicle or the AT₂R agonist compound 21 (C21), we observed a sixfold increase in AT₂R-B55α interaction in apical brush border membranes of renal proximal tubule cells (RPTCs) with C21 stimulation. In vitro binding of purified AT₂R and B55α supported a direct interaction between these two proteins. To test whether B55α is required for renal AT₂R signaling, we administered siRNA targeting B55α to rats in vivo by RI infusion, which resulted in a ∼70% decrease in B55α in proximal but not distal tubules. Remarkably, RPTC B55α knockdown abolished C21-induced natriuresis and simultaneously prevented C21-mediated AT₂R redistribution to apical brush border membranes and sodium transporter Na⁺/H⁺ exchanger-3 (NHE-3) retrieval. Furthermore, B55α knockdown prevented cellular Src (c-Src) phosphorylation with C21 stimulation, increased AT₂R colocalization with lysosomal marker lysosomal-associated membrane protein 1 (LAMP1) by four to sixfold, and reduced AT₂R colocalization with early and late endosomal markers early endosome antigen 1 (EEA1) and Rab7 by 50%. In conclusion, our results show that RPTC PP2A B55α binds to activated AT₂R and is required for AT₂R signaling to natriuresis and AT₂R intracellular trafficking. We thus establish RPTC PP2A B55α as a key AT₂R signaling intermediate and potential therapeutic target to promote sodium excretion in hypertensive individuals.NEW & NOTEWORTHY Protein phosphatase 2A (PP2A) B55α is required for angiotensin type 2 receptor (AT₂R) natriuretic signaling and AT₂R intracellular trafficking in renal proximal tubule cells (RPTCs). RPTC PP2A B55α is thus a key AT₂R signaling intermediate and potential therapeutic target to promote sodium excretion in hypertensive individuals. This study introduces knocking down B55α in vivo specifically in RPTCs using renal interstitial infusion of siRNA as a novel and unique approach to investigate physiological protein function in the kidney.

Proximal tubule Na⁺/H⁺ exchanger 3 (NHE3) is tightly regulated by factors controlling extracellular volume homeostasis, blood pressure, and acid-base balance. Emerging evidence suggests that glycemic control-related factors also influence NHE3, supporting the concept of integrated regulation of fluid and glucose handling in the proximal tubule. Accordingly, gliflozins, a class of antidiabetic drugs that inhibit the Na⁺/glucose cotransporter sodium-glucose cotransporter 2 (SGLT2), also inhibit NHE3. We previously demonstrated that NHE3 and SGLT2, but not sodium-glucose cotransporter 1 (SGLT1), colocalize in the proximal tubule apical membrane. However, whether NHE3 and SGLT2 physically associate within a multiprotein complex has remained unclear. This study investigated whether NHE3 and SGLT2 are indirectly linked through their accessory proteins PDZ domain containing 1 (PDZK1) and MAP17. Using nondenaturing electrophoresis, we found that SGLT2 comigrates with NHE3, MAP17, and PDZK1 in a ∼480 kDa complex in rat renal cortex (∼15% of total SGLT2 in the complex population). SGLT1 and Na⁺/K⁺-ATPase, included as negative controls, did not comigrate with NHE3. The NHE3-PDZK1-MAP17-SGLT2 complex is present in both sexes, although distinct NHE3 and SGLT2 banding patterns may reflect sex-associated differences in complex conformation. SGLT2-NHE3 complex formation occurred irrespective of NHE3 phosphorylation at serine 552. Acute empagliflozin infusion altered the relative migration and colocalization of NHE3 with SGLT2 and MAP17 but did not disrupt overall complex integrity, supporting a conformational mechanism underlying its effects on NHE3. Together, these findings suggest that NHE3 and SGLT2 assemble into a multiprotein complex through PDZK1 and MAP17, providing mechanistic insight into the coordinated regulation of sodium, fluid, and glucose reabsorption in the proximal tubule.NEW & NOTEWORTHY This study reveals that NHE3, SGLT2, and their accessory proteins, PDZK1 and MAP17, assemble into a multiprotein complex in the renal proximal tubule. The merged NHE3-SGLT2 signals show distinct intensity in females, suggesting potential sex-specific differences in complex composition or organization. Acute empagliflozin infusion modifies the colocalization patterns of NHE3 with SGLT2 and MAP17 without disrupting complex integrity, supporting a model in which SGLT2 inhibitors regulate NHE3 through conformational rearrangements within this native complex.

Plasma membrane repair is crucial for resealing membrane disruptions from physiological and pathological stimuli to preserve cell integrity and homeostasis. Tubular epithelial cells (TECs) die of unrepaired membrane injury induced by biochemical and immune factors, leading to the onset and progression of acute kidney injury (AKI). Indeed, mammalian cells are equipped with repair pathways and molecular machinery to safeguard cell viability. Depending on the severity and nature of plasma membrane injury, membrane disruptions can be resealed by vesicle-dependent and independent approaches. Besides, the process of membrane resealing is also important for the repair of damaged organelle membranes. Herein, different formats of plasma membrane damage were discussed, highlighting the membrane disruption induced by pore-forming proteins (PFPs), including MAC, perforin, and membrane-damaging proteins in regulated cell death (RCD). Moreover, the mechanisms of plasma and organelle membrane repair to guard against the death of TECs in AKI were discussed, with the aim of proposing novel strategies for AKI therapy.

Acute kidney injury (AKI) frequently progresses to chronic kidney disease (CKD), resulting in long-term renal dysfunction. Although traditional risk factors such as hypertension, diabetes, and aging contribute to this transition, endothelial dysfunction has emerged as a central mediator. In a murine model of severe ischemia-reperfusion injury (IRI), we observed persistent fibrosis with sustained activation of β-catenin signaling, especially when there is an endothelial nitric oxide synthase (eNOS) deficiency. Impaired nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling exacerbated fibrosis by failing to suppress β-catenin activity. RNA sequencing at day 7 post-IRI revealed upregulation of genes related to macrophage differentiation. Flow cytometry demonstrated a biphasic macrophage response: CD11b⁺F4/80^low (M1-like) macrophages predominated on day 1, shifting to CD11b⁺F4/80^high (M2-like) macrophages by day 3, and then resolving by day 7. However, in eNOS knockout mice, M2 macrophages persisted beyond day 3, indicating sustained fibrogenic signaling. In vitro, NO-cGMP-PKG signaling inhibited IL-4-induced M2 polarization via β-catenin degradation, linking endothelial dysfunction to prolonged M2 activation. In vivo, macrophage depletion in eNOS-deficient mice significantly reduced interstitial fibrosis and improved renal function, confirming an important pathogenic role of M2 macrophages in AKI-to-CKD progression. Furthermore, pharmacological enhancement of cGMP signaling using a phosphodiesterase-5 (PDE5) inhibitor from day 7 post-IRI ameliorated fibrosis. Together, these findings suggest that endothelial dysfunction promotes a profibrotic macrophage milieu via Wnt/β-catenin activation and highlights the therapeutic potential of targeting NO-cGMP-β-catenin signaling to prevent CKD progression following AKI.NEW & NOTEWORTHY Our study provides novel insights into the mechanisms underlying the transition from acute kidney injury (AKI) to chronic kidney disease (CKD), with a focus on the role of endothelial nitric oxide synthase (eNOS). We believe our findings, particularly their potential implications for developing new therapeutic strategies to prevent CKD progression, will be of significant interest to your readership and could significantly improve patient care.