American journal of physiology. Renal physiology最新文献

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.

Prostate inflammation and fibrosis are linked to lower urinary tract symptoms (LUTS) in men. Uropathogenic Escherichia coli (E. coli) infection of the mouse prostate triggers a cascade of immune responses that drive inflammation and fibrosis. A recent study found that lysosome 2-positive (LYZ2+) myeloid cells (fibrocytes) are recruited in a C-C motif chemokine receptor 2 (Ccr2)-dependent manner to the E. coli-infected prostate, where they produce collagen. This study aims to identify factors that drive collagen synthesis in LYZ2+ myeloid cells during E. coli infection. We show that lymphocyte antigen 6 family member (Ly6C)^hi monocytes and their maturation products, Ly6C^hi macrophages, infiltrate the E. coli infected prostate in a Ccr2-dependent manner, that monocytes in the infected prostate produce Tgfb1 RNA, and that E. coli infection activates TGFB signaling and collagen synthesis in LYZ2+ cells. Blockade of macrophage colony-stimulating factor (M-CSF), a factor required for monocyte differentiation into macrophages, reduces macrophage density, TGFB signaling in LYZ2+ cells, and collagen density in the E. coli-infected prostate. These findings highlight a critical role of macrophages in activating collagen synthesis in fibrocytes to drive a fibrotic response to E. coli infection in the mouse prostate.NEW & NOTEWORTHY Prostatic/urethral fibrosis is an emerging cause of urinary voiding dysfunction in aging men and has been linked to prostate inflammation, but cellular mediators and mechanisms of this process are incompletely understood. We provide evidence that Ly6C^hi monocytes and LYZ2+ myeloid cells are recruited to the E. coli infected mouse prostate. Macrophages in the infected prostate synthesize TGFB1 and stimulate collagen synthesis in LYZ2+ cells.

This study tested the hypothesis that ad libitum fluid intake during a 2-h occupational heat stress simulation attenuates increases in renal oxidative stress, inflammation, and acute kidney injury (AKI) risk compared with fluid restriction. Thirteen healthy adults (5 women) completed two 2-h occupational heat stress simulations consisting of eight circuits of treadmill walking and rowing exercise in a wet bulb globe temperature of 33.1 ± 0.2°C. In the drinking trial (Drink), participants were provided 237 mL of a noncaloric sport drink every 15 min and drank ad libitum. In the fluid restriction trial (No Drink), no fluid was provided. Urine and blood samples were analyzed for markers of oxidative stress (thioredoxin-1, TRX-1), inflammation (monocyte chemotactic protein-1, MCP-1), and AKI risk ([IGFBP7·TIMP-2]). During Drink, ad libitum fluid intake was 1,394 ± 316 mL, and reductions in body weight were greater in No Drink (1.3 ± 0.8% vs. 2.8 ± 0.9%, P < 0.001). Peak core temperature was not different between Drink (38.5 ± 0.4°C) and No Drink [38.6 ± 0.4°C, mean difference (upper, lower CI): 0.1 (0.4, -0.1)°C; P = 0.346]. Urine, but not serum TRX-1 (P = 0.254), was elevated at postrecovery and recovery (P < 0.001) but not different between trials (P = 0.743). Serum and urine MCP-1 were elevated at postrecovery and recovery (P < 0.001) but not different between trials (P ≥ 0.407). Urine [IGFBP7·TIMP-2] was elevated at postrecovery and recovery (P < 0.001) but not different between trials (P ≥ 0.096). Ad libitum fluid intake during a 2-h occupational heat stress simulation does not modify biomarkers of systemic and renal oxidative stress and inflammation, nor AKI risk, compared with when fluid is restricted.NEW & NOTEWORTHY Ad libitum fluid intake, consistent with occupational hydration recommendations, does not attenuate renal oxidative stress, inflammation, or acute kidney injury risk during simulated heat stress. These findings challenge the assumption that current hydration recommendations are protective, suggesting that additional strategies are needed to mitigate heat-induced kidney injury in occupational settings.

Lupus nephritis (LN) is the renal manifestation of the autoimmune disease systemic lupus erythematosus (SLE). LN is characterized by a dysregulated immune system, the presence of autoantibodies, and renal immune complex deposits, which collectively injure the kidney. However, novel nonimmune pathogenic mechanisms of human LN are continuously uncovered, presenting new challenges as well as opportunities for intervention. Iron accumulation and ferroptosis in the glomerular structure and renal tubules are relatively newly identified pathological features in LN. Ferroptosis is an iron-dependent nonapoptotic form of regulated cell death. Unlike generic oxidative stress mechanisms, ferroptosis occurs when the cellular antioxidative mechanism cannot suppress the oxidation of the cell membrane eventually leading to cell membrane rupture. Since iron absorption and recycling occur in the renal tubules, the renal tissue is particularly susceptible to ferroptosis. Ferroptosis inhibitors that reduce toxic phospholipid hydroperoxides to their corresponding nontoxic alcohols, or trap radicals in phospholipid bilayers, have improved disease outcomes in murine models of SLE/LN. In this review, we discuss mechanisms by which iron accumulation and ferroptosis perpetuate pathology in LN. These studies suggest that ferroptosis is very likely integral to parenchymal cell dysfunction in LN and a novel therapeutic target. The goal of this review is to introduce the fundamentals of iron biology and ferroptosis to clinicians and basic scientists and spur research to identify intracellular proferroptotic enzymes and their protein conjugates as potential targets to improve LN.