American journal of physiology. Renal physiology最新文献

Macrophages have been implicated in causing renal injury in diabetic kidney disease (DKD). Spleen tyrosine kinase (SYK) plays an important role in signaling via a number of cell surface receptors that promote recruitment and activation of myeloid cells, but whether SYK signaling is involved in DKD is unknown. Therefore, we examined the role of SYK in human and experimental DKD. Compared with control tissues, immunostaining of human DKD biopsies showed an accumulation of SYK⁺ cells and CD68⁺ macrophages with a very similar localization on serial sections. In a model of streptozotocin-induced type 1 diabetes in hypertensive Nos3^-/- mice, there was a significant increase in the number of glomerular SYK⁺ cells and CD68⁺ cells, with double staining showing that most SYK⁺ cells were CD68⁺ macrophages. After 15 wk of diabetes, Nos3^-/-Syk^f/f mice exhibited albuminuria, renal function impairment, glomerulosclerosis, tubular injury, and tubulointerstitial fibrosis. By contrast, diabetic mice with myeloid Syk gene deletion (Nos3^-/-Syk^f/fCsf1r^Cre) exhibited a significant reduction in glomerular SYK⁺ cell and CD68⁺ macrophage accumulation, glomerulosclerosis, and tubulointerstitial fibrosis-which were associated with reduced mRNA levels of Mmp12 and Arg1. However, albuminuria, renal function impairment, and tubular injury were unaffected. In conclusion, we have shown that SYK is predominantly expressed by macrophages in DKD and that SYK facilitates macrophage accumulation and activation in DKD resulting in glomerulosclerosis and tubulointerstitial fibrosis.NEW & NOTEWORTHY Spleen tyrosine kinase (SYK) was shown to be mainly expressed by infiltrating kidney macrophages in human and experimental diabetic kidney disease (DKD), suggesting a potential role for SYK in the progression of this disease. Furthermore, myeloid Syk deletion suppressed macrophage recruitment, expression of macrophage elastase (Mmp-12), and development of glomerular and interstitial fibrosis in a mouse model of hypertensive DKD.

Chronic kidney disease (CKD) is characterized by disruption of the native kidney architecture at the cellular and molecular levels, leading to eventual kidney fibrosis. To better resolve the spatial complexity of fibrotic remodeling, we applied spatial multiplexed immunostaining with signal tagging (Spatial MIST), a high-dimensional proteomic platform capable of quantifying protein expression at single-cell resolution across intact human kidney tissue specimens. Using kidney biopsies from control/low-grade and high-grade fibrosis, we profiled 22 protein markers to assess structural alterations, cell-type distribution, and spatial relationships across glomerular and interstitial compartments. Spatial proximity analysis revealed fibrosis-associated reorganization of endothelial and epithelial markers, including increased separation between CD31 and β-catenin and altered clustering of podocyte and immune markers. Integration with unsupervised uniform manifold approximation and projection (UMAP) clustering distinguished discrete cell populations, whereas correlation analysis with kidney function metrics revealed that vimentin and alpha smooth muscle actin (α-SMA) positively correlated with fibrosis severity, whereas Wilms tumor 1 (WT1) expression was inversely correlated with declining kidney function. A graph neural network (GNN) classifier trained on spatial proteomic features further identified megalin, WT1, and vimentin as a top predictor of fibrosis grade. Together, these findings demonstrate the utility of Spatial MIST for capturing the molecular heterogeneity of CKD and uncovering spatial signatures of disease progression. This integrative approach provides a foundation for biomarker discovery and spatially informed classification of kidney pathology.NEW & NOTEWORTHY In this study, we offer a novel spatial analysis of markers relevant to CKD, which may provide useful insights into disease progression. By using this spatial proximity data, we created a GNN model that is capable of classifying disease severity and identifying markers that are most important for its classification. This integrative approach offers a foundation for future studies aimed at developing clinically actionable tools for CKD diagnosis and prognosis.

A comprehensive spatial analysis of kidney metabolism is essential for advancing knowledge of both normal kidney physiology and pathophysiology. The kidney exhibits marked regional differences in bioenergetic demands and substrate utilization, reflecting the distinct functional profiles of each nephron segment. To complement existing approaches with freshly isolated tubules or primary cell cultures, we established and validated an ex vivo respirometry method using structurally preserved kidney slices on a Seahorse XFe24 platform. This protocol avoids tissue disruption or enzymatic digestion and enables simultaneous, region-specific measurements of metabolic fluxes in the cortex, outer medulla, and inner medulla. It provides an integrated readout of the metabolic properties of the cell types present within each anatomical region. We demonstrate the utility of this approach through proof-of-principle studies that profile region-specific metabolic fluxes under hyperglycemic conditions in a mouse model of obesity and type 2 diabetes, as well as the metabolic alterations that accompany the transition from acute ischemic injury to chronic kidney disease. Furthermore, to highlight its relevance for therapeutic discovery, we applied this method to assess the impact of pharmacological hypoxia-inducible factor activation on regional kidney bioenergetics. In summary, this protocol advances the study of kidney metabolism by providing a robust platform for region-specific analysis of kidney respiration and bioenergetics and holds promise for accelerating the development of novel therapies targeting metabolic pathways in kidney disease.NEW & NOTEWORTHY Assessment of regional metabolism in kidney tissue is crucial for understanding normal physiology and disease. We have developed a robust ex vivo method to measure respiration in structurally preserved kidney slices using a metabolic flux analyzer. This approach enables analysis of metabolic fluxes and substrate utilization in the kidney cortex, outer medulla, and inner medulla while maintaining tissue architecture, providing region-specific insights into kidney metabolism with broad applications in disease modeling and therapeutic discovery.

Arginine vasopressin (AVP) is a peptide hormone synthesized in the hypothalamus and secreted by the posterior pituitary. Previous studies toward understanding AVP physiology relied heavily on Brattleboro rats, which have a spontaneous mutation in the Avp gene and lack circulating AVP. However, these rats are difficult to breed due to high neonatal death and behavioral issues, causing commercial breeders to stop production. To address this, we developed a mouse line with tamoxifen-inducible deletion of Avp. We used CRISPR/Cas9 to insert loxP sites into the Avp gene. These mice were then bred with mice expressing a tamoxifen-inducible Cre recombinase. The resulting conditional knockout mice (Avp^flx/flxCre⁺) are viable, fertile, and healthy before induction. Administration of tamoxifen in 8-12-wk-old mice successfully deleted Avp, as confirmed by Sanger sequencing. This deletion caused a significant decrease in urine osmolality, a hallmark of AVP deficiency. The kidney structure remained normal, with no signs of medullary atrophy. In addition, these mice exhibited a substantially decreased expression of the aquaporin 2 water channel (AQP2), which is involved in water reabsorption in the kidney inner medulla. We illustrate the use of this model by using RNA-seq to profile the consequences of Avp deletion on gene expression in the kidney. The curated RNA-seq data can be browsed, searched, or downloaded at https://esbl.nhlbi.nih.gov/Databases/AVP-KO/. In conclusion, we successfully created an inducible Avp knockout mouse line that has been made available to the research community. This model will be valuable for studying water balance regulation, polycystic kidney disease, and the neural, vascular, and metabolic functions of vasopressin.NEW & NOTEWORTHY We developed an inducible Avp knockout mouse line that will be shared with the research community and is likely to be useful for further study of the regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin.

Mineralocorticoid receptor (MR) overactivation plays a crucial role in the pathogenesis of chronic kidney disease, as well as several cardiovascular and arterial diseases. Current studies determined the mechanisms of the beneficial kidney effects of the nonsteroidal MR antagonist finerenone (FN) in a mouse model of Western diet-induced obesity and insulin resistance. Ten-week-old male C57BL/6J mice were fed a low-fat (LF) or a Western diet (WD) for 12 weeks followed by treatment with either vehicle or FN for another 14 weeks (intervention studies) until they were 36 weeks old. Finerenone treatment prevented 1) the increased albuminuria and kidney injury molecule 1 (KIM1); 2) the expanded extracellular mesangial matrix and synaptopodin coverage; 3) fibronectin, collagen IV, CD45, and CD68 immunostaining; 4) glomerular basement membrane disruption, podocyte foot processes effacement, and mitochondrial structural abnormalities; 5) the proinflammatory cytokines [monocyte chemoattractant protein-1 (MCP-1)], innate immunity pathways [Toll-like receptor-2 (TLR2), stimulator of interferon genes (STING), signal transducer and activator of transcription 3 (STAT3)], and fibrosis markers fibronectin, transforming growth factor-β (TGFβ), and plasminogen activator inhibitor-1 (Pail); and 6) the increased kidney cholesterol levels. There was also reduced expression of nuclear receptor estrogen-related receptor-γ (ERRγ) without changes in ERRα in WD-fed mice, whereas both ERRα and ERRγ expression levels increased after finerenone treatment. NADH lifetime analysis showed decreased bound NADH, compatible with decreased mitochondrial oxidative phosphorylation (OXPHOS) in the kidneys of WD-fed mice compared to controls, which was prevented by finerenone treatment. In conclusion, finerenone treatment exhibits a renal protective role and prevents the progression of kidney disease by regulating mitochondrial function, most likely via ERRγ, and reducing lipid accumulation and inflammation. NEW & NOTEWORTHY Finerenone, a nonsteroidal mineralocorticoid receptor (MR) antagonist, has shown promise in protecting against kidney damage in obese, insulin-resistant mice. It effectively prevents albuminuria, inflammation, fibrosis, and mitochondrial dysfunction, while also restoring estrogen-related receptor-γ (ERRγ) expression. These results suggest that finerenone could play a key role in halting the progression of kidney disease by enhancing mitochondrial function and reducing harmful lipid accumulation, offering a potential therapeutic strategy for managing kidney complications in metabolic disorders.

Acute kidney injury (AKI) is prevalent among hospitalized patients. Novel biomarkers are needed to diagnose AKI and target therapies. Endotrophin (ETP) is a molecule released during collagen type VI formation that may promote injury and fibrosis. Although serum ETP elevation has been associated with adverse outcomes in AKI, urinary ETP has not been assessed in AKI, nor has ETP been evaluated in a pediatric population. Urine samples were collected from a tertiary children's hospital. Medical records were reviewed, and patients who met criteria were sorted into three categories: 1) AKI; 2) hospitalized controls; and 3) outpatient controls. ETP was measured using ELISA, and results were corrected to urine creatinine (uETP:Cre). A multivariate linear regression assessed whether demographic variables were independently associated with uETP:Cre. Odds of AKI were assessed in serial uETP:Cre tertiles using a multivariate logistic regression model that adjusted for patient variables. uETP:Cre was elevated in patients with AKI compared with hospitalized patients without AKI (P < 0.05) and outpatient controls (P < 0.0001). Multivariate analysis revealed that age, but not sex, race, or ethnicity independently correlated with uETP:Cre. After adjustment for these variables, the odds ratio for AKI increased with serial uETP:Cre tertiles. Noninvasive measurement of uETP may deliver meaningful information to aid AKI diagnosis. Given that ETP may be both a biomarker and a clinically actionable stimulus of inflammation and fibrosis, future studies are needed to understand the role of elevated ETP in AKI and whether existing ETP-neutralizing antibodies could represent a new avenue of AKI therapy.NEW & NOTEWORTHY Endotrophin (ETP) is a molecule released during the formation of type VI collagen that may promote fibrosis and inflammation. Serum ETP is elevated in acute kidney injury (AKI) and associates with adverse outcomes. Urine ETP during AKI has never been assessed. For the first time, this study demonstrates that urine ETP is also elevated during episodes of AKI, representing a novel, noninvasive AKI biomarker that may be clinically actionable.

The pregnane X receptor (PXR) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. PXR is constitutively expressed in the hypothalamus and kidney, with its physiological function incompletely understood. In this study, we found that treatment with pregnenolone-16α-carbonitrile (PCN), an endogenous PXR ligand, significantly reduced urine volume and increased urine osmolarity in C57BL/6 mice. In contrast, PXR gene knockout (PXR^-/-) mice exhibited impaired urine-concentrating ability, leading to a polyuria phenotype. In addition, treatment of mice with PCN significantly upregulated, whereas PXR gene deficiency substantially reduced, arginine vasopressin (AVP) expression in the hypothalamus. Bioinformatic analysis showed that the mouse AVP gene promoter contains a putative PXR response element (PXRE). The luciferase reporter, ChIP, and electrophoretic mobility shift assays further revealed that PXR can bind to the PXRE, resulting in a significant increase in AVP gene transcription. Collectively, the present study demonstrates that hypothalamic PXR plays a critical role in regulating urine volume, and its activation enhances urine-concentrating capacity primarily by upregulating the expression of AVP in the hypothalamus.NEW & NOTEWORTHY Activation of PXR enhances urine concentration, whereas PXR deficiency diminishes this capacity. PXR is coexpressed with AVP in the hypothalamus, where it upregulates AVP transcription to promote renal water reabsorption. These findings reveal a novel role for PXR in regulating urinary concentration and propose its potential as a therapeutic target for water metabolism disorders, such as diabetes insipidus.

In the kidney, the epithelial sodium channel (ENaC) facilitates sodium absorption in polarized epithelia and is required to maintain salt and water homeostasis. ENaC's apical membrane population is strictly controlled, with loss of this control leading to hyper- or hypotensive disorders such as Liddle's syndrome or pseudohypoaldosteronism type 1, respectively. Retromer and retriever are conserved endosome-localized protein trafficking complexes that mediate recycling of membrane proteins to the cell surface either directly, via recycling endosomes, or via the trans-Golgi network. Protein cargo for recycling is linked to retromer or retriever by sorting nexin (SNX) proteins. We hypothesized that SNX proteins are required for ENaC recycling. Using two epithelial cell lines [Fischer rat thyroid (FRT) and mouse cortical collecting duct clone 1 (mCCDcl1)] and the human embryonic kidney 293 (HEK293) cell line, with transient transfection of human ENaC-encoding plasmids into HEK293 and FRT epithelia, we found that siRNA knockdown of retromer- and retriever-associated sorting nexin (SNX1, 2, 3, 5, and 17) proteins reduced ENaC amiloride-sensitive short-circuit current and reduced ENaC cell surface population, observed using cell surface biotinylation. Coimmunoprecipitation experiments using transiently transfected human ENaC demonstrated a protein-protein interaction with SNX3 in FRT cells and with SNX17 in both FRT and HEK293 cells, suggesting that SNX3 and SNX17 act as cargo binding proteins between ENaC and the retromer and retriever complexes, respectively. Together, our findings suggest that SNX proteins associated with both the retromer and retriever recycling complexes play a role in maintaining ENaC cell surface populations in polarized epithelia.NEW & NOTEWORTHY Cell surface levels of the epithelial sodium channel, ENaC, are controlled by endocytosis and exocytosis pathways. Sorting nexin (SNX) family members facilitate the binding of protein cargo to recycling complexes for sequestration into recycling compartments. We show that knockdown of several SNX proteins decreases ENaC current and cell surface population, and both SNX3 and SNX17 coimmunoprecipitate with ENaC. Our data provide new insights into the control of ENaC cell surface levels and activity.