Renin synthesis and release is the rate-limiting step of the renin-angiotensin-aldosterone system (RAAS) that controls fluid homeostasis. A major activator of the RAAS is a decrease in perfusion pressure within the kidneys, suggesting a link between renal mechanotransduction and renin. However, the identity of the mechanosensor(s) in the kidneys and their physiological significance to the RAAS remain unclear. We find that loss of the force-gated nonselective cation channel PIEZO2 in cells of renin lineage dysregulates the RAAS by elevating renin. We observe that PIEZO2 is expressed in renin-producing juxtaglomerular granular cells and is required for their calcium dynamics in vivo. PIEZO2 deficiency in cells of renin lineage drives renin-dependent and MAS-receptor-dependent glomerular hyperfiltration and regulates the RAAS during acute and chronic blood volume challenges. Collectively, our study identifies PIEZO2 as an essential regulator of juxtaglomerular granular cell calcium activity and renin in vivo.

Key PointsCKD caused after acute cardiorenal syndrome is distinct from CKD due to kidney injury from the ischemia-reperfusion injury model.A cardiac-specific protein, cysteine-and-glycine-rich protein 3, transits plasma after heart injury and is partially responsible for distinct CKD.The cardiac-specific protein, cysteine-and-glycine-rich protein 3, is taken up in the proximal tubular epithelial cells, and interference with this uptake prevents specific changes.BackgroundThe heart and kidney are bidirectionally interacting organs. Because heart and kidney diseases are among the most common human diseases, investigating disease-causing interactions is important. Here, we identified a new role for cardiac-derived cardiac LIM protein, also known as cysteine-and-glycine-rich protein 3 (CSRP3), in acute cardiorenal syndrome.MethodsMice, both wild-type and genetically altered to remove CSRP3 from the myocardium, were subjected to a model of acute cardiorenal syndrome, cardiac arrest and cardiopulmonary resuscitation (CA/CPR), or ischemia-reperfusion injury. Recombinant CSRP3 was administered to mice subjected to ischemia-reperfusion injury, and CSRP3 uptake in the kidney was inhibited by pharmacologic means.ResultsWe found that CSRP3 transits plasma after CA/CPR, and we determined a kidney disease-modifying mechanism in which CSRP3 underwent megalin-dependent endocytosis in the renal proximal tubule and subsequently drove kidney fibrosis. Administration of CSRP3 to mice experiencing kidney injury exclusive of heart injury reproduced the kidney phenotype observed in CA/CPR mice. Genetic deletion of cardiac CSRP3 or proximal tubule megalin ameliorated cardiac arrest-induced chronic kidney injury. Translationally relevant pharmacologic megalin inhibition also ameliorated CSRP3-mediated kidney phenotypic change, and administration of CSRP3 caused transcriptional change in the kidney.ConclusionsWe describe the endocrine role of cardiac CSRP3 in a previously unknown heart-kidney interaction, which directs specific kidney dysfunction and renovascular remodeling after cardiac injury. These investigations elucidate a novel facet of the intricate coupling between the heart and kidney after acute cardiorenal syndrome.

Acute cardiorenal syndrome (CRS) represents a critical intersection of cardiac and renal dysfunction with profound clinical implications. Despite its significance, the molecular underpinnings that mediate cellular responses within the kidney during CRS remain inadequately understood. We used single nucleus RNA sequencing (snRNAseq) to dissect the cellular transcriptomic landscape of the kidney following a translational model of CRS, cardiac arrest/cardiopulmonary resuscitation (CA/CPR) in comparison to ischemia-reperfusion injury (IRI). In each dataset, we found that proximal tubule (PT) cells of the kidney undergo significant gene expression changes, with decreased expression of genes critically important for cell identity and function, indicative of dedifferentiation. Based on this, we created a novel score to capture the dedifferentiation state of each kidney cell population and found that certain epithelial cell populations, such as the PT S1 and S2 segments, as well as the distal convoluted tubule, exhibited significant dedifferentiation response. Interestingly, the dedifferentiation response in the distal nephron differed in magnitude between IRI and CA/CPR. Gene set enrichment analysis (GSEA) of PT response to IRI and CA/CPR revealed similarities between the two models and key differences, including enrichment of immune system process genes. Transcriptional changes in both mouse models of acute kidney injury (AKI) highly correlated with a dataset of human biopsies from patients diagnosed with AKI. This comprehensive single-nucleus transcriptomic profiling provides valuable insights into the cellular mechanisms driving CRS.NEW & NOTEWORTHY Cardiac dysfunction is a common cause of acute kidney injury in a malady called acute cardiorenal syndrome. In a mouse model of acute cardiorenal syndrome called cardiac arrest/cardiopulmonary resuscitation, we characterized, for the first time, the kidney transcriptional landscape at the single-cell level. We developed a novel method for quantifying cell response to injury and found that cells adapted through dedifferentiation, the magnitude of which varied depending on cell type.

Background: Hypertension remains a significant global health issue, contributing to widespread morbidity and mortality, and emphasizing the need for novel treatment strategies (PMID 32024986). At the core of blood pressure regulation lies the distal convoluted tubule (DCT) of the nephron, where sodium balance is carefully controlled by the NaCl cotransporter (NCC). NCC activity is regulated through phosphorylation: when phosphorylated, NCC enhances sodium reabsorption, contributing to higher blood pressure, whereas dephosphorylation promotes sodium excretion, lowering blood pressure. This fine-tuned balance is orchestrated by kinases such as WNK4 and SPAK (PMID 24855283). Current understanding highlights the impact of diet on NCC regulation, with Western diets—often low in potassium—leading to increased NCC activity and phosphorylation, thus contributing to hypertension (PMID 34788509). This highlights the complexity of hypertension and the need to explore additional regulatory mechanisms. In this search for new therapeutic targets, G-protein-coupled receptors (GPCRs), which are the focus of a third of FDA-approved drugs (PMID 29075003), emerge as promising candidates. Recent transcriptomic studies have identified multiple GPCRs in the DCT, suggesting they could play a key role in regulating NCC and, by extension, blood pressure (PMID 34029142). Notably, in vitro studies have shown that GPCRs, such as the calcium-sensing receptor (CaSR), can inhibit Kir4.1 channels (PMID 21084311). In the DCT, Kir4.1 channels function as K+ sensors that modulate NCC activity (PMID 28052988), raising the possibility that GPCR signaling might influence blood pressure regulation through their effects Kir4.1. These findings highlight the importance of exploring GPCR signaling modulation in the DCT as a potential novel therapeutic target for controlling sodium balance and treating hypertension. Methods: To investigate the physiological role of Gq-coupled GPCR activation in the DCT, we used chemogenetic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology (PMID 29351511). This approach enables selective GPCR activation in specific cell types, such as DCT cells, via Cre recombinase technology. We bred DCT-specific inducible Cre recombinase mice (NCC-creERT2) with Gq-coupled DREADD mice to generate DCT-specific DREADD mice. The DREADD receptors can be activated in vivo with the designer drug deschloroclozapine (DCZ), providing a precise tool to study GPCR signaling in the DCT. Results: Immunofluorescence (IF), western blot (WB), and hydrochlorothiazide (HCT) challenge confirmed DREADD expression on the basolateral membrane of DCT cells and preserved NCC function. WB and IF showed that Gq-GPCR activation with DCZ reduced NCC phosphorylation independently of WNK4-SPAK kinase pathway changes. This dephosphorylation was observed even during increased NCC activity/phosphorylation caused by a low-potassium (LK) diet. We then tested whether the effects are direct using ex vivo tubule suspensions from DREADD-expressing mice. These confirmed that DCZ rapidly reduced phosphorylated NCC abundance in normal and LK conditions, consistent with in vivo findings. Conclusions: Gq-coupled GPCR activation in the DCT downregulates NCC activity via a mechanism independent of the WNK4-SPAK pathway, suggesting an alternative GPCR-mediated regulatory pathway. We hypothesize that basolateral Gq-GPCRs in the DCT dephosphorylate NCC through a Kir4.1-involved mechanism. These findings highlight the potential for antihypertensive therapies by targeting GPCR signaling within the DCT, offering innovative strategies for blood pressure management. NIH R01 DK133220, DK51496, U54TR001628.Fondation LeDucq 17CVD05. JBL is supported by a Benjamin J. Lipps Research Fellowship, KidneyCure. JWN is supported by NIDDK DK121737. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in purine metabolism that may influence cellular responses to injury. We evaluated the effects of prophylactic MTAP inhibition in mouse models of ischemia-reperfusion and cisplatin-induced acute kidney injury (AKI). MTAP inhibition was confirmed by the accumulation of methylthioadenosine. Treated mice showed reduced renal injury and decreased tubular damage. Transcriptomic analysis revealed protection from inflammatory and stress pathways while maintaining oxidative phosphorylation, fatty acid metabolism, and epithelial integrity-related genes. Analysis of human single-cell RNA sequencing data from the Kidney Precision Medicine Project indicated that MTAP is highly expressed in kidney injury marker-positive adaptive proximal tubule cells, which display both reparative and maladaptive features during AKI. These findings highlight MTAP as a potential therapeutic target for modulating injury responses in AKI.NEW & NOTEWORTHY We show that prophylactic MTAP inhibition protects against experimental AKI in mice. Transcriptomic data indicate that MTAP inhibition suppresses epithelial stress and maladaptive repair-related gene programs. Single-cell analysis of human AKI biopsies supports a role for MTAP in injured proximal tubule subpopulations, identifying it as a potential therapeutic target in AKI.

Kidney thick ascending limb (TAL) cells reabsorb sodium, potassium, calcium, and magnesium and contribute to urinary concentration. These cells are typically viewed as a single type that recycles potassium across the apical membrane and generates a lumen-positive transepithelial voltage driving calcium and magnesium reabsorption, though variability in potassium channel expression has been reported. Additionally, recent transcriptomic analyses suggest that different cell types exist along this segment, but classifications have varied and have not led to a new consensus model. We used immunolocalization, electrophysiology, and enriched single-nucleus RNA-Seq to identify TAL cell types in rats, mice, and humans. We identified 3 major TAL cell types defined by expression of potassium channels and claudins. One has apical potassium channels, has low basolateral potassium conductance, and is bordered by a monovalent cation-permeable claudin. A second lacks apical potassium channels, has high basolateral potassium conductance, and is bordered by calcium- and magnesium-permeable claudins. A third type also lacks apical potassium channels and has high basolateral potassium conductance, but these cells are ringed by monovalent cation-permeable claudins. The recognition of diverse cell types may resolve longstanding questions about how solute transport can be modulated selectively and how disruption of these cells leads to human disease.

Acute kidney injury (AKI) increases mortality risk and predisposes individuals to chronic kidney disease. Metabolic pathways play a crucial role in AKI pathophysiology. Here, we investigate the potential of methylthioadenosine phosphorylase (MTAP) inhibition as a novel renoprotective strategy in AKI. Using AKI mouse models, we demonstrate that a small molecule MTAP inhibitor significantly reduces kidney injury markers and improves renal histology. RNA sequencing reveals that MTAP inhibition modulates pathways associated with inflammation, oxidative phosphorylation, and cell survival. Additionally, analysis of human single-cell RNA sequencing data links MTAP expression to kidney injury marker in AKI. This study provides evidence of MTAP inhibition as a potential therapeutic strategy for AKI, highlighting metabolic dysregulation as a target for future clinical interventions.

Sex differences in renal tubular salt and water transporters, channels, claudins and regulatory factors are evident all along the nephron. The influence of sex hormones on physiologic dimorphisms has been established in studies removing, inhibiting or restoring sex hormones and their receptors. The influence of the sex chromosome complement (SCC, XY vs. XX) on renal transporter abundance and activity is an open question. We employed the Four Core Genotypes (FCG) mouse model (in which the testis determining SRY gene is deleted from the Y chromosome and inserted onto an autosomal chromosome) to compare abundance of more than fifty renal transporters and regulators in: FXX gonadal females, FXY gonadal females, MXX Sry males, and MXY XYSry males using semi-quantitative immunoblots. In addition to establishing the significant influence of gonadal hormones, we show, for the first time, that SCC contributes to sexual dimorphisms in abundance of renal transporters including: NHE3, SGLT1 and 2, AQP1, mNKAα1 and β1, NCC, and ENaC β and γ subunits. The findings in this FCG model analysis provide the foundation for future studies of the role of sex hormones vs. chromosomes on physiologic parameters including filtration and flow, on transporter covalent modifications, and trafficking in both heath and disease.

Background The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. Methods To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining “Isolation of Nuclei TAgged in specific Cell Types” and NCC (sodium chloride cotransporter)-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting, and performed single nucleus transcriptomics. Results Among 25,183 DCT cells, 70% were from DCT1 and 30% from DCT2. Additionally, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a, Cenpp, and Mki67. Both DCT1 and DCT2 express NCC, magnesium transport genes are more abundant along DCT1; whereas calcium, electrogenic sodium and potassium transport genes are more abundant along DCT2. The transition between these two segments are gradual with a transitional zone where DCT1 and DCT2 cells are interspersed. The expression of the homeobox genes is not consistent between all DCT cells, suggesting that they develop along different trajectories. Conclusion Transcriptomics analysis of an enriched rare cell population using genetically targeted approach offers better clarification of the function and classification. The DCT segment is short, yet, can be separated into two sub-types that serve distinct functions, and are speculated to derive from different origins during development. Significance Statement High-resolution snRNAseq data indicate a clear separation between primary sites of calcium and magnesium handling within DCT. Both DCT1 and DCT2 express Slc12a3 , but these subsegments serve distinctive functions, with more abundant magnesium handling genes along DCT1 and more calcium handling genes along DCT2. The data also provides insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand.

Calcium homeostasis is maintained by the coordination of the intestines, bones, and kidneys. Dietary calcium is absorbed in the intestines, enters the bloodstream, and eventually finds its place in the bone reservoir or undergoes filtration by the glomerulus before being reabsorbed throughout the nephron. The late distal convoluted tubule (DCT2) and connecting tubule (CNT) determine the final rate of urinary calcium excretion, since no calcium reabsorption takes place beyond the connecting tubule (CNT). When excess calcium is excreted in the urine, also known as hypercalciuria, it contributes to the development of osteoporosis and the formation of kidney stones. High dietary potassium intake is strongly associated with a lower risk of kidney stone formation. Yet, the precise mechanism by which potassium intake modulates calcium excretion is not clear. To examine the transcriptional changes linking high dietary potassium intake and calcium transport, we applied single-nucleus RNA-sequencing (snRNA-Seq) in enriched distal nephron cells. To achieve the enrichment, we crossed a Calbindin 1 (Calb1)-driven Cre mouse line with the INTACT (for Isolation of Nuclei TAgged in Specific Cell Types) reporter line, which allows the GFP reporter to be expressed at the nuclear envelope of all calbindin 1-expressing cells. As the Calb1-Cre is constitutively expressed, all cells that have calbindin 1 expression at any time point during development will express the reporter. We examined the GFP expression by immunofluorescence, and found that it is expressed along the entire distal nephron from the distal convoluted tubule (DCT), CNT, to the collecting duct (CD). Male Calb1-Cre-INTACT mice were provided either a normal (NK, 1.05%) or a high (HK, 2%) potassium diet for 4 days and kidneys were snap-frozen for targeted snRNA-Seq using 10X Chromium (n=3 mice per group, targeting 10,000 nuclei per mouse). Our snRNA-seq dataset showed 6 major clusters, including DCT (DCT1 and DCT2), CNT (3 subclusters), CD principal cells (3 subclusters), ɑ-intercalated cells (2 subclusters), β-intercalated cells (2 subclusters), and proliferating cells. We curated a calcium score from the expression of known calciotropic genes ( Slc8a1, Vdr, Trpv5, Calb1, S100g, Ryr2, Trpv6) and used it as an index of calcium handling capacity. The calcium score is the highest in DCT2 and CNT along the distal nephron. In all CNT subclusters of the HK-treated animals, the calcium score is higher compared to the NK-treated animals, suggesting more calcium transport in CNT. Given that HK stimulates aldosterone and causes CNT hypertrophy, we compared the results with mice subjected to low dietary potassium (LK) treatment and metolazone (thiazide) treatment. The aim was to delineate the effects of dietary potassium and aldosterone A comprehensive summary of plasma potassium levels, urinary calcium excretion, aldosterone levels, calcium scores, and CNT morphology has been presented in the table below. Notably, the comparative analysis reveals a significant correlation between aldosterone levels and CNT size, both of which exhibit an inverse relationship with urinary calcium excretion: [Formula: see text] Our results suggest that DCT2 and CNT are the major sites for calcium transport. Yet, CNT exhibit more heightened adaptability in response to physiological or pharmacological perturbations at both transcriptional and morphological levels. Calcium homeostasis coincides with the regulation of aldosterone levels and CNT remodeling. DK51496 and DK133220 to DHE; K01DK121737 and AHA 20CDA35320169 to JWN. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na2EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25-50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. NEW & NOTEWORTHY Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility.

Study objective, hypothesis: The Thick Ascending Limb (TAL) in the kidney is crucial for reabsorbing Na+ and divalent cations like Ca2+ and Mg2+. This reabsorption depends on the apical cotransporter NKCC2, encoded by the Slc12a1 gene. The TAL extends from the outer medulla to the cortex. While typically considered a single functional cell type, recent evidence suggests the presence of two distinct TAL cell types, distinguished by the expression of Claudin 10 and Claudin 16 (PMID: 28028216). Despite these insights, the lack of correlation with functional and morphological data and the absence of a comprehensive model for the cooperative regulation of NaCl and Ca2+/Mg2+ transport remain significant knowledge gaps. Our objective is to couple immunohistochemistry with single nuclei (sn) RNA sequencing technology to uncover unique cell features that are obscure, when a single approach is employed. Methodology: Immunofluorescence analysis was performed on samples from mice, rats, and humans. Additionally, Slc12a1-positive cells were analyzed using sn-RNA sequencing datasets that contained spatial information about cell localization. These datasets were sourced from published studies on mouse (PMID: 31689386) and human samples (PMID: 37468583). Data Immunofluorescence images from mice, rats, and humans revealed mutually exclusive expression patterns of Kir4.1 and ROMK. Apical ROMK was observed only in cells that express Claudin 10, while Kir4.1 was expressed only in cells that express Claudin 16. phospho NKCC2 and CaSR displayed a mosaic pattern, with expression in the Claudin 16 cells throughout the entire TAL and, in both Claudin 10 and Claudin 16 cells only in the cortex. Unsupervised clustering of mouse sn-RNA seq data identified two distinct clusters, TAL Cldn10 and TAL Cldn16, present in both the cortex and medulla. Key Differentially Expressed Genes (DEG) for TAL Cld10 included Avp2r, Wnk4, Spak, Kcnt1 (encoding KCa4.1 or Slo2.2), and genes related to arachidonic acid signaling such as Ptger3 (encoding Ep3), Pla2g7, Cox7a1. Kcnj1 (encoding ROMK) transcripts were found in both clusters, even though ROMK is located apically only in TAL Claudin 10 cells. TAL Claudin 16 cells showed a higher expression of calcium-related genes, including Casr, Pth1r, Vdr, Kl (encoding Klotho), as well as transcripts for Kcnj10 (encoding Kir4.1), Kcnj16 (encoding Kir5.1), Wnk1, Clcnkb, and Bsnd (encoding Barttin). This transcriptional heterogeneity was also observed in human TAL cells. Summary of results Combining morphological and transcriptomic analyses identified two functionally distinct cell types within the TAL, present in both cortex and medulla. Each cell type appears to be associated with specific transport pathways: TAL Claudin 10 cells, express ROMK apically and are thus responsible for generating the transepithelial voltage and for paracellular Na+ reabsorption. They also express more Na+ transport-regulatory genes, including Avp2r and Wnk4, along with genes related to arachidonic acid signaling. TAL Claudin 16 cells mediate Ca2+ & Mg2+ transport via the paracellular pathway, driven by the voltage generated by claudin 10 cells; they express calcium-regulatory genes, including Casr, Pth1r, and Vdr. Interestingly, they also express genes associated with transcellular Na+, K+, and Cl− transport, including Kcnj10 (Kir4.1), Kcnj16 (Kir5.1), Wnk1, and Clcnkb. Together, these results present a new model of discrete but coupled ion transport pathways in the TAL. NIH DK51496 | NIH DK054983 | VA 1I01BX002228 | Global Research Network of Excellence, Leducq Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic demyelination and oligodendrocyte loss deprive neurons of crucial support. It is the degeneration of neurons and their connections that drives progressive disability in demyelinating disease. However, whether chronic demyelination triggers neurodegeneration and how it may do so remain unclear. We characterize two genetic mouse models of inducible demyelination, one distinguished by effective remyelination and the other by remyelination failure and chronic demyelination. While both demyelinating lines feature axonal damage, mice with blocked remyelination have elevated neuronal apoptosis and altered microglial inflammation, whereas mice with efficient remyelination do not feature neuronal apoptosis and have improved functional recovery. Remyelination incapable mice show increased activation of kinases downstream of dual leucine zipper kinase (DLK) and phosphorylation of c-Jun in neuronal nuclei. Pharmacological inhibition or genetic disruption of DLK block c-Jun phosphorylation and the apoptosis of demyelinated neurons. Together, we demonstrate that remyelination is associated with neuroprotection and identify DLK inhibition as protective strategy for chronically demyelinated neurons.