Collecting Duct Cells Research Articles

Abstract A011: Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples

Abstract Introduction: The study aims to identify the progenitor of clear-cell renal cell carcinoma (ccRCC). Background: Inactivation of tumor suppressor gene VHL is the driver for the pathogenesis of ccRCC, the majority subtype of kidney tumors. However, it has also been observed that not all VHL-deficient cells develop into tumors. It is therefore likely that additional characteristics are required for ccRCC formation. Accumulating evidence suggests that tumor formation is initiated by a small pool of cells with tumor-initiating capacity, which are termed tumor progenitor cells (TPCs). Yet, the concept of TPCs in ccRCC has not been fully validated. Methods: LSL-tdTomato reporter was used to trace Vhlh mutant cells in the prior pre-malignant ccRCC mouse model, in which Vhlh was conditionally knocked out (VhlhKO) in renal epithelial cells under the control of Hoxb7-Cre. We also utilized single-cell RNA sequencing (scRNA-seq) to investigate the diversity of Vhlh-deficient renal tubule cells. Results: We show that the tubule phenotypes (traced by the tdTomato reporter) are associated with Vhlh inactivation; the abnormal tdTomato+ cells in Vhlh KO mice exhibit clear-cell phenotype, hyperplasia and multi-layered epithelial cell clusters. scRNA-seq was then used to analyze isolated tdTomato+ cells from Vhlh KO and wild-type (WT) kidneys. The results reveal that inactivation of Vhlh generates an expanded population of principal collecting duct cells characterized by the expression of keratin 19 (Krt19). These Krt19+ cells highly express stem cell-related genes (Wnt4, Sox4, Klf4, Ly6a). They also express multiple tubule segment-specific markers, suggesting the process of dedifferentiation. The expression of Krt19 is strikingly increased and colocalized with a subpopulation of tdTomato+ cells in Vhlh KO kidney, compared with WT where Krt19+ cells are a rare population scattered in tubule epithelia. Colocalization of KRT19 and CAIX in ccRCC samples confirms the ccRCC identity of these KRT19+ cells. High expression of KRT19 is significantly associated with poor overall survival of ccRCC patients. On kidney tissue microarray, KRT19 is strongly expressed in the early-stage peri-tumor tissue with the presence of immune cell infiltration and lipid droplets in the vicinity. In the high-grade ccRCC, KRT19 is expressed in the typical ccRCC cells and in multi-layered adenomas. Notably, Krt19+ progenitor-like cells are found in the fibrotic region with immune cell infiltration in Vhlh mutant kidney. The direct contact between macrophages and Krt19+ cells in mutant kidney, but not in WT, implies that macrophages may be a component in the progenitor-cell niche when Vhlh is conditionally knocked out. Conclusion: We suspect that VHL loss-of-function in the rare tissue progenitor cells may generate a tumor progenitor subset. Elucidation of the role of KRT19 in the context of VHL inactivation and characterization of the stem/progenitor cell niche are of importance as they will shed new lights on early diagnosis and therapeutic intervention. Citation Format: Dinh-Xuan Pham, Chen-Yun Chen, Hieu-Huy Nguyen-Tran, Tien Hsu. Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A011.

A spatially anchored transcriptomic atlas of the human kidney papilla identifies significant immune injury in patients with stone disease

Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.

Absence of E-Cadherin and β-Catenin in the Basal Plasma Membrane of Collecting Duct Cells During NDI Development and Recovery.

Lithium (Li) induces severe polyuria and polydipsia in up to 40% of patients undergoing Li treatment. In rats, Li treatment induces a reversible cellular remodeling of the collecting duct (CD), decreasing the fraction of principal-to-intercalated cells. To investigate the potential role of adherens junction proteins, we performed immunohistochemistry on kidney cross-sections from rats treated with Li as well as rats undergoing recovery on a normal diet following 4 weeks of Li-treatment. We performed immunoelectron microscopy on cryosections to determine the ultrastructural localizations. Immunohistochemistry showed that E-cadherin and β-catenin were present in both the lateral and basal plasma membrane domains of CD cells. Immunoelectron microscopy confirmed that β-catenin was localized both to the lateral and the basal plasma membrane. The basal localization of both proteins was absent from a fraction of mainly principal cells after 10 and 15 days of Li-treatment. After 4 weeks of Li-treatment few to no cells were absent of E-cadherin and β-catenin at the basal plasma membrane. After 12 and 19 days of recovery some cells exhibited an absence of basal localization of both proteins. Thus, the observed localizational changes of E-cadherin and β-catenin appear before the cellular remodeling during both development and recovery from Li-NDI.

402-P: ENaC Regulation in Mouse Cortical Collecting Duct Cells by EVs Released during the Inactive and Active Cycles of Salt-Loaded Hypertensive Diabetic db/db Mice

The downregulation of renal epithelial sodium channel (ENaC) activity in response to salt-loading in the diabetic kidney is important to prevent the exacerbation of diabetic kidney disease. An inflammatory response in diabetic db/db mice is associated with constitutively high ENaC activity even after salt-loading. During salt-loading, diabetic db/db mice release extracellular vesicles (EVs) into the urine during the active and inactive cycles that are enriched in bioactive molecules. A vast majority of these EVs are released across the luminal membrane of renal epithelial cells from each segment of the nephron and have the potential to module ENaC activity in recipient collecting duct cells. Our hypothesis was the distinct cargo that is enriched in EVs released during the inactive and active cycles of salt-loaded hypertensive diabetic db/db mice is responsible for increased ENaC membrane protein expression and activity in recipient collecting duct cells. In this study, we measured blood pressure by telemetry during the light (inactive) or dark (active) cycles. In addition, osmolality, electrolytes, and creatinine were measured and EV were isolated from urine samples collected during both cycles. Western blotting and densitometric analysis showed greater ENaC alpha protein expression in membrane fractions of mouse cortical collecting duct (mpkCCD) cells while single-channel patch clamp studies show greater ENaC activity in mpkCCD cells treated with uEVs isolated from the active cycle compared to the inactive cycle of hypertensive diabetic db/db mice. Various proteins and metabolites were found to be enriched in each of the two groups of EVs. Taken together, these data suggest the packaged cargo within EVs released after salt-loading diabetic db/db mice changes over a 24 hour cycle, and various molecules enriched in active cycle EVs contribute to the upregulation of renal ENaC protein expression and activity. Disclosure L.Yu: None. M.F.Gholam: None. A.A.Alli: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK123078-01A1 to A.A.A.)

404-P: Single-Cell RNA Profiling Changes in Glomerular Cells in Diabetic Nephropathy (DN)

Hyperglycemia, the main risk factor for DN, causes complex pathological damages in glomeruli. To characterize detail changes in cell differentiation and gene expressions induced by diabetes, we performed unbiased single-cell RNA sequencing (scRNA-seq) on glomerular cells from diabetic and nondiabetic mice of long duration. Glomerular cells were isolated from C57 mice after 7-months of STZ-induced diabetes, when clear glomerular pathologies have been documented. Total number of 48,337 high-qualitied glomerular cells were isolated and sequenced from WT and STZ-treated mice. Based on clustering analysis and cell-type specific markers, 12 different cell types were identified, including endothelial cells (EC), podocytes, mesangial cells (MC), proximal tubule cells, parietal epithelial cells (PEC), collecting duct cells, ascending loop of Henle, macrophages, B cells, T cells, NK cells and neutrophils. Over 90% of the cells were from glomeruli (41% EC, 34 % podocytes, 14% MC, 2% of PEC), and 6% immune cells. Interestingly, gene profiling identified 9 clusters of EC, 5 clusters of MC and 7 clusters of podocytes. Diabetes did not change the total cell numbers for EC, MC and podocytes, although macrophage and B cell numbers were increased from 2.2% to 4.8% and 0.35% to 1%, respectively. Cluster subset analysis of EC, MC and podocytes showed that diabetes induced substantial changes. For podocytes, diabetes increased those clusters (5% to 37%) with high expressions of stressed proteins such as Hspa1a, Hspa1b and Hsp90aa1, while decreased those clusters with high expressions of ECM production and CDC42 GTPase cycle, that were involved in cytoskeleton stabilization and foot process formation. Findings from scRNA-seq of glomeruli showed there were multiple subsets of podocytes, EC and MC, which appear to have different functions and their distributions were changed by diabetes. Understanding the mechanism for the distribution changes of the subsets may provide new insight in the pathogenesis of DN. Disclosure J. Fu: None. Q. Li: None. K. Park: None. Q. Huang: None. I. Wu: None. G.L. King: Research Support; Janssen Research & Development, LLC.

#4082 TAZ-KNOCKDOWN AFFECTS THE VASOPRESSIN-INDUCED AQUAPORIN-2 (AQP2) TRAFFICKING AND PROTEIN ABUNDANCE IN KIDNEY COLLECTING DUCT CELLS

Abstract Background and Aims Transcriptional coactivator with PDZ-binding motif (TAZ), a downstream effector of the hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ KO mice were known to display multicystic kidneys with polyuria. We aimed to study the role of TAZ in vasopressin-induced AQP2 regulation. Method 1) siRNA-mediated knockdown of TAZ in mpkCCDc11 cells; 2) RT-qPCR, semiquantitative immunoblotting, immunocytochemistry of AQP2; 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, the mouse collecting duct cell line. Results Endogenous AQP2 expression was induced in mpkCCDc11 cells by dDAVP (10−9M) treatment. After starvation for 24 h, dDAVP (10−9M) stimulation for 15 and 30 min induced a significant AQP2 translocation to the cell membrane. In contrast, the dDAVP-induced AQP2 translocation was markedly attenuated in the cells with siRNA-mediated TAZ knockdown (TAZ-KD), despite no changes in cAMP production. Phalloidin staining demonstrated the excessive stress fiber formation in the TAZ-KD. dDAVP (10−9 M) treatment for 24 h induced AQP2 mRNA (12,608 ± 177% of the control) and AQP2 protein abundance (270 ± 18%). In contrast, dDAVP-induced increase of AQP2 mRNA (273 ± 14% of the control) and protein abundance (99 ± 17%) was significantly attenuated in TAZ-KD. Unchanged TonEBP protein abundance was observed in TAZ-KD. NGS identified several potential AQP2 transcription factors (TF), and Klf6, Irf3, Cebpb, and Nr4a1 were selected based on previous in silico database. Among them, Nr4a1 was chosen for further studies due to a significant decrease in the mRNA expression levels in TAZ-KD, as demonstrated by RT-qPCR. Conclusion TAZ is likely to affect dDAVP-induced AQP2 trafficking. This is not mediated by the changes in cAMP/PKA pathway, but other non-canonical pathways are involved. TAZ could regulate AQP2 abundance, possibly via an interaction with several TF.

Structural analysis of the water channel AQP2 by single-particle cryo-EM

Water channels, which are small membrane proteins almost entirely buried in lipid membranes, are challenging research targets for single-particle cryo-electron microscopy (cryo-EM), a powerful technique routinely used to determine the structures of membrane proteins. Because the single-particle method enables structural analysis of a whole protein with flexible parts that interfere with crystallization, we have focused our efforts on analyzing water channel structures. Here, utilizing this system, we analyzed the structure of full-length aquaporin-2 (AQP2), a primary regulator of vasopressin-dependent reabsorption of water at the renal collecting ducts. The 2.9 Å resolution map revealed a cytoplasmic extension of the cryo-EM density that was presumed to be the highly flexible C-terminus at which the localization of AQP2 is regulated in the renal collecting duct cells. We also observed a continuous density along the common water pathway inside the channel pore and lipid-like molecules at the membrane interface. Observations of these constructions in the AQP2 structure analyzed without any fiducial markers (e.g., a rigidly bound antibody) indicate that single-particle cryo-EM will be useful for investigating water channels in native states as well as in complexes with chemical compounds.

Retraction: The Role for HNF-1β-Targeted Collectrin in Maintenance of Primary Cilia and Cell Polarity in Collecting Duct Cells.

Retraction: The Role for HNF-1β-Targeted Collectrin in Maintenance of Primary Cilia and Cell Polarity in Collecting Duct Cells.

Lithium sensitizes P2Y2-dependent intracellular Ca2+ signaling in the collecting duct cells

INTRODUCTION. Lithium salts are used as mood stabilizers to treat bipolar disorder and depression. A common renal side effect of lithium therapy is nephrogenic diabetes insipidus (NDI), manifesting in polyuria. Lithium decreases the numbers of aquaporin 2 (AQP2) water channels in collecting duct cells reducing water reabsorption in response to vasopressin (AVP). Stimulation of purinergic P2Y2 receptors (P2Y2R) can impair AVP signaling and deletion of P2Y2R in mice slows down the progression of lithium-induced NDI. Interestingly, stimulation of P2Y2R triggers Ca2+ release from the endoplasmic reticulum (ER) in collecting duct cells. The interplay between P2Y2R and intracellular Ca2+ ([Ca2+]i) signaling in lithium-induced NDI has not been tested. We hypothesize that lithium facilitates P2Y2-mediated Ca2+ signaling in collecting duct cells, attenuating AVP-dependent water transport and disrupting [Ca2+]i balance. METHODS. This project combines water permeability tests in cultured cell monolayers, immunoblotting and ratiometric Fura-2 Ca2+-imaging to study the effects of lithium on P2Y2-dependent [Ca2+]i mobilization in a culture of mouse renal cortical collecting duct cells, mpkCCDcl4. RESULTS. Upon stimulation with 1 nM AVP, mpkCCDcl4 cells incubated in the medium containing 10 mM LiCl for 4 days exhibited a 2-fold lower abundance of membrane resident AQP2, phosphorylated at serine 269, when compared to control cells. Transepithelial water transport was also significantly slower in LiCl-treated than in control cells, further validating the detrimental effects of Li+ in our cell model. Saturating (10-100 uM) concentrations of a P2Y2R-selective ligand, uridine-5'-triphosphate (UTP), induced comparable [Ca2+]i responses in in control and LiCl-treated cells. When we assessed the P2Y2-mediated [Ca2+]i responses to a range of UTP concentrations (0.1-100 uM), the dose response curve was shifted to the left in LiCl-treated cells when compared to controls. The half-maximal effective UTP concentration, EC50, was 0.4±0.03 uM and 2.0±0.07 uM in Li-treated and control cells, respectively, indicating higher sensitivity to UTP in LiCl-treated cells. Next, we evaluated how P2Y2-dependent depletion of the ER Ca2+ stores, affects store-operated calcium entry (SOCE) in mpkCCDcl4 cells. The magnitude of SOCE-reporting Ca2+ signal was significantly (~25%) higher in LiCl-treated cells than in controls. CONCLUSIONS. Lithium facilitates P2Y2-mediated [Ca2+]i mobilization, reduces AVP-dependent transepithelial water transport and AQP2 trafficking in collecting duct cells. Sensitization of P2Y2-dependent Ca2+ signaling causes excessive depletion of the ER stores and requires higher activity of SOCE to compensate for Ca2+ loss and restore the balance. Our findings reveal previously unrecognized signaling determinants underlying the pathophysiological effects of lithium, pertaining to Ca2+ homeostasis in collecting duct cells. This work was supported by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) R01DK125464. This is the full abstract presented at the American Physiology Summit 2023 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.

Piezo1 and TRPV4 complementary contribute to [Ca2+]i homeostasis in collecting duct cells

Mechanical stress arising from elevated fluid flow is an important regulator of the water-electrolyte transport in the collecting duct (CD). It is well established that mechano-sensitive TRPV4 Ca2+-permeable channel mediates intracellular Ca2+ ([Ca2+]i) responses to high flow in these cells. Piezo1, a Ca2+-permeable channel directly activated by the plasma membrane stretch, is a recent arrival at this scene. Piezo1 expression has been reported in the CD. However, its functional significance is yet to be determined. Here, we applied a combination of Ca2+ imaging and confocal microscopy in freshly isolated split-opened CDs to investigate a potential interplay between TRPV4 and Piezo1 in mediating [Ca2+]i signaling in the CD. Immunofluorescent analysis of split-opened CDs showed presence of Piezo1 in both AQP2-positive (principal cells, PC) and -negative (intercalated cells, IC) with higher intensity in the former. Importantly, Piezo1 was apparent on both apical and basolateral membrane of CD cells. Piezo1 agonists, Yoda-1 (20μM) and Jedi2 (500μM) acutely increase [Ca2+]i in both principal and intercalated cells. The responses were abolished when extracellular Ca2+ was buffered with EGTA suggesting a direct Ca2+ influx via Piezo1. Interestingly, we detected two disparate calcium responses to Piezo1 activation with the majority (70%) cells showing a fast transient response with a sustained plateau, whereas the remaining (30%) population had much slower gradual calcium increase to the same plateau level. Subsequent immunofluorescent analysis revealed that fast Ca2+ responses were attributed to PC, whereas responses with slow kinetics corresponded to IC. Consistently, PCs have ~3 fold higher Piezo-1 signal when compared to neighbor ICs. We next tested whether Piezo1 activity depends on TRPV4 status. Genetic deletion and pharmacological inhibition of TRPV4 with GSK2193874 did not affect Piezo1-dependent elevations in [Ca2+]i. However, high K+ diet, which is known to elevate the flow in the CD and augment both TRPV4 activity and expression, led to diminished Piezo1 responses to Yoda-1. In summary, our results are consistent with the view that Piezo1 and TRPV4 are functional in both principal and intercalated cells of the collecting duct and operate independently to modulate mechanosensitive [Ca2+]i signaling. We hypothesize that augmented TRPV4-dependent [Ca2+]i is compensated by the diminished activity of Piezo1 to account for elevated flow rate upon increased K+ intake. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-19CDA34660148 (to V. N. Tomilin). This is the full abstract presented at the American Physiology Summit 2023 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.

Aberrant adaptation of the inner medullary collecting ducts results in sodium retention and hypernatremia in a thick ascending limb specific Myh9 and Myh10 conditional knockout mouse model

Sodium reabsorption in the thick ascending limb (TAL) epithelium is essential to generate and sustain the interstitial osmotic gradient, which facilitates water reabsorption in the adjacent collecting duct system. Impaired TAL function can compromise this gradient and lead to electrolyte imbalance. Our previous work demonstrated that pan renal tubular conditional genetic inactivation of nonmuscle myosin II genes Myh9&10 in adult mice results in progressive kidney disease of TAL origin with aberrant regulation of TAL proteins U MOD and NKCC2 (Otterpohl et al; 2020). To confirm the cell-autonomous role for NM2 proteins in adult TAL epithelial cells, we generated a TAL-specific conditional knockout mouse model of Myh9&10 using the UMOD->CreERT2 mouse line (cKO). Genetic inactivation of Myh9&10 in the TAL was induced at 5 weeks of age with tamoxifen injections. Age matched control and cKO littermates were part of every cohort. Mice underwent metabolic, histologic, immunohistochemical, and biochemical analyses at varying time points until moribund. Development of progressive kidney disease in cKO mice was indicated by increasing levels of blood urea nitrogen, serum creatinine, and the tubular injury marker NGAL. Our results show that cKO females experience significant loss of weight at 12 weeks of age and are moribund between 13 – 14 weeks, while cKO males survive up to 25 weeks. Interestingly, female cKO mice exhibit hypernatremia at 12 weeks of age and male cKO mice between 16 - 20 weeks of age. We analyzed NKCC2 protein levels in whole kidney lysates and a drastic reduction was evident in both female and male cKO mice by 9 weeks of age, therefore hypernatremia was unexpected. We examined if the hypernatremia in TAL-Myh9&10 cKO mice is due to tubular adaptation in the distal nephron and/or the collecting duct segments. Systematic analysis of sodium cotransporters and channels in the distal nephron and collecting duct segments revealed an increase in NCC/pNCC levels in males and a decrease in females when compared with age matched control littermates. No apparent volume loss (increased urine output) has been observed in the female mice. Additionally, immunostaining experiments showed aberrant increase in expression of ENaC gamma in the inner medullary collecting duct cells in female and not male mice by 9-weeks of age. ENaC expression is limited to cortical collecting duct and the connecting segment under normal conditions in the mouse kidney. Consistent with this, our control mice do not have ENaC gamma expression in the inner medullary collecting ducts. Further experiments are underway to determine the cellular state/identity of these ENaC expressing inner medullary collect duct cells. These experiments confirm the critical role for Myh9&10 proteins in TAL tubule NKCC2 transport, and reveal novel, sex specific, adaptive mechanisms in distal nephron and collecting ducts in response to compromised NKCC2 transport within the TAL. NIDDK RO1 DK131020-01A1 This is the full abstract presented at the American Physiology Summit 2023 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.

Expression of human sPRR in renal collecting duct cells increases blood pressure in female mice independently of its circulating levels

Elevated circulating sPRR has been associated with essential hypertension, obesity-related hypertension, heart failure and chronic kidney disease in humans. However, the exact contribution of tissue-specific human sPRR in blood pressure control remains to be investigated. Therefore, we evaluated the effect of human sPRR on circulating sPRR and blood pressure control when expressed in mouse kidney, adipose tissue or liver from both male and female mice.Human sPRR-Myc-tag transgenic mice were bred with mice expressing either Hoxb7/Cre, Adiponectin/Cre or Albumin/Cre to selectively express human sPRR (HsPRR) in the collecting duct of the kidney (Kid-HsPRR), adipose tissue (Adi-HsPRR) or liver (Liv-HsPRR), respectively. The presence of human sPRR-Myc-tag (28 KDa) was validated in each mouse model by detecting the presence of C-myc. HsPRR and control (WT) male and female mice were fed a control diet throughout the study (n=8-12/group). At 6 months, baseline and acute blood pressure responses were assessed by radio-telemetry.Tissue expression of sPRR increased in kidney-derived HsPRR (M: 1.0±0.2 vs. 1.6±0.3 AU, F: 1.0±0.1 vs. 1.7±0.2 AU p<0.05), adipose-derived HsPRR (M: 1.0±0.3 vs. 4.7±1.5 AU, F: 1.0±0.2 vs. 3.2±1.4 AU p<0.05) and liver-derived HsPRR mice (M: 1.0±0.2 vs. 9.7±0.8 AU, F: 1.0±0.1 vs. 8.7±0.9 AU p<0.05) but only liver-derived HsPRR mice elevated circulating sPRR (M: 7.0±2.4 vs. 125.0±7.9 ng/ml, F: 2.2±0.4 vs. 93.9±13.4 ng/ml p<0.05). Human sPRR increased blood pressure only in female mice expressing HsPRR in kidney (F: 111±2 vs. 121±4 mmHg p<0.05). Also, only female mice expressing HsPRR in kidney displayed a blunted acute response to losartan injection (M: -30.1±3.3 vs. -22.1±3.7, F: -16.3±2.4 vs. 14.1±5.8 Δ mmHg p<0.05) and Ang II-induced pressor response in acute (M: 28.9±5.1 vs. 47.6±4.4 mmHg p<0.05, F: 35.2±7.2 vs. 26.9±7.5 mmHg) and chronic fashion (M: 33.9±3.6 vs. 30.7±5.0, F: 35.1±6.8 vs.17.6±3.2 Δ mmHg, p<0.05).Overall, our data show that increased intrarenal human sPRR expression contributes to increase blood pressure in a sex-specific manner. Taken together, increased HsPRR in kidney is associated with hypertension and impaired response to AT1R blockade and AngII-induced pressor response suggesting a dysfunctional AT1R-mediated signaling that could be due to ligand competition. These studies were supported by grants from the American Heart Association (13SDG17230008), the National Institute of General Medical Sciences (P30 GM127211), the Center for Clinical and Translational Sciences (UL1TR000117) and the National Heart Lung and Blood Institute (R01HL142969-01) This is the full abstract presented at the American Physiology Summit 2023 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.

Uncovering the functional role of GPR39 in the renal collecting duct

G protein-coupled receptors (GPCRs) are seven transmembrane domain receptors that comprise the largest class of proteins in the mammalian genome. GPR39 is part of the ghrelin subfamily of GPCRs, and is well-expressed in the kidney but has no reported function in renal physiology. To probe the renal role of GPR39, we first sought to localize it; in the absence of a validated immunofluorescence (IF) antibody, we used RNAscope. We found that Gpr39 localizes to nephron segments in the cortex and medulla which are positive for DBA (Dolichos Biflorus Agglutinin), a collecting duct marker. RNAScope expression of Gpr39 was highest in the inner medullary collecting duct, with lower levels in the outer medullary and cortical collecting ducts. Within the collecting duct, Gpr39 transcripts colocalized with Aqp2, indicating that Gpr39 is expressed specifically in principal cells. To address whether GPR39 is apical or basolateral, we cloned murine Gpr39 with a C-terminal EGFP tag and transiently transfected this construct into either polarized M-1 cells (murine collecting duct cell line) or polarized MDCK cells (Madin-Darby canine kidney cells); in both cases, cells were grown to confluence on permeable supports. In both cell lines, heterologous GPR39 trafficked to the basolateral membrane. Based on the localization of GPR39 to principal cells, we hypothesized that GPR39 activation may alter AQP2 expression or trafficking. To test this hypothesis in vitro, we first validated mpkCCD (mouse principal cell kidney cortical collecting duct) cells as a model. We cultured mpkCCD on transwells until polarized (TER >5kΩ·cm 2 ). In the absence of dDAVP, mpkCCD do not express AQP2 by western or IF. Basolateral treatment with 1nM dDAVP for 4 days induced AQP2 expression (western blot) and apical trafficking (confocal IF). We also confirmed that withdrawal of dDAVP (4h) resulted in a cytosolic accumulation of AQP2, while the re-introduction of dDAVP (1h) returned AQP2 to the apical membrane by IF. Moreover, we found that Gpr39 expression was unaffected by dDAVP by qPCR (n=3, ΔCt ± SEM: control = 7.9±0.3, dDAVP = 7.2±0.1) and western blot. In a preliminary study, we treated mpkCCD with 13nM of a GPR39 selective agonist, cpd1324, or with a vehicle control, and probed for changes in AQP2 by IF. Both groups were treated with 1nM dDAVP and with 10μM ZnCl2 (a GPR39 cofactor). We found that cpd1324 treatment decreased AQP2 by IF (mean integrated fluorescence density ± SEM (A.U.): cpd1324 = 233.5 ± 20.8, control = 373.8 ± 13.0). Additionally, cpd1324 treatment reduced the apical AQP2 abundance (mean integrated fluorescence density ± SEM (A.U.) of the most apical Z-slice: cpd1324 = 6.05e+05 ± 3.9e+04, control = 2.6e+06 ± 4.3e+05). These results suggest that GPR39 agonism may antagonize vasopressin-induced AQP2 trafficking and/or expression. NIH T32HL007534 This is the full abstract presented at the American Physiology Summit 2023 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.

Sex Modulates Response to Renal-Tubule-Targeted Insulin Receptor Deletion in Mice.

Insulin facilitates renal sodium reabsorption and attenuates gluconeogenesis. Sex differences in this regulation have not been well characterized. Using tetracycline-inducible Cre-lox recombination, we knocked out (KO) the insulin receptor (InsR) from the renal tubule in adult male (M) and female (F) mice (C57Bl6 background) with a paired box 8 (PAX8) promoter. Body weights were not affected by the KO, but mean kidney weights were reduced in the KO mice (13 and 3%, in M and F, respectively, relative to wild-type (WT) mice). A microscopic analysis revealed 25 and 19% reductions in the proximal tubule (PT) and cortical collecting duct cell heights, respectively, in KOMs relative to WTMs. The reductions were 5 and 11% for KOFs. Western blotting of renal cortex homogenates showed decreased protein levels for the β and γ subunits of the epithelial sodium channel (ENaC) and the sodium-potassium-2-chloride cotransporter type 2 (NKCC2) in both sexes of KO mice; however, α-ENaC was upregulated in KOMs and downregulated in KOFs. Both sexes of KO mice cleared exogenously administered glucose faster than the WT mice and had lower semi-fasted, anesthetized blood glucose levels. However, KOMs (but not KOFs) demonstrated evidence of enhanced renal gluconeogenesis, including higher levels of renal glucose-6-phosphatase, the PT's production of glucose, post-prandial blood glucose, and plasma insulin, whereas KOFs exhibited downregulation of renal high-capacity sodium glucose cotransporter (SGLT2) and upregulation of SGLT1; these changes appeared to be absent in the KOM. Overall, these findings suggest a sex-differential reliance on intact renal tubular InsR signaling which may be translationally important in type 2 diabetes, obesity, or insulin resistance when renal insulin signaling is reduced.

Increased Renal Medullary NOX-4 in Female but Not Male Mice during the Early Phase of Type 1 Diabetes: Potential Role of ROS in Upregulation of TGF-β1 and Fibronectin in Collecting Duct Cells

Chronic diabetes mellitus (DM) can lead to kidney damage associated with increased reactive oxygen species (ROS), proteinuria, and tubular damage. Altered protein expression levels of transforming growth factor-beta 1 (TGF-β1), fibronectin, and renal NADPH oxidase (NOX-4) are associated with the profibrotic phenotype in renal tubular cells. NOX-4 is one of the primary sources of ROS in the diabetic kidney and responsible for the induction of profibrotic factors in collecting duct (CD) cells. The renal medulla is predominantly composed of CDs; in DM, these CD cells are exposed to high glucose (HG) load. Currently there is no published literature describing the expression of these markers in the renal medulla in male and female mice during the early phase of DM, or the role of NOX-4-induced ROS. Our aim was to evaluate changes in transcripts and protein abundances of TGF-β1, fibronectin, and NOX-4 along with ROS levels in renal medullary tissues from male and female mice during a short period of streptozotocin (STZ)-induced type 1 DM and the effect of HG in cultured CD cells. CF-1 mice were injected with or without a single dose of STZ (200 mg/kg) and euthanized at day 6. STZ females showed higher expression of fibronectin and TGF-β1 when compared to control mice of either gender. Interestingly, STZ female mice showed a >30-fold increase on mRNA levels and a 3-fold increase in protein levels of kidney medullary NOX-4. Both male and female STZ mice showed increased intrarenal ROS. In primary cultures of inner medullary CD cells exposed to HG over 48 h, the expression of TGF-β1, fibronectin, and NOX-4 were augmented. M-1 CD cells exposed to HG showed increased ROS, fibronectin, and TGF-β1; this effect was prevented by NOX-4 inhibition. Our data suggest that at as early as 6 days of STZ-induced DM, the expression of profibrotic markers TGF-β1 and fibronectin increases in renal medullary CD cells. Antioxidants mechanisms in male and female in renal medullary tissues seems to be differentially regulated by the actions of NOX-4.

Metformin Alleviates Diabetes-Associated Hypertension by Attenuating the Renal Epithelial Sodium Channel.

Diabetic nephropathy is the primary cause of morbidity in type 2 diabetes mellitus (T2DM) patients. New data indicate that hypertension, a common comorbidity in T2DM, can worsen outcomes of diabetic nephropathy. While metformin is a commonly prescribed drug for treating type 2 diabetes, its blood pressure regulating ability is not well documented. The aim of this study was to investigate the effect of metformin on normalizing blood pressure in salt-loaded hypertensive diabetic db/db mice. Sixteen-week-old male and female diabetic db/db mice were individually placed in metabolic cages and then randomized to a control vehicle (saline) or metformin treatment group. We evaluated the blood pressure reducing ability of metformin in salt-induced hypertension and progression of nephropathy in db/db mice. We observed that metformin- normalized systolic blood pressure in hypertensive diabetic mice. Mechanistically, metformin treatment reduced renal cathepsin B expression. Low cathepsin B expression was associated with reduced expression and activity of the epithelial sodium channel (ENaC), sodium retention, and thus control of hypertension. In addition, we identified that urinary extracellular vesicles (EVs) from the diabetic mice are enriched in cathepsin B. Compared to treatment with urinary EVs of vehicle-treated hypertensive diabetic mice, the amiloride-sensitive transepithelial current was significantly attenuated upon exposure of renal collecting duct cells to urinary EVs isolated from metformin-treated db/db mice or cathepsin B knockout mice. Collectively, our study identifies a novel blood pressure reducing role of metformin in diabetic nephropathy by regulating the cathepsin B-ENaC axis.

TRPML1-Induced Lysosomal Ca2+ Signals Activate AQP2 Translocation and Water Flux in Renal Collecting Duct Cells.

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.

Transcription factor Ap2b regulates the mouse autosomal recessive polycystic kidney disease genes, Pkhd1 and Cys1.

Transcription factor Ap2b (TFAP2B), an AP-2 family transcription factor, binds to the palindromic consensus DNA sequence, 5'-GCCN3-5GGC-3'. Mice lacking functional Tfap2b gene die in the perinatal or neonatal period with cystic dilatation of the kidney distal tubules and collecting ducts, a phenotype resembling autosomal recessive polycystic kidney disease (ARPKD). Human ARPKD is caused by mutations in PKHD1, DZIP1L, and CYS1, which are conserved in mammals. In this study, we examined the potential role of TFAP2B as a common regulator of Pkhd1 and Cys1. We determined the transcription start site (TSS) of Cys1 using 5' Rapid Amplification of cDNA Ends (5'RACE); the TSS of Pkhd1 has been previously established. Bioinformatic approaches identified cis-regulatory elements, including two TFAP2B consensus binding sites, in the upstream regulatory regions of both Pkhd1 and Cys1. Based on reporter gene assays performed in mouse renal collecting duct cells (mIMCD-3), TFAP2B activated the Pkhd1 and Cys1 promoters and electromobility shift assay (EMSA) confirmed TFAP2B binding to the in silico identified sites. These results suggest that Tfap2b participates in a renal epithelial cell gene regulatory network that includes Pkhd1 and Cys1. Disruption of this network impairs renal tubular differentiation, causing ductal dilatation that is the hallmark of recessive PKD.

Single-Cell Transcriptome Identifies the Renal Cell Type Tropism of Human BK Polyomavirus.

BK polyomavirus (BKPyV) infection is the main factor affecting the prognosis of kidney transplant recipients, as no antiviral agent is yet available. A better understanding of the renal-cell-type tropism of BKPyV can serve to develop new treatment strategies. In this study, the single-cell transcriptomic analysis demonstrated that the ranking of BKPyV tropism for the kidney was proximal tubule cells (PT), collecting duct cells (CD), and glomerular endothelial cells (GEC) according to the signature of renal cell type and immune microenvironment. In normal kidneys, we found that BKPyV infection-related transcription factors P65 and CEBPB were PT-specific transcription factors, and PT showed higher glycolysis/gluconeogenesis activities than CD and GEC. Furthermore, in the BKPyV-infected kidneys, the percentage of late viral transcripts in PT was significantly higher than in CD and GEC. In addition, PT had the smallest cell-cell interactions with immune cells compared to CD and GEC in both normal and BKPyV-infected kidneys. Subsequently, we indirectly demonstrated the ranking of BKPyV tropism via the clinical observation of sequential biopsies. Together, our results provided in-depth insights into the renal cell-type tropism of BKPyV in vivo at single-cell resolution and proposed a novel antiviral target.

Kidney collecting duct cells make vasopressin in response to NaCl-induced hypertonicity.

Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The questions of whether vasopressin could be produced outside of the brain and if the kidney could be a source of vasopressin are raised by the syndrome of inappropriate antidiuretic hormone secretion (vasopressin). We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects preprovasopressin, we found that immunoreactive preprovasopressin protein was found in mouse and human kidneys. Moreover, we found that murine collecting duct cells made biologically active vasopressin, which increased in response to NaCl-mediated hypertonicity, and that water restriction increased the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.