Collecting Duct Cells Research Articles

Bayesian analysis of dynamic phosphoproteomic data identifies protein kinases mediating GPCR responses

BackgroundA major goal in the discovery of cellular signaling networks is to identify regulated phosphorylation sites (“phosphosites”) and map them to the responsible protein kinases. The V2 vasopressin receptor is a G-protein coupled receptor (GPCR) that is responsible for regulation of renal water excretion through control of aquaporin-2-mediated osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on protein kinase A (PKA), but events downstream from PKA are still obscure.MethodsHere, we used: 1) Tandem mass tag-based quantitative phosphoproteomics to experimentally track phosphorylation changes over time in native collecting ducts isolated from rat kidneys; 2) a clustering algorithm to classify time course data based on abundance changes and the amino acid sequences surrounding the phosphosites; and 3) Bayes’ Theorem to integrate the dynamic phosphorylation data with multiple prior “omic” data sets covering expression, subcellular location, known kinase activity, and characteristic surrounding sequences to identify a set of protein kinases that are regulated secondary to PKA activation.ResultsPhosphoproteomic studies revealed 185 phosphosites regulated by vasopressin over 15 min. The resulting groups from the cluster algorithm were integrated with Bayes’ Theorem to produce corresponding ranked lists of kinases likely responsible for each group. The top kinases establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) mitogen-activated protein kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling.ConclusionsOur findings identify a novel set of downstream small GTPase effectors and calcium/calmodulin-dependent kinases with potential roles in the regulation of water permeability through actin cytoskeleton rearrangement and aquaporin-2 trafficking. The proposed signaling network provides a stronger hypothesis for the kinases mediating V2 vasopressin receptor responses, encouraging future targeted examination via reductionist approaches. Furthermore, the Bayesian analysis described here provides a template for investigating signaling via other biological systems and GPCRs.7VJHPzA8Cu6VMRYsgYqSsgVideo abstract

Glucocorticoid Receptor Maintains Vasopressin Responses in Kidney Collecting Duct Cells.

Water permeability of the kidney collecting ducts is regulated in part by the amount of the molecular water channel protein aquaporin-2 (AQP2), whose expression, in turn, is regulated by the pituitary peptide hormone vasopressin. We previously showed that stable glucocorticoid receptor knockdown diminished the vasopressin-induced Aqp2 gene expression in the collecting duct cell model mpkCCD. Here, we investigated the pathways regulated by the glucocorticoid receptor by comparing transcriptomes of the mpkCCD cells with or without stable glucocorticoid receptor knockdown. Glucocorticoid receptor knockdown downregulated 5,394 transcripts associated with 55 KEGG pathways including “vasopressin-regulated water reabsorption,” indicative of positive regulatory roles of these pathways in the vasopressin-induced Aqp2 gene expression. Quantitative RT-PCR confirmed the downregulation of the vasopressin V2 receptor transcript upon glucocorticoid receptor knockdown. Glucocorticoid receptor knockdown upregulated 3,785 transcripts associated with 42 KEGG pathways including the “TNF signaling pathway” and “TGFβ signaling pathway,” suggesting the negative regulatory roles of these pathways in the vasopressin-induced Aqp2 gene expression. Quantitative RT-PCR confirmed the upregulation of TNF and TGFβ receptor transcripts upon glucocorticoid receptor knockdown. TNF or TGFβ inhibitor alone, in the absence of vasopressin, did not induce Aqp2 gene transcription. However, TNF or TGFβ blunted the vasopressin-induced Aqp2 gene expression. In particular, TGFβ reduced vasopressin-induced increases in Akt phosphorylation without inducing epithelial-to-mesenchymal transition or interfering with vasopressin-induced apical AQP2 trafficking. In summary, our RNA-seq transcriptomic comparison revealed positive and negative regulatory pathways maintained by the glucocorticoid receptor for the vasopressin-induced Aqp2 gene expression.

Statins attenuate cholesterol-induced ROS via inhibiting NOX2/NOX4 and mitochondrial pathway in collecting ducts of the kidney

BackgroundStatins therapy has been primarily recommended for the prevention of cardiovascular risk in patients with chronic kidney diseases. Statins has also been proved some benefits in lipid-induced kidney diseases. The current study aims to investigate the protection and underlying mechanisms of statins on renal tubular injuries induced by cholesterol overloaded.MethodsWe used tubular suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys and mouse collecting duct cell line mpkCCD cells to investigate the effect of statins on reactive oxygen species (ROS) production induced by cholesterol. Protein and mRNA expression of NADPH oxidase 2 (NOX2) /NOX4 was examined by Western blot and RT-PCR in vitro studies and in rats with 5/6 nephrectomy and high-fat diet. Mitochondrial morphology and membrane potential was observed by Mito-tracker and JC-1.ResultsStatins treatment was associated with decreased NOX2 and NOX4 protein expression and mRNA levels in 5/6Nx rats with high-fat diet. Statins treatment markedly reduced the ROS production in IMCD suspensions and mpkCCD cells. Also, statins reduced NOX2 and NOX4 protein expression and mRNA levels in cholesterol overload mpkCCD cells and improved mitochondrial morphology and function.ConclusionStatins prevented ROS production induced by cholesterol in the kidney, likely through inhibiting NOXs protein expression and improving mitochondrial function. Statins may be a therapeutic option in treating obesity-associated kidney diseases.

Single Gene Mutations in Pkd1 or Tsc2 Alter Extracellular Vesicle Production and Trafficking.

Simple SummaryExtracellular vesicles shed from primary cilia may be involved in renal cystogenesis. The disruption of the Pkd1 gene in our cell culture system increased the production of EVs in a similar way that occurs when the Tsc2 gene is disrupted. Disruption of the primary cilia depresses EV production, and this may be the reason that the combined Kif3A/Pkd1 mutant mouse has a less severe phenotype than the Pkd1 mutant alone. We initiated studies aimed at understanding the renal trafficking of renally-derived EVs and found that single gene disruptions can alter the EV kinetics based on dye tracking studies. These results raise the possibility that EV features, such as cargo, dose, tissue half-life, and targeting, may be involved in the disease process, and these features may also be fertile targets for diagnostic, prognostic, and therapeutic investigation.Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.

Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

SummaryAutosomal dominant polycystic kidney disease is the most common monogenic disease that causes end-stage renal failure. It primarily results from mutations in the PKD1 gene that encodes for Polycystin-1. How loss of Polycystin-1 translates into bilateral renal cyst development is mostly unknown. cAMP is significantly involved in cyst enlargement but its role in cyst initiation has remained elusive. Deletion of Polycystin-1 in collecting duct cells resulted in a switch from tubule to cyst formation and was accompanied by an increase in cAMP. Pharmacological elevation of cAMP in Polycystin-1-competent cells caused cyst formation, impaired plasticity, nondirectional migration, and mis-orientation, and thus strongly resembled the phenotype of Polycystin-1-deficient cells. Mis-orientation of developing tubule cells in metanephric kidneys upon loss of Polycystin-1 was phenocopied by pharmacological increase of cAMP in wildtype kidneys. In vitro, cAMP impaired tubule formation after capillary-induced injury which was further impaired by loss Polycystin-1.

Lithium‐associated nephrotoxicity: role of GDF15‐mediated collecting duct cells proliferation

Lithium (Li+) salts are the most effective treatment of bipolar disorder (BD), affecting 1 to 4% of the population worldwide. However, this efficacy is counterbalanced by adverse long‐term events, mostly renal effects. Lithium treatment discontinuation is usually not an option as it substantially increases suicidal risk. Understanding the sequence of events in the kidneys might thus improve therapeutic strategies while maintaining this treatment.Li+‐induced nephrogenic diabetes insipidus (NDI) is the earliest and the most frequent side effect of chronic lithium use, leading to polyuria and polydipsia. Li+ treatment decreases AQP2 expression both in vivo and in vitro. Li+ treatment also induces a microcystic tubulointerstitial nephropathy eventually leading to end‐stage kidney disease. The mechanisms of tubular toxicity and microcyst formation have been related to a proliferative effect of Li+. The time course, the site and the mechanisms of this proliferation are still a matter of debate.Previous studies from our lab have shown that acidosis‐induced collecting duct cell proliferation depended on the overexpression of GDF15 by principal cells, especially in the early phase of linear proliferation along the tubular axis.Our objective during this project is to determine the pathways leading to planar and cystic proliferation of collecting duct cells, in particular GDF15 signaling, and their role in lithium nephrotoxicity, and to identify potential biomarkers that could help to predict the course of Li‐induced nephropathy.We have established a rat and a mouse experimental model treated with therapeutic concentrations of Li+, displaying as expected polyuria and polydipsia, with microcystic tubular dilations and a global proliferation of collecting duct cells (both principal and intercalated) on kidney slices and on microdissected collecting ducts, with an increase in the intercalated/principal cells ratio (figure). We examined the effect of Li+ in GDF15‐/‐ and wild type (WT) mice and found significantly lower polyuria and polydipsia in GDF15‐/‐ mice, along with a higher urine osmolality, and decreased intercalated/principal cell ratio compared to WT mice (figure).As collecting duct cell proliferation is mediated by GDF15, we studied urinary GDF15 levels (GDF ELISA kit) in Li+‐treated patients with proven diabetes insipidus and found them significantly increased compared to control patients (p<0.01) (figure).In conclusion, these results suggest a direct relationship between lithium‐induced proliferation signals, collecting duct cell dysfunction and GDF15 expression. Further investigation is needed to determine if GDF15 signaling might represent a therapeutic target in Li+‐induced nephrotoxicity.

Dynamic Compartmentalisation of Intracellular Sodium in Collecting Duct Cells

In Principal cells (PC) of the cortical collecting duct (CCD), the highly regulated and co‐ordinated reabsorption of sodium occurs through the epithelial sodium channel (ENaC) at the apical membrane and Na/K ATPase at the basolateral membrane. However, it is not known how sodium ions (Na+) are transported across the cell and if it is a regulated mechanism. We investigated trans‐cellular transport in the mouse collecting duct cell line, mCCDcl1 cells using a fluorescent sodium dye, Corona Green AM. The dye was found to accumulate in fluid‐filled domes forming between the single cell layer and the plastic dish, indicating that the cells are polarised and that Na+ ions are transported across and concentrated beneath the principal cells. Dye uptake was stimulated by aldosterone, blocked by amiloride (an ENaC inhibiter), and basolateral transport was prevented by ouabain (an Na/K ATPase blocker) thus validating the dye’s apparently faithful replication of sodium transport across the cells. Live cells exhibited a consistent pattern of sodium‐containing vesicles, of various sizes, surrounded by cytoskeleton and lipid membrane. While the smallest vesicles (~0.5µm) co‐stained with lysotracker, larger vesicles (up to 6.4µm) did not co‐stain with either lysosomal‐ or mitochondrial‐specific dyes and appeared to have internal structure, suggesting that they were multivesicular bodies. Time‐lapse confocal imaging of cells cultured on a 3D polyHYPE scaffold and subsequent movies of intracellular dynamics showed a subset of these multivesicular bodies release or take up sodium dye in a controlled manner, over the course of a few minutes. Our novel data reveal intracellular sodium compartmentalisation, which may offer new insights into intracellular sodium dynamics, possibly in response to stress, in the collecting duct.

Increase in Salt Concentration in the Culture Media Enhances Protein Expression of Tumor Necrosis Factor‐Alpha Receptor Type 1 in Cultured Renal Cortical Collecting Duct Cells But Not in Proximal Tubular Cells

Tumor necrosis factor‐alpha (TNFα) exerts its biological responses via interaction with its’ two cell surface receptors, type 1 (TNFR1) and type 2 (TNFR2) which are differentially expressed and regulated in the kidney. Previously, we have demonstrated that circulating TNFα exerts natriuresis via its action on TNFR1 but not on TNFR2 and such TNFR1 activation exerts inhibitory action on distal tubular sodium transport, particularly tubular ENaC activity. As TNFα level is elevated during dietary intake of high salt (HS), we hypothesized that increases in salt concentration in renal tissue enhances TNFα ‐TNFR1 activity in the renal tubules to induce natriuretic response to HS intake. To test this hypothesis, we examined the protein expression of TNFR1 and TNFR2 in cultured proximal (human kidney [HK‐2] cell line) and distal (murine cortical collecting duct [M‐1] cell line) tubular cells at different concentrations of NaCl in the culture media. Both these HK‐2 and M‐1 cells were incubated for 24 hours in control media with normal NaCl concentration (120 mM) and at media containing higher NaCl (140 mM and 160 mM) concentrations. TNF receptors protein expressions were analyzed by the Western Blot technique using appropriate antibodies for TNFR1 (rabbit anti‐TNFR1; Abcam cat # 19139) and TNFR2 (rabbit anti‐TNFR2; Abcam cat # 109322), which were normalized by expression of β‐actin or GAPDH proteins using appropriate antibodies. The blot image was quantified and measured as band intensity density with Image J software. The quantitative immunoblot analysis in HK‐2 cells showed that there were no significant changes in the band intensity unit (IU) in TNFR1 (120 mM, 70.1±12.8 IU; 140 mM, 73.7±9.9 IU; 160 mM, 57.2±9.8 IU; n=6 in each condition) or TNFR2 protein (120 mM, 83.9 ±6.3 IU; 140 mM, 66.8±14.1 IU; 160 mM, 77.8±2.3 IU; n=6 in each condition) levels. On the other hand, TNFR1 protein expression was significantly higher in M‐1 cells incubated in 160 mM NaCl conc. (135.6 ±7.8 IU, n=7; P< 0.005) than that in 120 mM (109.3±2.8 IU, n=7) and in 140 mM (91.1±10.8 IU, n=7) NaCl conc. However, TNFR2 protein expression in M‐1 cells did not significantly alter with the changes in NaCl conc. in the media (120 mM, 137±20.1 IU; 140 mM, 128±7.2 IU; 160 mM, 143±7.2 IU; n=7 in each condition). These data indicate that TNFR1 activity is upregulated in renal collecting duct cells (CCD) in response increases in tissue NaCl concentration in the kidney. Such upregulation of TNFR1 activity in the CCD induced by high salt concentration, exerts inhibitory action on distal sodium transport and plays a role in enhancing urinary sodium excretion during dietary HS intake.

Forty‐five Vasopressin‐Regulated Phosphoproteins Involved in Control of Collecting Duct Water Transport

Vasopressin regulates renal water excretion through actions to regulate aquaporin 2 trafficking and transcription. These actions are dependent on vasopressin‐induced phosphorylation changes in collecting duct cells. Although some phosphorylation targets are well known including aquaporin 2 itself, the full gamut of vasopressin‐induced phosphorylation changes is unexplored. Our prior studies using quantitative phosphoproteomics have identified phosphorylation changes in cultured mouse mpkCCD cells (PMID: 33346914) and native inner medullary collecting duct cells (PMID: 31313956). Here, we have curated a list of vasopressin‐regulated phosphoproteins based on a consensus of the two studies. This includes 51 phosphorylation sites in 45 proteins, archived at https://esbl.nhlbi.nih.gov/Databases/AVP‐Phos/. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. Fifteen downregulated phosphorylation sites are associated with decreased activation of the MAP kinases, ERK1 and ERK2. The remaining 13 sites are likely to be phosphorylated by other kinases. Regulated phosphoproteins include several protein kinases, namely Mink1, Camkk2, Cdk18, Prkar2a, Erbb3, and Src, which play likely roles downstream from PKA. Gene Ontology term analysis reveals multiple vasopressin‐regulated phosphoproteins involved in known vasopressin‐regulated processes: “transmembrane transport” (Aqp2, Itpr1, Itpr2, Itpr3), “actin cytoskeleton organization” (Bin1, Myo9b, Pdlim5, Slc9a3r1, Specc1l), “regulation of GTPase activity” (Als2, Arfgef1, Ralgapa2), “exocytosis” (Exoc7, Stxbp5, Itsn1), “endocytosis” (Bin1, Itsn1), “cell cycle” (Sept9, Arhgef2, Specc1l, Src), “regulation of transcription” (Hdac4, Ctnnb1, Lrrfip1, Tsc22d4), “Wnt signaling pathway” (Ctnnb1, Slc9a3r1, Src). Overall, this study provides a list of vasopressin‐regulated phosphoproteins likely to be involved in vasopressin‐mediated regulation of collecting duct water transport, providing grist for future studies.

FOXI1 Promotes Expression of V‐ATPase and GPR116 in M‐1 Cells

G protein‐coupled receptors (GPCRs) are a diverse family of integral membrane proteins that have significant roles in numerous physiological systems. We previously reported that GPR116 (ADGRF5), an adhesion‐class GPCR, is a critical regulator of vacuolar‐type H+‐ATPase (V‐ATPase) surface expression in A‐type intercalated cells (AICs) in mouse kidney cortical collecting ducts. The V‐ATPase is a multi‐subunit proton pump that localizes to the plasma membrane in specialized acid‐secreting epithelial cells. FOXI1 is a transcription factor that regulates V‐ATPase expression in ICs and other mitochondria‐rich cells. Recently, FOXI1 was identified as a key regulator of CFTR‐rich pulmonary ionocytes which express both V‐ATPase and GPR116. Therefore, we hypothesized that FOXI1 is a transcription factor in AICs that upregulates V‐ATPase as well as GPR116. To test this hypothesis, we cloned FOXI1 from whole mouse kidney into a pME18s expression vector (pFOXI1). Transfection with pFOXI1 significantly increased GPR116 expression (qPCR) in M‐1 mouse cortical collecting duct cells (n=3, ΔCt ±SEM: untransfected = 13.8±0.6, pFoxi1 = 10.7±0.2, p<0.05), but not in HEK293T cells (n=3, not significant). RNA‐scope hybridization on murine kidney sections and pFOXI1‐transfected M‐1 cells revealed colocalization of FOXI1 and GPR116 mRNA in the same cell. Treating pFOXI1‐transfected M‐1 cells with a synthetic agonist peptide for GPR116 results in an increase of [Ca2+]iin a subset of cells (n=3, ΔF340/380 ±SEM: pFoxi1 = 0.296±0.017). Internal calcium mobilization is never seen with the agonist peptide in untransfected control cells, indicating that pFOXI1 is sufficient for the production of physiologically responsive GPR116 on the cell surface (n=3, ΔF340/380±SEM: untransfected = 0 ±0.0009). Moreover, only M‐1 cells transfected with pFOXI1 express GPR110 (ADGRF1), an adhesion‐GPCR with unknown function in the kidney, as well as three V‐ATPase subunits, ATP6V1B1, ATP6V1G3, ATP6V0D2, indicating a larger transcriptional program initiated by pFOXI1 (n=3, untransfected: Ct ~ 40 for all genes, pFoxi1 ΔCt±SEM: ADGRF1 = 13.2±0.21, ATP6V1B1 = 11.4±0.3, ATP6V1G3 = 10±0.1, ATP6V0D2 = 12.1±0.03). Additionally, these pFOXI1 cells also upregulate the expression of SLC26A4 (pendrin), a known marker of B‐type intercalated cells, suggesting that FOXI1‐driven regulation is upstream of AIC fate determination (n=3, ΔCt ± SEM: untransfected = 19.3±0.7, pFoxi1 = 16.1±0.2, p<0.01). These data reveal that FOXI1 upregulates both GPR116 and V‐ATPase in M‐1 cells, generating IC‐like cells. Finally, our study suggests that GPR116 may be a universal and genetically‐coded regulator of V‐ATPase surface expression in FOXI1‐positive cells.

Interrogation of Novel Aldosterone‐induced Genes Associated With Acute Stimulation Of ENaC In Primary Collecting Duct Cells

Aldosterone (ALDO) is a key volume‐regulating hormone which promotes Na+ reabsorption in the distal nephron. ALDO sensitivity is conferred by the enzyme 11βHSD2 which breaks down the related more abundant steroid, cortisol. Mutations in Hsd11b2 give rise to the syndrome of apparent mineralocorticoid excess, where hypertension is associated with excessive renal Na+ reabsorption. In the distal nephron corticosteroid‐induced Na+ transport, via the epithelial Na+ channel ENaC, is due to transcriptional processes and previous studies have identified key genes including Sgk1. Our lab carried out transcriptomic analysis of mCCDcl1 cells following acute corticosteroid induced ENaC‐mediated Na+ transport, identifying several novel ALDO induced genes. The present study aimed to elucidate the effects of corticosteroids on these target genes in the collecting duct (CD), utilising an in vitro and in vivo approach.A CD‐specific reporter mouse was generated using Aqp2Cre to express eGFP. Primary cells were isolated by FACS and either grown in culture or RNA was extracted immediately. Cells were grown on filter membranes to allow development of polarised monolayers and electrometric properties were assessed using an epithelial volt‐ohm‐meter. Target gene expression was determined by qRT‐PCR.Assessment of nephron segment‐specific gene expression revealed an enrichment of genes encoding α‐ENaC, ROMK and 11βHSD2 but not NHE3 or NKCC2. NCC was detected but parvalbumin was absent, demonstrating isolation of cells from the late distal tubule onwards. In addition to genes associated with principal cells we also detected genes encoding pendrin and AE1, indicative of intercalated cells therefore this population reflects CD cells. When grown in culture for 9‐11 days, these cells exhibited Vt and Rt of ‑20.3±3.1 mV and 5.1 ±0.1 kΩ∙cm2, respectively. This gave rise to an Ieq of ‑3.9±0.6 μA·cm‑2 which was near abolished with 10 µM amiloride – indicating Na+ transport via ENaC.Acute application of steroid hormones to cells for 3h: ALDO (3 nM), DEX (100 nM) and CORT (10 nM) – the latter only when 11βHSD2 activity was inhibited using carbenoxolone (CBX, 10 µM) – stimulated amiloride‐sensitive currents by 2.0‐, 3.6‐ and 2.4‐fold, respectively. Analysis of ALDO‐induced genes revealed both ALDO and CORT upregulated Sgk1, Zbtb16 and Rasd1. ALDO also upregulated Sult1d1 whereas CORT also upregulated an unannotated gene Gm43305. DEX upregulated all of these as well as 2 additional genes previously detected: Defb1 and Gm16178.To determine the relevance of these findings in vivo reporter mice were administered ALDO, vehicle or CORT, by ip injection, the latter under control conditions or following CBX treatment for 8 days. Cells were isolated after 3h. The previously identified Zbtb16, Rasd1, Gm43305 and Sult1d1 were upregulated in both ALDO and CBX+CORT groups, though no change was detected in either Sgk1 or Defb1. Scnn1a was also upregulated in both groups and additionally Per1 and Sult1a1 were upregulated in mice treated with CBX‐CORT.Together these data confirm 4 novel corticosteroid‐induced genes in primary CD cells associated with acute stimulation of ENaC activity.

Sphingomyelin‐enriched extracellular vesicles from hypertensive diabetic mice regulate calcium mobilization and amiloride‐sensitive transepithelial current in mouse cortical collecting duct cells

Uncontrolled hypertension (HTN) and diabetes mellitus (DM) are two major public health burdens contributing to increased hospitalization, readmission costs, and adverse clinical outcomes. Moreover, DM is a leading cause of death and long‐term morbidity. Overexpression and activity of epithelial sodium channels (ENaC) in the kidney results in hypertension, but the mechanism for its regulation in the diabetic kidney is not entirely understood. Cholesterol is known to increase ENaC activity in cultured kidney cells in a calcium and PI(4,5)P2‐dependent mechanism. Presumably ENaC is regulated by other lipids in pathophysiology. Extracellular vesicles (EVs) are nano‐sized vesicles that play an important role in intercellular communication and intracellular signaling. Here, we investigated whether EVs isolated from the urine of hypertensive diabetic db/db mice or diabetic db/db mice augment ENaC activity by inhibiting calcium mobilization in mpkCCD cells. Adult diabetic db/db mice were salt loaded (4% NaCl) for 7 days to induce hypertension. Blood pressure was measured by tail‐cuff, and urine from db/db mice with systolic blood pressure of greater than 140mmHg or from normotensive db/db mice were used to isolate EVs. Nanoparticle tracking analysis was performed to determine EV concentration. EVs from hypertensive diabetic db/db mice (N=5) compared to diabetic db/db mice (N=5) suppressed ionomycin‐induced calcium mobilization in mpkCCD cells cultured on glass bottom 35 mm dishes (N=3 experiments, p<0.05) which was measured by confocal microscopy and augmented amiloride‐sensitive transepithelial current in mpKCCD cells cultured in 12mm diameter permeable supports (N=6 permeable supports per group) measured by an epithelial volt ohm meter (EVOM). Sphingomyelin were found to be enriched in urinary EVs from hypertensive diabetic db/db mice compared to diabetic only db/db mice. Exogenous application of sphingomyelin to mpkCCD cells mimicked the suppression of calcium mobilization and increase in amiloride‐sensitive transepithelial current in these cells. Taken together, these data suggest differences in the packaged cargo of urinary EVs between HTN and DM mice that presumably plays a role in the regulation of ENaC activity.

HDAC3 Inhibition Improved Urinary Concentrating Defect in Hypokalemia by Promoting AQP2 Transcription

Hypokalemia is a common electrolyte disturbance frequently encountered in clinical medicine. Animals with hypokalemia had a significant arginine vasopressin (AVP)‐resistant polyuria with reduced expression of aquaporin 2 (AQP2) levels in the collecting ducts of the kidneys. The precise molecular mechanisms of hypokalemia‐induced nephrogenic diabetes insipidus remain unresolved. AVP induced a marked increase in histone H3K27 acetylation (H3K27ac) across the body of the AQP2 gene, in the promoter and in upstream of the AQP2 transcriptional start site in mouse mpkCCD cell line (PMID: 29572403). The purpose of the present study was to investigate whether enhanced histone acetylation prevented decreased AQP2 expression and improved urinary concentrating defect in rats with hypokalemia. Male Wistar rats were fed potassium‐free diet with or without 4‐phenylbutyric acid (4‐PBA) or a selective inhibitor of histone deacetylase 3 (HDAC3) RGFP966 for 4 days. 4‐PBA or RGFP966 markedly increased AQP2 mRNA and protein levels in kidney inner medulla of rats with hypokalemia, which was associated with decreased urine output and increased urinary osmolality. Protein expression of H3K27ac was markedly decreased in kidney inner medulla of hypokalemic rats, which was significantly prevented by 4‐PBA or RGFP966. Expression of H3K27ac was decreased in primary cultured inner medullary collecting duct cells (IMCD) or mpkCCD cells cultured in potassium‐deprivation media, in particular, H3K27ac in the promoter region of AQP2 was markedly decreased, which was associated with reduced mRNA levels of AQP2. Protein expression of HDAC3 was significantly upregulated in both mpkCCD and IMCD cells in response to potassium deprivation; the binding of HDAC3 to the promoter of AQP2 was also dramatically increased. RGFP966 significantly increased expression of H3K27ac and AQP2 protein and enhanced the binding of H3K27ac to AQP2 in mpkCCD cells. In conclusion, HDAC3 inhibition prevented downregulation of AQP2 and improved urinary concentrating defect induced by potassium deprivation, likely through enhancing acetylation of H3K27 and transcription of AQP2.

Effects of flow and polycystin‐1 dysfunction on ATP release in the inner medullary collecting duct of the kidney

After filtration of blood by the kidney, the remaining pro‐urine flows through the renal tubular system with a flow rate that can vary considerably. These variations in pro‐urinary flow can be sensed by mechanosensing proteins on the epithelial cells lining the renal tubules. An important mechanosensing protein is polycystin‐1 (PC1), which is thought to be a key sensor of fluid flow. Mutations in PC1 give rise to autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid‐filled cysts in the kidney. Previous research has highlighted a role for the ATP release channel pannexin‐1 (PANX1) in the increased flow‐induced ATP release in a Pkd1‐/‐ cell model of the distal convoluted tubule. However, it remains unclear if other purinergic signaling components are also involved, especially in the collecting duct.Using the mouse inner medullary collecting duct 3 (mIMCD3) cell line in combination with microfluidic experiments, we show here that a similar flow‐induced ATP release is present in Pkd1‐/‐ mIMCD3 cells under low (0.32 mL/min) or high (1.27 mL/min) flow application for 1 minute compared to wildtype (WT) mIMCD3 cells. This is despite a six‐fold increase in the expression of Panx1 observed in Pkd1‐/‐ mIMCD3 cells. Moreover, application of the specific PANX1‐inhibitor BB‐FCF revealed that PANX1 did not drive the high flow‐induced increased ATP release. Accordingly, intermediate flow application (0.47 mL/min) for 3 hours did not increase Panx1 expression in WT mIMCD3 cells compared to the static condition. Flow did not increase the expression of any of the known purinergic receptors in the collecting duct cells compared to the static condition. We did observe a two‐fold increase in gene expression of the putative ATP release channel connexin‐30.3 (CX30.3) compared to static conditions. Moreover, we also report a three‐fold increase in CX30.3 gene expression in Pkd1‐/‐ mIMCD3 cells compared to WT cells.In conclusion, we show here that flow‐induced purinergic signaling in the collecting duct greatly differs from previously published work on the distal convoluted tubule. Specifically, in the inner medulla of the collecting duct, PANX1 may not be the key flow‐regulated ATP release channel. Instead, CX30.3 may facilitate flow‐induced ATP release in this segment in health and in ADPKD.

Expression of Pannexin 1 in the Human Kidney during Embryonal, Early Fetal and Postnatal Development and Its Prognostic Significance in Diabetic Nephropathy

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman’s capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th–10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes.

Effect of waterborne exposure to perfluorooctanoic acid on nephron and renal hemopoietic tissue of common carp Cyprinus carpio

Per- and poly-fluoroalkyl substances (PFAS) are synthetic contaminants of global concern for environmental and public health. Perfluorooctanoic acid (PFOA) is an important PFAS, and considerable attention has been paid to its hepatotoxicity and reproductive and developmental impact, while potential nephrotoxic effects are largely ignored, especially in fish. This study documents the structural and ultrastructural effects on kidney of common carp Cyprinus carpio exposed to waterborne PFOA at an environmentally relevant concentration of 200 ng L−1 and at 2 mg L−1. Dilation of the glomeruli capillary bed, increased vesiculation in the proximal tubular segment, compromised mitochondria, apical blebbing, and sloughing of collecting duct cells occurred in exposed fish, primarily at 2 mg L−1. Perfluorooctanoic acid exposure resulted in higher numbers of rodlet cells (RC), putative immune cells exclusive to fish, mainly in the renal interstitium, than seen in controls, increased association with cells of myeloid lineage and modifications to ultrastructure. No differences in other cells of innate immunity were observed.Despite the absence of severe histological lesions, PFOA was shown to affect both nephron and hemopoietic interstitium at high concentration, raising concern of the impact on renal and immune function in fish. The response of RCs to PFOA concentration of 200 ng L−1 suggests a potential role as a biomarker of PFOA exposure.

Functional role of histamine receptors in the renal cortical collecting duct cells.

Histamine is an important immunomodulator, as well as a regulator of allergic inflammation, gastric acid secretion, and neurotransmission. Although substantial histamine level has been reported in the kidney, renal pathological and physiological effects of this compound have not been clearly defined. The goal of this study was to provide insight into the role of histamine-related pathways in the kidney, with emphasis on the collecting duct (CD), a distal part of the nephron important for the regulation of blood pressure. We report that all four histamine receptors (HRs) as well as enzymes responsible for histamine metabolism and synthesis are expressed in cultured mouse mpkCCDcl4 cells, and histamine evokes a dose-dependent transient increase in intracellular Ca2+ in these cells. Furthermore, we observed a dose-dependent increase in cAMP in the CD cells in response to histamine. Short-circuit current studies aimed at measuring Na+ reabsorption via ENaC (epithelial Na+ channel) demonstrated inhibition of ENaC-mediated currents by histamine after a 4-h incubation, and single-channel patch-clamp analysis revealed similar ENaC open probability before and after acute histamine application. The long-term (4 h) effect on ENaC was corroborated in immunocytochemistry and qPCR, which showed a decrease in protein and gene expression for αENaC upon histamine treatment. In summary, our data highlight the functional importance of HRs in the CD cells and suggest potential implications of histamine in inflammation-related renal conditions. Further research is required to discern the molecular pathways downstream of HRs and assess the role of specific receptors in renal pathophysiology.

Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel.

Simple SummaryHere, we investigated the regulation of the renal epithelial sodium channel (ENaC) by iron in two established cell lines and a transgenic animal model of iron overload. We show that iron availability inhibits ENaC protein expression and its activity. We provide a discussion for putative mechanistic pathways that may be essential in this feedback inhibition.Hypertension is associated with an increased renal expression and activity of the epithelial sodium channel (ENaC) and iron deficiency. Distal tubules absorb iron, causing perturbations that may influence local responses. In this observational study, we investigated the relationship between iron content and ENaC expression and activity using two cell lines and hepcidin knockout mice (a murine model of iron overload). We found that iron did not transcriptionally regulate ENaC in hepcidin knockout mice or in vitro in collecting duct cells. However, the renal tubules of hepcidin knockout mice have a lower expression of ENaC protein. ENaC activity in cultured Xenopus 2F3 cells and mpkCCD cells was inhibited by iron, which could be reversed by iron chelation. Thus, our novel findings implicate iron as a regulator of ENaC protein and its activity.

Apelin-13 Decreases Epithelial Sodium Channel (ENaC) Expression and Activity in Kidney Collecting Duct Cells.

Apelin and its G protein-coupled receptor APLNR (also known as APJ) are widely expressed within the central nervous system and peripheral organs including heart, lung and kidney. Several studies have shown that the apelin/APJ system is involved in various important physiological processes such as energy metabolism, cardiovascular functions and fluid homeostasis. In the kidney, the apelin/APJ system performs a wide range of activities. We recently demonstrated that apelin antagonises the hydro-osmotic effect of vasopressin on aquaporin-2 water channel (AQP-2) expression by reducing its mRNA and protein levels in collecting duct principal cells. The central role of these cells in water and sodium transport is governed by AQP-2 and the epithelial sodium channel (ENaC). The coordination of these channels is essential for the control of extracellular fluid volume, sodium homeostasis and blood pressure. This study aimed at investigating the role of apelin in the regulation of sodium balance in the distal nephron, and more specifically its involvement in modulating the expression and activity of ENaC in collecting duct principal cells. mpkCCD cells were incubated in the presence of aldosterone and treated with or without apelin-13. Transepithelial Na+ current was measured and the changes in ENaC expression determined by RT-PCR and immunoblotting. Our data show that apelin-13 reduces the transepithelial sodium amiloride-sensitive current in collecting duct principal cells after 8h and 24h treatment. This effect was associated with a decrease in αENaC subunit expression and mediated through the ERK pathway as well as SGK1 and Nedd4-2. Our findings indicate that apelin is involved in the fine regulation of sodium balance in the renal collecting duct by opposing the effects of aldosterone, likely by activation of ENaC ubiquitination.

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.