Cortical Collecting Duct Cells Research Articles

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.

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.

Abstract 09: Mtor Inhibition Decreases Ligand-induced Mineralocorticoid Receptor Activation

Introduction: ULK1 phosphorylates the MR at S843, decreasing its ligand binding and transcriptional activity. Angiotensin II-induced mTOR phosphorylation of ULK1 inactivates ULK1, preventing its phosphorylation of MR. Aim: Further elucidate the role of mTOR in the regulation of MR transcriptional activity. Methods: M1 mouse cortical collecting duct cells stably transduced with the rat MR cDNA and a MMTV- Gaussia luciferase reporter gene, were incubated with an mTOR activator and several inhibitors, +/- aldosterone or corticosterone. Similar studies were done after lentiviral transduction of CRISPR/gRNA for raptor and rictor genes or mutated MR (mu/S843A) cDNA. Results: mTOR inhibition significantly decreased ligand activation of the MR reporter gene, while the mTOR activator MHY1485 had no effect suggesting that mTOR is tonically active. MR activation induced by aldosterone and corticosterone was also decreased by CRISPR/gRNA gene knockdown of raptor and rictor, the adaptors of mTOR complex 1 and 2, respectively, supporting a role for mTOR. The mTOR inhibitor AZD8055 (AZD) reduced phospho-ULK1 and attenuated ligand-mediated MR transactivation in a dose-dependent manner. The ULK1 inhibitor MRT68921 increased MR transactivation. We speculated that mTOR decreased ULK1 activity by phosphorylating it, thereby preventing ULK1 phosphorylation of the MR at Serine (S843). However, when M1 cells were transduced with an MR cDNA in which S843 was replaced with Alanine that cannot be phosphorylated, ligand-induced activation of the mu/S843A MR was still decreased by AZD, but unchanged by MRT68921. This suggests that mTOR has an additional effect on MR activity unrelated to ULK1 activity. AZD also decreased P70S6K and AKT phosphorylation in these cells. Conclusions: mTOR phosphorylation of ULK1 prevents its phosphorylation of the MR and reduction of MR transcriptional activity. mTOR inhibitors and deletion of raptor and rictor decreased MR transcriptional activity. mTOR has additional positive effects on MR activity possibly related to its phosphorylation of AKT and P70S6K. Inhibition of mTOR action may be a useful target for mitigating excessive MR activation.

The Angiotensin II Type 1 Receptor-Associated Protein Attenuates Angiotensin II-Mediated Inhibition of the Renal Outer Medullary Potassium Channel in Collecting Duct Cells.

Adjustments in renal K+ excretion constitute a central mechanism for K+ homeostasis. The renal outer medullary potassium (ROMK) channel accounts for the major K+ secretory route in collecting ducts during basal conditions. Activation of the angiotensin II (Ang II) type 1 receptor (AT1R) by Ang II is known to inhibit ROMK activity under the setting of K+ dietary restriction, underscoring the role of the AT1R in K+ conservation. The present study aimed to investigate whether an AT1R binding partner, the AT1R-associated protein (ATRAP), impacts Ang II-mediated ROMK regulation in collecting duct cells and, if so, to gain insight into the potential underlying mechanisms. To this end, we overexpressed either ATRAP or β-galactosidase (LacZ; used as a control), in M-1 cells, a model line of cortical collecting duct cells. We then assessed ROMK channel activity by employing a novel fluorescence-based microplate assay. Experiments were performed in the presence of 10−10 M Ang II or vehicle for 40 min. We observed that Ang II-induced a significant inhibition of ROMK in LacZ, but not in ATRAP-overexpressed M-1 cells. Inhibition of ROMK-mediated K+ secretion by Ang II was accompanied by lower ROMK cell surface expression. Conversely, Ang II did not affect the ROMK-cell surface abundance in M-1 cells transfected with ATRAP. Additionally, diminished response to Ang II in M-1 cells overexpressing ATRAP was accompanied by decreased c-Src phosphorylation at the tyrosine 416. Unexpectedly, reduced phospho-c-Src levels were also found in M-1 cells, overexpressing ATRAP treated with vehicle, suggesting that ATRAP can also downregulate this kinase independently of Ang II-AT1R activation. Collectively, our data support that ATRAP attenuates inhibition of ROMK by Ang II in collecting duct cells, presumably by reducing c-Src activation and blocking ROMK internalization. The potential role of ATRAP in K+ homeostasis and/or disorders awaits further investigation.

Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells.

The kidney cortical collecting duct (CCD) comprises principal cells (PCs), intercalated cells (IC), and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess revealed plasticity of the CCD, with altered PC:intermediate cell:IC ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs, but displays a range of cell types, including intermediate cells, making it ideal to study plasticity. We knocked out Adam10, a key component of the Notch pathway, in mCCDcl1 cells, using CRISPR-Cas9 technology, and isolated independent clones, which exhibited severely affected sodium transport capacity and loss of aldosterone response. Single-cell RNA sequencing revealed significantly reduced expression of major PC-specific markers, such as Scnn1g (γ-ENaC) and Hsd11b2 (11βHSD2), but no significant changes in transcription of components of the Notch pathway were observed. Immunostaining in the knockout clone confirmed the decrease in expression of γ-ENaC and importantly, showed an altered, diffuse distribution of PC and IC markers, suggesting altered trafficking in the Adam10 knockout clone as an explanation for the loss of polarization.

Angiotensin II Elicits Intracellular Ca 2+ Responses in Rat Cortical Collecting Ducts

Regulation of sodium transport in the cortical collecting duct (CCD) is pivotal for the long-term blood pressure control and excessive Na+ retention in this nephron segment is a known pathogenic determinant of hypertension. Sodium reabsorption in the CCD cells can be stimulated by volume-regulatory hormones, such as angiotensin II (AngII). AngII reportedly binds to AT1 receptors and increases the activity of the epithelial sodium channel (ENaC) via generation of reactive oxygen species (ROS). Yet, our understanding of the exact molecular determinants mediating AngII-induced intracellular signaling in the CCD cells remains limited. Numerous studies show that AngII induces intracellular Ca2+ mobilization in the vasculature, cardiomyocytes and glomerular podocytes. Here, we used ratiometric Fura-2 Ca2+-imaging to test if AngII employs Ca2+ as a second messenger in the CCDs freshly isolated from male and female Sprague-Dawley (SD) rats. We found that application of 500 nM of AngII to a split-opened CCD results in a transient elevation of [Ca2+]i in the CCD cells. The amplitude of the observed [Ca2+]i increase is about 150 nM and is not significantly different in the CCDs isolated from male and female rats. AngII-dependent [Ca2+]i signal is not affected by removal of Ca2+ from the extracellular bathing solution, but is practically abolished, when the tubules are pre-incubated in 3 μM thapsigargin, indicative of Ca2+ release from the intracellular stores. Inhibition of PLC with 10 μM U73122 markedly diminishes AngII-induced [Ca2+]i transient. Chronic AngII infusion moderately impairs [Ca2+]i responses to AngII in the CCDs isolated from male rats by approximately 30%. In contrast, AngII-induced [Ca2+]i elevation is attenuated by 70% in the CCDs from AngII-infused females. We have previously reported a stronger antihypertensive effect of aldosterone antagonism in AngII-infused females, when compared to males. Here, we found that systemic administration of eplerenone prevents the decline of AngII-stimulated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension, but not in males. Overall, our data demonstrate that AngII triggers Ca2+ release from the intracellular stores of the CCD cells in a PLC-dependent manner. This AngII-activated [Ca2+]i mobilization is impaired in AngII-infused SD rats of both sexes, but the attenuation is remarkably more pronounced in females. Systemic aldosterone antagonism with eplerenone restores the AngII-mediated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension. Future studies are granted to investigate the interplay between AngII-dependent [Ca2+]i signal and ROS generation in the CCD cells.

Investigating the Transitional Cell Type in the Kidney Collecting Duct

The kidney cortical collecting duct (CCD) has long been thought to contain two distinct cell types, principal cells (PC) and intercalated cells (IC), which facilitate 4-5% of sodium and 10% of water reabsorption in the kidney. Recent single cell RNA-Seq data has identified a distinct transitional cell expressing both PC and IC markers and a number of distinct markers. In this study, the presence of CCD cell types was investigated in mice and rats under the disease model of the syndrome of apparent mineralocorticoid excess (SAME, conferred by a mutation of the Hsd11b2gene, expressed by PCs) and also in two CCD derived cell lines. Inactivation of HSD11B2in humans results in a severe, and often fatal, phenotype in early neonatal life, whilst milder mutations, with residual enzyme activity cause hypertension, sodium retention and hypokalemia in adulthood. The Hsd11b2 gene knockout (KO) model of SAME in mice and rats was studied in the context of CCD cell plasticity and it was hypothesised to reduce the proportion of PCs within the CCD. Cells in the CCD were counted in WT and Hsd11b2 KO animals following immunofluorescent staining for PC- and IC-specific markers (Aqp2 and V-ATPase B1, respectively) in paraffin-embedded kidney sections. ImageJ and GraphPadPrism were used to analyse data, which are expressed as mean ± SEM. Significance was assessed using Tukey's multiple comparisons test, p<0.0001. PC proportion was significantly reduced in KO mice CCDs compared to controls at 4 weeks (52.1 ± 1.9 % vs. 73.0 ± 1.9 %) and 17 weeks (54.1 ± 1.8 % vs. 72.2 ± 1.3 %). A significant increase in the number of cells expressing both PC and IC markers was observed between WT and KO at 4 weeks (8.4 ± 1.2 % vs.25.9 ± 2.0 %) and at 17 weeks (6.5 ± 0.8 % vs. 22.1 ± 2.0 %). Similarly, in the rat KO a significant reduction in the proportion of PCs was noted compared to controls at 5 weeks (60.69 ± 2.51 % vs. 73.30 ± 1.84 %) and 24 weeks (38.40 ± 2.07 % vs. 66.80 ± 1.94 %). This study shows CCD cell plasticity in models of SAME, but also provides further evidence for a PC+/IC+cell in the kidney. The mCCDcl1­ and mpkCCDcl4­ cell lines are typically used as models of PC function. Immunofluorescent staining using antibodies against PC, IC and transitional cell markers (Aqp2, V-ATPase B1 and Parm1, respectively) was carried out on fixed, polarised cells and images analysed using ImageJ software. It was found that both cell line lines contained cells that co-expressed PC and IC markers, similar to the cell type seen in mouse and rat kidneys. Furthermore, a large proportion of cells in both cell lines expressed the transitional cell marker, Parm1. These results provide further support for the presence of a transitional cell type in the CCD, especially in pathophysiological states. The observation of similar cells in CCD cell lines also indicates their use as in vitro models for investigating the role and plasticity of CCD cell types.

Knock‐out of Adam10 in mCCD cl1 cells gives insight into the effects of Notch pathway disruption for kidney collecting duct cell plasticity

The kidney cortical collecting duct (CCD) is responsible for the fine tuning of sodium, volume, and acid-base homeostasis, and comprises of principal and intercalated cells (PCs and ICs) as well as a newly discovered ‘intermediate’ cell type (1). Plasticity between PCs and ICs has been reported previously in mice in which genes for factors implicated in the Notch pathway, such as Adam10, have been knocked out (KO) (2). In vivo, disruption of the Notch pathway leads to a decreasing PC:IC ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs and a response to physiological concentrations of hormones such as aldosterone; it also has been shown to express both PC and IC markers, a phenotype transmitted through single cell cloning (3). These characteristics make mCCDcl1 cells a useful platform for the study of plasticity in the CCD. Adam10 was knocked out in mCCDcl1 cells using CRISPR-Cas9 technology and independent clones were isolated and expanded for study. Despite mCCDcl1 cells being polyploid, our strategy enabled all alleles for each gene to be successfully targeted. Clones were cultured on Transwell filters over 10 days and their electrophysiological properties measured (trans-epithelial resistance Rte, voltage Vte, calculated short-circuit current Isc). Both Adam10 KO clones showed a slightly reduced baseline Rte compared to control (0.90±0.19 kΩ.cm2 vs 1.12±0.18 kΩ.cm2) but a complete reduction in baseline Isc (0.3±0.1 μA/cm2 vs -8.1±0.4 μA/cm2 for control) as well as aldosterone response, decreased by 95% (fold change 0.19±0.03 down 3.70±0.11 for control). Single-cell RNA sequencing principal component analysis shows different clustering of KO clones compared to WT. Potentially explaining the loss of sodium transport function, a significant drop in expression for the gamma subunit of the epithelial sodium channel (γ-ENaC) (between ~40% and ~60% depending on the cluster) was detected in the KO clones, as well as a decrease in the expression of 11ßHSD2 (between ~20% and ~60%). Immunostaining in KO clones confirmed the decrease in expression of γ-ENaC and showed an altered distribution and localisation of γ-ENaC, Aqp2, and V-ATPase B1 compared to WT mCCDcl1, overall suggesting a loss of polarisation. These data suggest that genomic modification of mCCDcl1 cells provides an effective means to investigate the mechanisms through which CCD cells mediate their plasticity and can identify new pathways involved in regulating cell phenotype as well as specific effects of pathway disruption on collecting duct cells functions. (1) Park et al.; Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease; Science; 360: 758-763, 2018. (2) Guo et al.; Adam10 Mediates the Choice between Principal Cells and Intercalated Cells in the Kidney; JASN; 26: 149-159, 2015. (3) Assmus et al.; mCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells; AJP-renal; 314 (5): 820-831, 2018.

The ubiquitin ligase NEDD4-2/NEDD4L regulates both sodium homeostasis and fibrotic signaling to prevent end-stage renal disease

Kidney disease progression can be affected by Na+ abundance. A key regulator of Na+ homeostasis is the ubiquitin ligase NEDD4-2 and its deficiency leads to increased Na+ transport activity and salt-sensitive progressive kidney damage. However, the mechanisms responsible for high Na+ induced damage remain poorly understood. Here we show that a high Na+ diet compromised kidney function in Nedd4-2-deficient mice, indicative of progression toward end-stage renal disease. Injury was characterized by enhanced tubule dilation and extracellular matrix accumulation, together with sustained activation of both Wnt/β-catenin and TGF-β signaling. Nedd4-2 knockout in cortical collecting duct cells also activated these pathways and led to epithelial–mesenchymal transition. Furthermore, low dietary Na+ rescued kidney disease in Nedd4-2-deficient mice and silenced Wnt/β-catenin and TGF-β signaling. Our study reveals the important role of NEDD4-2-dependent ubiquitination in Na+ homeostasis and protecting against aberrant Wnt/β-catenin/TGF-β signaling in progressive kidney disease.

Unilateral Nephrectomy Stimulates ERK and Is Associated With Enhanced Na Transport.

Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (Isc). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, n > 6 per group, p < 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive Isc in static mpkCCD cells was 25.3 ± 1.7 μA/cm2 (n = 21), but after exposure to 24 h of FSS the Isc increased to 41.4 ± 2.8 μA/cm2 (n = 22; p < 0.01) and returned to 19.1 ± 2.1 μA/cm2 (n = 18, p < 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. In vitro studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.

Glutathione depletion induces oxidative injury and apoptosis via TRPM2 channel activation in renal collecting duct cells

Oxidative stress (OS)-induced glutathione (GSH) depletion plays an essential role in several kidney diseases such as chronic kidney disease and nephrotoxicity. The OS-dependent activation of TRPM2 cation channel in several neurons and cells were modulated by the concentration of intracellular GSH. However, the effects of GSH alteration on TRPM2 activation, OS, and apoptosis in the cortical collecting duct (mpkCCDc14) cells still remain elusive. We investigated the effects of GSH supplementation on OS-induced TRPM2 activation, mitochondrial oxidative stress, and apoptosis in the human embryonic kidney 293 (HEK293) and mpkCCDc14 cells treated with buthionine-sulfoximine (BSO), a GSH synthase inhibitor. The HEK293 and mpkCCDc14 cells were divided into five groups as control, GSH (10 mM for 2 h), BSO (0.5 mM for 6 h), BSO + GSH, and BSO + TRPM2 channel blockers. Apoptosis, cell death, mitochondrial OS, caspase −3, caspase −9, cytosolic free Zn2+, and Ca2+ concentrations were increased in the BSO group of the TRPM2 expressing mpkCCDc14 cells, although they were diminished by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2-APB). The BSO-induced decreases in the levels of cell viability and cytosolic GSH were increased by the treatments of GSH, ACA, and 2-APB. However, the effects of BSO and GSH were not observed in the non-TRPM2 expressing HEK293 cells. Current results show that maintaining GSH homeostasis is not only important for quenching OS in the cortical collecting duct cells but equally critical to modulate TRPM2 activation. Thus, suppressing apoptosis and mitochondrial OS responses elicited by oxidant action of GSH depletion.

Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II-Induced Hypertension in Mice.

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

20-HETE downregulates Na/K-ATPase α1 expression via the ubiquitination pathway

In this article, we found that 20-Hydroxyeicosatetraenoic acid (20-HETE) reduced Na/K-ATPase α1 expression via the ubiquitin-proteasome pathway. The ubiquitination level of Na/K-ATPase α1 protein was increased in 20-HETE-treated mouse cortical collecting duct cells and the kidney tissues of CYP4F2 transgenic mice. We also demonstrated that 20-HETE-induced high level phosphorylation of Na/K-ATPase α1 was necessary for its ubiquitination.The protein kinase C inhibitor sotrastaurin significantly reduced the phosphorylation of Na/K-ATPase α1 and increased the expression of Na/K-ATPase α1 although 20-HETE stimulus being applied at the same time. Moreover, high level of 20-HETE increased the expression and neddylation of Cullin3,which is an important ubiquitin E3 ligase in kidney. MLN4924, an inhibitor of NEDD8-activating enzyme, inhibited neddylation of Cullin3 and reversed the reduction of Na/K-ATPase α1 expression caused by 20-HETE. Thus, 20-HETE downregulates Na/K-ATPase α1 via the ubiquitination pathway, and phosphorylation of Na/K-ATPase α1 is a prerequisite to ubiquitination. Additionally, 20-HETE regulates Cullin3 expression via neddylation.

Knockout of the circadian clock protein PER1 results in sex-dependent alterations of ET-1 production in mice in response to a high-salt diet plus mineralocorticoid treatment.

Previously, we showed that global knockout (KO) of the circadian clock transcription factor PER1 in male, but not female, mice fed a high-salt diet plus mineralocorticoid treatment (HS/DOCP) resulted in nondipping hypertension and decreased night/day ratio of sodium (Na) excretion. Additionally, we have shown that the endothelin-1 (ET-1) gene is targeted by both PER1 and aldosterone. We hypothesized that ET-1 would exhibit a sex-specific response to HS/DOCP treatment in PER1 KO. Here we show that male, but not female, global PER1 KO mice exhibit a decreased night/day ratio of urinary ET-1. Gene expression analysis revealed significant genotype differences in ET-1 and endothelin A receptor (ETA) expression in male, but not female, mice in response to HS/DOCP. Additionally, both wild-type and global PER1 KO male mice significantly increase endothelin B receptor (ETB) expression in response to HS/DOCP, but female mice do not. Finally, siRNA-mediated knockdown of PER1 in mouse cortical collecting duct cells (mpkCCDc14) resulted in increased ET-1 mRNA expression and peptide secretion in response to aldosterone treatment. These data suggest that PER1 is a negative regulator of ET-1 expression in response to HS/DOCP, revealing a novel mechanism for the regulation of renal Na handling in response to HS/DOCP treatment.

Release of ATP by TRPV4 activation is dependent upon the expression of AQP2 in renal cells.

Increasing evidence indicates that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the transient receptor potential vanilloid 4 (TRPV4) channel. We previously found that TRPV4 physically and functionally interacts with AQP2 in cortical collecting ducts (CCD) cells, favoring cell volume regulation and cell migration. Because TRPV4 was implicated in ATP release in several tissues, we investigated the possibility that TRPV4/AQP2 interaction influences ATP release in CCD cells. Using two CCD cell lines expressing or not AQP2, we measured extracellular ATP (ATPe) under TRPV4 activation and intracellular Ca2+ under ATP addition. We found that AQP2 is critical for the release of ATP induced by TRPV4 activation. This ATP release occurs by an exocytic and a conductive route. ATPe, in turn, stimulates purinergic receptors leading to ATPe-induced ATP release by a Ca2+ -dependent mechanism. We propose that AQP2 by modulating Ca2+ and ATP differently could explain AQP2-increased cell migration.

A novel method for assessing the renal biopsy specimens using an activatable fluorescent probe

Gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) is an activatable fluorescent probe that can be activated by γ-glutamyltranspeptidase (GGT). The expression of GGT in the kidney, which is one of the major organs exhibiting enhanced GGT expression, is exclusively localised to the cortex. Here, we aimed to investigate the feasibility of gGlu-HMRG as a probe for the on-site assessment of renal biopsy specimens. gGlu-HMRG fluorescent probe was applied to the renal proximal tubular epithelial cells and cortical collecting duct cells in vitro, mouse kidneys ex vivo, and human biopsy specimens. In addition, the fluorescence intensities in the cortex and the medulla were comparatively evaluated in the biopsy specimens. The fluorescence signal was rapidly detected in the renal proximal tubular epithelial cells, whereas that in the cortical collecting duct cells was not detected. The fluorescence signal was detected in the mouse kidneys ex vivo without markedly affecting the tissue morphology. In the human biopsy specimens, the fluorescence signal in the cortex was significantly distinct from that in the medulla (p < 0.05). Thus, this fluorescent probe can be used to distinctly identify the renal cortex in the biopsy specimens.

Resveratrol diminishes bisphenol A-induced oxidative stress through TRPM2 channel in the mouse kidney cortical collecting duct cells

Bisphenol A (BisPH-A) is a latent danger that threatens our health, which we frequently exposure in our modern life (e.g. the widespread use of drinking water in plastic pet bottles). But the BisPH-A induced transient receptor potential melastatin 2 (TRPM2)-mediated oxidative stress and apoptosis in these cells has not been studied yet. Calcium (Ca2+) plays an important role in a versatile intracellular signal transduction that works over a wide range to regulate oxidative stress processes. TRPM2 is activated by oxidative stress and it has emerged as an important Ca2+ signaling mechanism in a variety of cells, contributing many cellular functions including cell death. Resveratrol (RESV), which belongs to the polyphenol group, acts as an antioxidant, eliminating cellular oxidative stress and increasing the body’s resistance to diseases. The current study aimed to elucidate the effect of antioxidant resveratrol on TRPM2-mediated oxidative stress induced by BisPH-A exposure in the mouse kidney cortical collecting duct cells (mpkCCDcl4). The cells were divided into four groups as control, resveratrol (50 µM for 24 h), BisPH-A (100 µM for 24 h) and BisPH-A + RESV. Intracellular free Ca2+ concentrations and TRPM2 channel currents were high in BisPH-A treated cells, but decreased with resveratrol treatment. In addition, BisPH-A induced mitochondrial membrane depolarization, reactive oxygen species (ROS), caspase 3, caspase 9 and apoptosis values were decreased by the resveratrol treatment. In conclusion, resveratrol protected cells from BisPH-A induced oxidative damage. In this study, we showed that TRPM2 channel mediates this protective effect of resveratrol.

Prostaglandin E2 stimulates the epithelial sodium channel (ENaC) in cultured mouse cortical collecting duct cells in an autocrine manner.

Prostaglandin E2 (PGE2) is the most abundant prostanoid in the kidney, affecting a wide range of renal functions. Conflicting data have been reported regarding the effects of PGE2 on tubular water and ion transport. The amiloride-sensitive epithelial sodium channel (ENaC) is rate limiting for transepithelial sodium transport in the aldosterone-sensitive distal nephron. The aim of the present study was to explore a potential role of PGE2 in regulating ENaC in cortical collecting duct (CCD) cells. Short-circuit current (ISC) measurements were performed using the murine mCCDcl1 cell line known to express characteristic properties of CCD principal cells and to be responsive to physiological concentrations of aldosterone and vasopressin. PGE2 stimulated amiloride-sensitive ISC via basolateral prostaglandin E receptors type 4 (EP4) with an EC50 of ∼7.1 nM. The rapid stimulatory effect of PGE2 on ISC resembled that of vasopressin. A maximum response was reached within minutes, coinciding with an increased abundance of β-ENaC at the apical plasma membrane and elevated cytosolic cAMP levels. The effects of PGE2 and vasopressin were nonadditive, indicating similar signaling cascades. Exposing mCCDcl1 cells to aldosterone caused a much slower (∼2 h) increase of the amiloride-sensitive ISC. Interestingly, the rapid effect of PGE2 was preserved even after aldosterone stimulation. Furthermore, application of arachidonic acid also increased the amiloride-sensitive ISC involving basolateral EP4 receptors. Exposure to arachidonic acid resulted in elevated PGE2 in the basolateral medium in a cyclooxygenase 1 (COX-1)-dependent manner. These data suggest that in the cortical collecting duct, locally produced and secreted PGE2 can stimulate ENaC-mediated transepithelial sodium transport.

Chloroquine attenuates lithium-induced NDI and proliferation of renal collecting duct cells.

Lithium is widely used in psychiatry as the golden standard for more than 60 yr due to its effectiveness. However, its adverse effect has been limiting its long-term use in clinic. About 40% of patients taking lithium develop nephrogenic diabetes insipidus (NDI). Lithium can also induce proliferation of collecting duct cells, leading to microcyst formation in the kidney. Lithium was considered an autophagy inducer that might contribute to the therapeutic benefit of neuropsychiatric disorders. Thus, we hypothesized that autophagy may play a role in lithium-induced kidney nephrotoxicity. To address our hypothesis, we fed mice with a lithium-containing diet with chloroquine (CQ), an autophagy inhibitor, concurrently. Lithium-treated mice presented enhanced autophagy activity in the kidney cortex and medulla. CQ treatment significantly ameliorated lithium-induced polyuria, polydipsia, natriuresis, and kaliuresis accompanied with attenuated downregulation of aquaporin-2 and Na+-K+-2Cl- cotransporter protein. The protective effect of CQ on aquaporin-2 protein abundance was confirmed in cultured cortical collecting duct cells. In addition, we found that lithium-induced proliferation of collecting duct cells was also suppressed by CQ as detected by proliferating cell nuclear antigen staining. Moreover, both phosphorylated mammalian target of rapamycin and β-catenin expression, which have been reported to be increased by lithium and associated with cell proliferation, were reduced by CQ. Taken together, our study demonstrated that CQ protected against lithium-induced NDI and collecting duct cell proliferation possibly through inhibiting autophagy.

Interaction between Epithelial Sodium Channel γ-Subunit and Claudin-8 Modulates Paracellular Sodium Permeability in Renal Collecting Duct.

Water and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient. To investigate whether transcellular sodium transport controls tight-junction composition and paracellular permeability via modulating expression of the transmembrane protein claudin-8, we used cultured mouse cortical collecting duct cells to see how overexpression or silencing of epithelial sodium channel (ENaC) subunits and claudin-8 affect paracellular permeability. We also used conditional kidney tubule-specific knockout mice lacking ENaC subunits to assess the ENaC's effect on claudin-8 expression. Overexpression or silencing of the ENaC γ-subunit was associated with parallel and specific changes in claudin-8 abundance. Increased claudin-8 abundance was associated with a reduction in paracellular permeability to sodium, whereas decreased claudin-8 abundance was associated with the opposite effect. Claudin-8 overexpression and silencing reproduced these functional effects on paracellular ion permeability. Conditional kidney tubule-specific ENaC γ-subunit knockout mice displayed decreased claudin-8 expression, confirming the cell culture experiments' findings. Importantly, ENaC β-subunit or α-subunit silencing or kidney tubule-specific β-ENaC or α-ENaC knockout mice did not alter claudin-8 abundance. Our data reveal the specific coupling between ENaC γ-subunit and claudin-8 expression. This coupling may play an important role in preventing the backflow of reabsorbed solutes and water to the tubular lumen, as well as in coupling paracellular and transcellular sodium permeability.