Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct.

The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli.

Using CRISPR-Cas9/phosphoproteomics to identify substrates of calcium/calmodulin-dependent kinase 2δ.

Ca2+/Calmodulin-dependent protein kinase 2 (CAMK2) family proteins are involved in the regulation of cellular processes in a variety of tissues including brain, heart, liver, and kidney. One member, CAMK2δ (CAMK2D), has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water excretion through regulation of the water channel aquaporin-2 (AQP2). To identify CAMK2D target proteins in renal collecting duct cells (mpkCCD), we deleted Camk2d and carried out LC-MS/MS-based quantitative phosphoproteomics. Specifically, we used CRISPR/Cas9 with two different guide RNAs targeting the CAMK2D catalytic domain to create multiple CAMK2D KO cell lines. AQP2 protein abundance was lower in the CAMK2D KO cells than in CAMK2D-intact controls. AQP2 phosphorylation at Ser256 and Ser269 (normalized for total AQP2) was decreased. However, trafficking of AQP2 to and from the apical plasma membrane was sustained. Large-scale quantitative phosphoproteomic analysis (TMT-labeling) in the presence of the vasopressin analog dDAVP (0.1 nM, 30 min) allowed quantification of 11,570 phosphosites of which 169 were significantly decreased, while 206 were increased in abundance in CAMK2D KO clones. These data are available for browsing or download at https://esbl.nhlbi.nih.gov/Databases/CAMK2D-proteome/. Motif analysis of the decreased phosphorylation sites revealed a target preference of -(R/K)-X-X-p(S/T)-X-(D/E), matching the motif identified in previous in vitro phosphorylation studies using recombinant CAMK2D. Thirty five of the significantly downregulated phosphorylation sites in CAMK2D KO cells had exactly this motif and are judged to be likely direct CAMK2D targets. This adds to the list of known CAMK2D target proteins found in prior reductionist studies.

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.

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells.

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.

Expression of claudin-8 is induced by aldosterone in renal collecting duct principal cells.

Fine tuning of Na+ reabsorption takes place along the aldosterone-sensitive distal nephron, which includes the collecting duct (CD), where it is mainly regulated by aldosterone. In the CD, Na+ reabsorption is mediated by the epithelial Na+ channel and Na+ pump (Na+-K+-ATPase). Paracellular ion permeability is mainly dependent on tight junction permeability. Claudin-8 is one of the main tight junction proteins expressed along the aldosterone-sensitive distal nephron. We have previously shown a coupling between transcellular Na+ reabsorption and paracellular Na+ barrier. We hypothesized that aldosterone controls the expression levels of both transcellular Na+ transporters and paracellular claudin-8 in a coordinated manner. Here, we show that aldosterone increased mRNA and protein levels as well as lateral membrane localization of claudin-8 in cultured CD principal cells. The increase in claudin-8 mRNA levels in response to aldosterone was prevented by preincubation with 17-hydroxyprogesterone, a mineralocorticoid receptor antagonist, and by inhibition of transcription with actinomycin D. We also showed that a low-salt diet, which stimulated aldosterone secretion, was associated with increased claudin-8 abundance in the mouse kidney. Reciprocally, mice subjected to a high-salt diet, which inhibits aldosterone secretion, or treated with spironolactone, a mineralocorticoid receptor antagonist, displayed decreased claudin-8 expression. Inhibition of glycogen synthase kinase-3, Lyn, and Abl signaling pathways prevented the effect of aldosterone on claudin-8 mRNA and protein abundance, suggesting that signaling of protein kinases plays a permissive role on the transcriptional activity of the mineralocorticoid receptor. This study shows that signaling via multiple protein kinases working in concert mediates aldosterone-induced claudin-8 expression in the CD.NEW & NOTEWORTHY In this study, we showed that aldosterone modulates claudin-8 expression in cultured collecting duct principal cells and in the mouse kidney. The upregulation of claudin-8 expression in response to aldosterone is dependent on at least glycogen synthase kinase-3, Lyn, and Abl signaling pathways, indicating the participation of multiple protein kinases to the effect of aldosterone.

Connecting tubules develop from the tip of the ureteric bud in the human kidney.

The connecting tubule (CNT) is a unique segment of the nephron connecting the metanephric mesenchyme (MM)-derived distal convoluted tubule (DCT) and ureteric bud (UB)-derived collecting duct (CD). Views on the cellular origin of the CNT in the human kidney are controversial. It was suggested that in mice, the connecting segment arises from the distal compartment of the renal vesicle (RV). However, there are several differences in embryonic development between the mouse and human kidney. The aim of our study was to establish the possible origin of the CNT in the human kidney. We analysed the expression of markers defining distinct cells of the CNT CD in foetal and adult human kidneys by immunohistochemistry. Based on microscopic observation, we suggest that CNT differentiates from the outgrowth of cells of the UB tip, and therefore the CNT is an integral part of the CD system. In the adult kidney, the CNT and CD consist of functionally and morphologically similar cells expressing α- and ß-intercalated cell (IC) and principal cell (PC) markers, indicating their common origin.

Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium.

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5-5 µmol/l) and/or Fe2+ (50-100 µmol/l) for 4-24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors.

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney's collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney's collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.

Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells.

Aquaporin-2-4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2-4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2-3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.

Inward Ca2+ current through the polycystin-2-dependent channels of renal primary cilia.

In 15% of cases, autosomal dominant polycystic kidney disease arises from defects in polycystin-2 (PC2). PC2 is a member of the polycystin transient receptor potential subfamily of cation-conducting channels and is expressed in the endoplasmic reticulum and primary cilium of renal epithelial cells. PC2 opposes a procystogenic influence of the cilium, and it has been proposed that this beneficial effect is mediated in part by a flow of Ca2+ through PC2 channels into the primary cilium. However, previous efforts to determine the permeability of PC2 channels to Ca2+ have yielded widely varying results. Here, we report the mean macroscopic Ca2+ influx through native PC2 channels in the primary cilia of mIMCD-3 cells, which are derived from the murine inner medullary collecting duct. Under conditions designed to isolate inward Ca2+ currents, a small inward Ca2+ current was detected in cilia with active PC2 channels but not in cilia lacking those channels. The current was activated by the addition of 10 µM internal Ca2+, which is known to activate ciliary PC2 channels. It was blocked by 10 µM isosakuranetin, which blocks the same channels. On average, the current amplitude was -1.8 pA at -190 mV; its conductance from -50 to -200 mV averaged 20 pS. Thus, native PC2 channels of renal primary cilia are able to conduct a small but detectable Ca2+ influx under the conditions tested. The possible consequences of this influx are discussed.NEW & NOTEWORTHY In autosomal dominant polycystic kidney disease, it is proposed that Ca2+ entering the primary cilium through polycystin-2 (PC2) channels may limit the formation of cysts. Recent studies predict that any macroscopic Ca2+ influx through these channels should be small. We report that the native PC2 channels in primary cilia of cultured renal epithelial cells can allow a small macroscopic calcium influx. This may allow a significant accumulation of Ca2+ in the cilium in vivo.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Kidney intercalated cells are phagocytic and acidify internalized uropathogenic Escherichia coli.

Kidney intercalated cells are involved in acid-base homeostasis via vacuolar ATPase expression. Here we report six human intercalated cell subtypes, including hybrid principal-intercalated cells identified from single cell transcriptomics. Phagosome maturation is a biological process that increases in biological pathway analysis rank following exposure to uropathogenic Escherichia coli in two of the intercalated cell subtypes. Real time confocal microscopy visualization of murine renal tubules perfused with green fluorescent protein expressing Escherichia coli or pHrodo Green E. coli BioParticles demonstrates that intercalated cells actively phagocytose bacteria then acidify phagolysosomes. Additionally, intercalated cells have increased vacuolar ATPase expression following in vivo experimental UTI. Taken together, intercalated cells exhibit a transcriptional response conducive to the kidney's defense, engulf bacteria and acidify the internalized bacteria. Intercalated cells represent an epithelial cell with characteristics of professional phagocytes like macrophages.

Olive Leaf Extract (OLE) impaired vasopressin-induced aquaporin-2 trafficking through the activation of the calcium-sensing receptor.

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption.

Involvement of Klotho, TNF­α and ADAMs in radiation­induced senescence of renal epithelial cells.

While radiation nephropathy is a major problem associated with radiotherapy, the exact mechanisms underlying its pathogenesis and the mediators involved in kidney deterioration remain to be elucidated. In view of the finding that senescence is typically increased post­irradiation, the present study examined whether ionizing radiation may cause kidney injury by enhancing premature senescence. The present study explored the relevance of the aging suppressor, Klotho, which has anti­aging activity and is highly expressed in murine renal cells/kidney tissues, under irradiation conditions. Firstly, the effects of radiation on mouse inner medullary collecting duct­3 (mIMCD­3) cells and kidney tissues of mice were assessed. Subsequently, the mRNA expression levels of Klotho, TNF­α and ADAM metallopeptidase domain (ADAM)9/10/17 were analyzed by reverse transcription­quantitative PCR following exposure to radiation. In addition, the levels of these proteins were measured by western blotting or ELISA. The results revealed that irradiation of mIMCD­3 cells clearly triggered cellular senescence. Notably, Klotho gene expression was considerably decreased in radiation­exposed mIMCD­3 cells and in the kidney tissues of irradiated BALB/c mice, and the corresponding translated protein was consistently expressed following radiation exposure. Moreover, expression of TNF­α, a negative regulator of Klotho, was significantly increased, whereas ADAM9/10/17, an ectodomain shedding enzyme of Klotho, was decreased in irradiated mIMCD­3 cells and in the kidney tissues of BALB/c mice. Collectively, these data suggested that TNF­α­mediated inhibition of Klotho expression and blockage of soluble Klotho formation via decreased ADAM expression following irradiation may contribute to the development of renal dysfunction through acceleration of radiation­induced cellular senescence.

Regulation of renal pendrin activity by aldosterone.

PURPOSE OF REVIEW: Pendrin resides on the luminal membrane of type B intercalated cells in the renal collecting tubule system mediating the absorption of chloride in exchange for bicarbonate. In mice or humans lacking pendrin, blood pressure is lower, and pendrin knockout mice are resistant to aldosterone-induced hypertension. Here we discuss recent findings on the regulation of pendrin. RECENT FINDINGS: Pendrin activity is stimulated during alkalosis partly mediated by secretin. Also, angiotensin II and aldosterone stimulate pendrin activity requiring the mineralocorticoid receptor in intercalated cells. Angiotensin II induces dephosphorylation of the mineralocorticoid receptor rendering the receptor susceptible for aldosterone binding. In the absence of the mineralocorticoid receptor in intercalated cells, angiotensin II does not stimulate pendrin. The effect of aldosterone on pendrin expression is in part mediated by the development of hypokalemic alkalosis and blunted by K-supplements or amiloride. Part of the blood pressure-increasing effect of pendrin is also mediated by its stimulatory effect on the epithelial Na-channel in neighbouring principal cells. SUMMARY: These findings identify pendrin as a critical regulator of renal salt handling and blood pressure along with acid--base balance. A regulatory network of hormones fine-tuning activity is emerging. Drugs blocking pendrin are being developed.

Collecting duct cells show differential retinoic acid responses to acute versus chronic kidney injury stimuli.

Retinoic acid (RA) activates RA receptors (RAR), resulting in RA response element (RARE)-dependent gene expression in renal collecting duct (CD). Emerging evidence supports a protective role for this activity in acute kidney injury (AKI) and chronic kidney disease (CKD). Herein, we examined this activity in RARE-LacZ transgenic mice and by RARE-Luciferase reporter assays in CD cells, and investigated how this activity responds to neurotransmitters and mediators of kidney injury. In RARE-LacZ mice, Adriamycin-induced heavy albuminuria was associated with reduced RA/RAR activity in CD cells. In cultured CD cells, RA/RAR activity was repressed by acetylcholine, albumin, aldosterone, angiotensin II, high glucose, cisplatin and lipopolysaccharide, but was induced by aristolochic acid I, calcitonin gene-related peptide, endothelin-1, gentamicin, norepinephrine and vasopressin. Compared with age-matched normal human CD cells, CD-derived renal cystic epithelial cells from patients with autosomal recessive polycystic kidney disease (ARPKD) had significantly lower RA/RAR activity. Synthetic RAR agonist RA-568 was more potent than RA in rescuing RA/RAR activity repressed by albumin, high glucose, angiotensin II, aldosterone, cisplatin and lipopolysaccharide. Hence, RA/RAR  in CD cells is a convergence point of regulation by neurotransmitters and mediators of kidney injury, and may be a novel therapeutic target.

Activation of farnesoid X receptor (FXR) induces crystallin zeta expression in mouse medullary collecting duct cells.

Crystallin zeta (CRYZ) is a phylogenetically restricted water-soluble protein and provides cytoprotection against oxidative stress via multiple mechanisms. Increasing evidence suggests that CRYZ is high abundantly expressed in the kidney where it acts as a transacting factor in increasing glutaminolysis and the Na+/K+/2Cl- cotransporter (BSC1/NKCC2) expression to help maintain acid-base balance and medullary hyperosmotic gradient. However, the mechanism by which CRYZ is regulated in the kidney remains largely uncharacterized. Here, we show that CRYZ is a direct target of farnesoid X receptor (FXR), a nuclear receptor important for renal physiology. We found that CRYZ was ubiquitously expressed in mouse kidney and constitutively expressed in the cytoplasm of medullary collecting duct cells (MCDs). In primary cultured mouse MCDs, CRYZ expression was significantly upregulated by the activation and overexpression of FXR. FXR-induced CRYZ expression was almost completely abolished in the MCD cells with siRNA-mediated FXR knockdown. Consistently, treatment with FXR agonists failed to induce CRYZ expression in the MCDs isolated from mice with global and collecting duct-specific FXR deficiency. We identified a putative FXR response element (FXRE) on the CRYZ gene promoter. The luciferase reporter and ChIP assays revealed that FXR can bind directly to the FXRE site, which was further markedly enhanced by FXR activation. Furthermore, we found CRYZ overexpression in MCDs significantly attenuated hypertonicity-induced cell death possibly via increasing Bcl-2 expression. Collectively, our findings demonstrate that CRYZ is constitutively expressed in renal medullary collecting duct cells, where it is transcriptionally controlled by FXR. Given a critical role of FXR in MCDs, CRYZ may be responsible for protective effect of FXR on the survival of MCDs under hypertonic condition during dehydration.

Intracellular location of aquaporin-2 serine 269 phosphorylation and dephosphorylation in kidney collecting duct cells.

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for water reabsorption by the kidney collecting ducts. Under control conditions, most AQP2 resides in the recycling endosomes of principal cells, where it answers to vasopressin with trafficking to the apical plasma membrane to increase water reabsorption. Upon vasopressin withdrawal, apical AQP2 retreats to the early endosomes before joining the recycling endosomes for the next vasopressin stimulation. Prior studies have demonstrated a role of AQP2 S269 phosphorylation in reducing AQP2 endocytosis, thereby prolonging apical AQP2 retention. Here, we studied where in the cells S269 was phosphorylated and dephosphorylated in response to vasopressin versus withdrawal. In mpkCCD collecting cells, vacuolar protein sorting 35 knockdown slowed vasopressin-induced apical AQP2 trafficking, resulting in AQP2 accumulation in the recycling endosomes where S269 was phosphorylated. Rab7 knockdown, which impaired AQP2 trafficking from the early to recycling endosomes, reduced vasopressin-induced S269 phosphorylation. Rab5 knockdown, which impaired AQP2 endocytosis, did not affect vasopressin-induced S269 phosphorylation. Upon vasopressin withdrawal, S269 was not dephosphorylated in Rab5 knockdown cells. In contrast, S269 dephosphorylation upon vasopressin withdrawal was completed in Rab7 or vacuolar protein sorting 35 knockdown cells. We conclude that S269 is dephosphorylated during Rab5-mediated AQP2 endocytosis before AQP2 joins the recycling endosomes upon vasopressin withdrawal. While in the recycling endosomes, AQP2 can be phosphorylated at S269 in response to vasopressin before apical trafficking.

Aldosterone-induced microRNAs act as feedback regulators of mineralocorticoid receptor signaling in kidney epithelia.

The final steps in the Renin-Angiotensin-Aldosterone signaling System (RAAS) involve binding of the corticosteroid hormone, aldosterone to its mineralocorticoid receptor (MR). The bound MR interacts with response elements to induce or repress the transcription of aldosterone-regulated genes. A well characterized aldosterone-induced gene is the serum and glucocorticoid-induced kinase (SGK1), which acts downstream to increase sodium transport in distal kidney nephron epithelial cells. The role of microRNAs (miRs) induced by extended aldosterone stimulation in regulating MR and SGK1 has not been reported. In these studies, miRs predicted to bind to the 3'-UTR of mouse MR were profiled by qRT-PCR after aldosterone stimulation. The miR-466a/b/c/e family was upregulated in mouse kidney cortical collecting duct epithelial cells. A luciferase reporter assay confirmed miR-466 binding to both MR and SGK1 3'-UTRs. Inhibition of miR-466 increased MR and SGK1 mRNA and protein levels. Inhibiting miR-466b and preventing its upregulation after aldosterone stimulation increased amiloride-sensitive sodium transport and sensitivity to aldosterone stimulation. In vivo upregulation of miR-466 was confirmed in distal nephrons of mice on low Na+ diets. Repression of MR and SGK1 by aldosterone-induced miRs may represent a negative feedback loop that contributes to a form of aldosterone escape in vivo.