The study of intercalated cells using ex vivo techniques: primary cell culture, cell lines, kidney slices, and organoids.

This review summarizes methods to study kidney intercalated cell (IC) function ex vivo. While important for acid-base homeostasis, IC dysfunction is often not recognized clinically until it becomes severe. The advantage of using ex vivo techniques is that they allow for the differential evaluation of IC function in controlled environments. Although in vitro kidney tubular perfusion is a classical ex vivo technique to study IC, here we concentrate on primary cell cultures, immortalized cell lines, and ex vivo kidney slices. Ex vivo techniques are useful in evaluating IC signaling pathways that allow rapid responses to extracellular changes in pH, CO2, and bicarbonate (HCO3-). However, these methods for IC work can also be challenging, as cell lines that recapitulate IC do not proliferate easily in culture. Moreover, a "pure" IC population in culture does not necessarily replicate its collecting duct (CD) environment, where ICs are surrounded by the more abundant principal cells (PCs). It is reassuring that many findings obtained in ex vivo IC systems signaling have been largely confirmed in vivo. Some of these newly identified signaling pathways reveal that ICs are important for regulating NaCl reabsorption, thus suggesting new frontiers to target antihypertensive treatments. Moreover, recent single-cell characterization studies of kidney epithelial cells revealed a dual developmental origin of IC, as well as the presence of novel CD cell types with certain IC characteristics. These exciting findings present new opportunities for the study of IC ex vivo and will likely rediscover the importance of available tools in this field.NEW & NOTEWORTHY The study of kidney intercalated cells has been limited by current cell culture and kidney tissue isolation techniques. This review is to be used as a reference to select ex vivo techniques to study intercalated cells. We focused on the use of cell lines and kidney slices as potential useful models to study membrane transport proteins. We also review how novel collecting duct organoids may help better elucidate the role of these intriguing cells.

Trimethoprim inhibits renal H+-K+-ATPase in states of K+ depletion.

There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.

TNF inhibits AQP2 expression via a miR137-dependent pathway.

As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased ∼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgH2O medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by ∼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function.NEW & NOTEWORTHY An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na+-K+-2Cl- cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems.

Fluid shear stress stimulates ATP release without regulating purinergic gene expression in the renal inner medullary collecting duct.

In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1-/- IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1-/- IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1-/- IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release.

Data resource: vasopressin-regulated protein phosphorylation sites in the collecting duct.

Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressin-regulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/ and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. 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. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressin-induced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies.NEW & NOTEWORTHY Vasopressin regulates renal water excretion through control of the aquaporin-2 water channel in collecting duct cells. Studies of vasopressin-induced protein phosphorylation have focused mainly on the phosphorylation of aquaporin-2. This study describes 44 phosphoproteins other than aquaporin-2 that undergo vasopressin-mediated phosphorylation changes and summarizes potential physiological roles of each.

The proximal tubule through an NBCe1-dependent mechanism regulates collecting duct phenotypic and remodeling responses to acidosis.

The renal response to acid-base disturbances involves phenotypic and remodeling changes in the collecting duct. This study examines whether the proximal tubule controls these responses. We examined mice with genetic deletion of proteins present only in the proximal tubule, either the A variant or both A and B variants of isoform 1 of the electrogenic Na+-bicarbonate cotransporter (NBCe1). Both knockout (KO) mice have spontaneous metabolic acidosis. We then determined the collecting duct phenotypic responses to this acidosis and the remodeling responses to exogenous acid loading. Despite the spontaneous acidosis in NBCe1-A KO mice, type A intercalated cells in the inner stripe of the outer medullary collecting duct (OMCDis) exhibited decreased height and reduced expression of H+-ATPase, anion exchanger 1, Rhesus B glycoprotein, and Rhesus C glycoprotein. Combined kidney-specific NBCe1-A/B deletion induced similar changes. Ultrastructural imaging showed decreased apical plasma membrane and increased vesicular H+-ATPase in OMCDis type A intercalated cell in NBCe1-A KO mice. Next, we examined the collecting duct remodeling response to acidosis. In wild-type mice, acid loading increased the proportion of type A intercalated cells in the connecting tubule (CNT) and OMCDis, and it decreased the proportion of non-A, non-B intercalated cells in the connecting tubule, and type B intercalated cells in the cortical collecting duct (CCD). These changes were absent in NBCe1-A KO mice. We conclude that the collecting duct phenotypic and remodeling responses depend on proximal tubule-dependent signaling mechanisms blocked by constitutive deletion of proximal tubule NBCe1 proteins.NEW & NOTEWORTHY This study shows that the proximal tubule regulates collecting duct phenotypic and remodeling responses to acidosis.

Role of microRNAs in aquaporin 2 regulation.

PURPOSE OF REVIEW: The current review aims to present the most recent achievements on the role of microRNAs (miRNAs) on the kidney function to stimulate research in the field and to expand new emerging concepts. RECENT FINDINGS: The focus is on the role of miRNAs in intercellular communication along the segments of the nephron and on the epi-miRNAs, namely the possibility of some miRNAs to modulate the epigenetic machinery and so gene expression. Indeed, recent evidence showed that miRNAs included in exosomes and released by proximal tubule cells can modulate ENaC activity on cells of collecting duct. These data, although, from in-vitro models open to a novel role for miRNAs to participate in paracrine signaling pathways. In addition, the role of miRNAs as epigenetic modulators is expanding not only in the cancer field, but also in the other kidney diseases. Recent evidence identified three miRNAs able to modulate the AQP2 promoter metilation and showing an additional level of regulation for the AQP2. SUMMARY: These evidence can inspire novel area of research both for renal physiology and drug discovery. The diseases involving the collecting duct are still missing disease modifying agents and the expanding miRNAs field could represent an opportunity.

Expression of active B-Raf proto-oncogene in kidney collecting ducts induces cyst formation in normal mice and accelerates cyst growth in mice with polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by the formation and progressive enlargement of fluid-filled cysts due to abnormal cell proliferation. Cyclic AMP agonists, including arginine vasopressin, stimulate ERK-dependent proliferation of cystic cells, but not normal kidney cells. Previously, B-Raf proto-oncogene (BRAF), a MAPK kinase kinase that activates MEK-ERK signaling, was shown to be a central intermediate in the cAMP mitogenic response. However, the role of BRAF on cyst formation and enlargement in vivo had not been demonstrated. To determine if active BRAF induces kidney cyst formation, we generated transgenic mice that conditionally express BRAFV600E, a common activating mutation, and bred them with Pkhd1-Cre mice to express active BRAF in the collecting ducts, a predominant site for cyst formation. Collecting duct expression of BRAFV600E (BRafCD) caused kidney cyst formation as early as three weeks of age. There were increased levels of phosphorylated ERK (p-ERK) and proliferating cell nuclear antigen, a marker for cell proliferation. BRafCD mice developed extensive kidney fibrosis and elevated blood urea nitrogen, indicating a decline in kidney function, by ten weeks of age. BRAFV600E transgenic mice were also bred to Pkd1RC/RC and pcy/pcy mice, well-characterized slowly progressive PKD models. Collecting duct expression of active BRAF markedly increased kidney weight/body weight, cyst number and size, and total cystic area. There were increased p-ERK levels and proliferating cells, immune cell infiltration, interstitial fibrosis, and a decline in kidney function in both these models. Thus, our findings demonstrate that active BRAF is sufficient to induce kidney cyst formation in normal mice and accelerate cystic disease in PKD mice.

Altered arginine vasopressin-cyclic AMP-aquaporin 2 pathway in patients with chronic kidney disease.

BACKGROUND: In the collecting ducts of the kidney, arginine vasopressin (AVP), cyclic adenosine monophosphate (cAMP), and aquaporin 2 (AQP2) play a pivotal role in maintaining fluid volume and serum osmolality in humans. However, their association among those with chronic kidney disease (CKD) remains uncertain. METHODS: We prospectively included the out-patients with CKD and measured osmolality-related biomarkers including plasma AVP, urine cAMP, urine AQP2, and urine osmolality levels. Association among these parameters at each CKD stage was investigated. RESULTS: A total of 121 patients were included (median age 71 years old [61-78], 89 men, estimated glomerular filtration ratio 28.6 [16.4-45.3] mL/min/1.73 m2). Serum osmolality increased as CKD progression, accompanying incremental plasma AVP levels, whereas urine cAMP, urine AQP2, and urine osmolality decreased as CKD progression. At advanced CKD stage, urine cAMP remained low irrespective of the AVP stimulation, whereas urine cAMP levels varied according to the levels of plasma AVP at less advanced CKD stage. The associations between urine cAMP and urine AQP2 and between urine AQP2 and urine osmolality remained preserved irrespective of the CKD stages. CONCLUSIONS: Vasopressin type-2 receptor seems to be particularly impaired in patients with advanced CKD, whereas the signal cascade of the downstream of vasopressin type-2 receptor is relatively preserved. Urine cAMP might be a promising marker to estimate the residual function of the collecting duct.

Aqp2+ Progenitor Cells Maintain and Repair Distal Renal Segments.

BACKGROUND: Adult progenitor cells presumably demonstrate clonogenicity, self-renewal, and multipotentiality, and can regenerate cells under various conditions. Definitive evidence demonstrating the existence of such progenitor cells in adult mammalian kidneys is lacking. METHOD: We performed in vivo lineage tracing and thymidine analogue labeling using adult tamoxifen-inducible (Aqp2ECE/+ RFP/+, Aqp2ECE/+ Brainbow/+, and Aqp2ECE/+ Brainbow/Brainbow) and WT mice. The tamoxifen-inducible mice were analyzed between 1 and 300 days postinduction. Alternatively, WT and tamoxifen-induced mice were subjected to unilateral ureteral obstruction and thymidine analogue labeling and analyzed 2-14 days post-surgery. Multiple cell-specific markers were used for high-resolution immunofluorescence confocal microscopy to identify the cell types derived from Aqp2+ cells. RESULTS: Like their embryonic counterparts, adult cells expressing Aqp2 and V-ATPase subunits B1 and B2 (Aqp2+ B1B2+) are the potential Aqp2+ progenitor cells (APs). Adult APs rarely divide to generate daughter cells, either maintaining the property of the AP (self-renewal) or differentiating into DCT2/CNT/CD cells (multipotentiality), forming single cell-derived, multiple-cell clones (clonogenicity) during tissue maintenance. APs selectively and continuously regenerate DCT2/CNT/CD cells in response to injury resulting from ureteral ligation. AP proliferation demonstrated direct correlation with Notch activation and was inversely correlated with development of kidney fibrosis. Derivation of both intercalated and DCT2 cells was found to be cell division-dependent and -independent, most likely through AP differentiation which requires cell division and through direct conversion of APs and/or regular principal cells without cell division, respectively. CONCLUSION: Our study demonstrates that Aqp2+ B1B2+ cells behave as adult APs to maintain and repair DCT2/CNT1/CNT2/CD segments.

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.

MAGED2 controls vasopressin-induced aquaporin-2 expression in collecting duct cells.

Mutations in the Melanoma-Associated Antigen D2 (MAGED2) cause antenatal Bartter syndrome type 5 (BARTS5). This rare disease is characterized by perinatal loss of urinary concentration capability and large urine volumes. The underlying molecular mechanisms of this disease are largely unclear. Here, we study the effect of MAGED2 knockdown on kidney cell cultures using proteomic and phosphoproteomic analyses. In HEK293T cells, MAGED2 knockdown induces prominent changes in protein phosphorylation rather than changes in protein abundance. MAGED2 is expressed in mouse embryonic kidneys and its expression declines during development. MAGED2 interacts with G-protein alpha subunit (GNAS), suggesting a role in G-protein coupled receptors (GPCR) signalling. In kidney collecting duct cell lines, Maged2 knockdown subtly modulated vasopressin type 2 receptor (V2R)-induced cAMP-generation kinetics, rewired phosphorylation-dependent signalling, and phosphorylation of CREB. Maged2 knockdown resulted in a large increase in aquaporin-2 abundance during long-term V2R activation. The increase in aquaporin-2 protein was mediated transcriptionally. Taken together, we link MAGED2 function to cellular signalling as a desensitizer of V2R-induced aquaporin-2 expression. SIGNIFICANCE: In most forms of Bartter Syndrome, the underlying cause of the disease is well understood. In contrast, the role of MAGED2 mutations in a newly discovered form of Bartter Syndrome (BARTS5) is unknown. In our manuscript we could show that MAGED2 modulates vasopressin-induced protein and phosphorylation patterns in kidney cells, providing a broad basis for further studies of MAGED2 function in development and disease.

Activation of the Hypoxia-Inducible Factor Pathway Inhibits Epithelial Sodium Channel-Mediated Sodium Transport in Collecting Duct Principal Cells.

BACKGROUND: Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxia-inducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)-dependent sodium transport. METHODS: We investigated HIF's effect on ENaC expression in mpkCCD cl4 cells (a model of collecting duct principal cells) using real-time PCR and western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule-specific Hif1α knockout mice. RESULTS: In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of ß ENaC and γ ENaC, as well as of Na,K-ATPase. HIF1 α silencing increased ß ENaC and γ ENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1 α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γ ENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γ ENaC abundance under high sodium intake. CONCLUSIONS: This study reveals that γ ENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.

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.

Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2-Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

Metabolic and Lipidomic Assessment of Kidney Cells Exposed to Nephrotoxic Vancomycin Dosages.

Vancomycin is a glycopeptide antibiotic used against multi-drug resistant gram-positive bacteria such as Staphylococcus aureus (MRSA). Although invaluable against resistant bacteria, vancomycin harbors adverse drug reactions including cytopenia, ototoxicity, as well as nephrotoxicity. Since nephrotoxicity is a rarely occurring side effect, its mechanism is incompletely understood. Only recently, the actual clinically relevant concentration the in kidneys of patients receiving vancomycin was investigated and were found to exceed plasma concentrations by far. We applied these clinically relevant vancomycin concentrations to murine and canine renal epithelial cell lines and assessed metabolic and lipidomic alterations by untargeted and targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses. Despite marked differences in the lipidome, both cell lines increased anabolic glucose reactions, resulting in higher sorbitol and lactate levels. To the best of our knowledge, this is the first endometabolic profiling of kidney cells exposed to clinically relevant vancomycin concentrations. The presented study will provide a valuable dataset to nephrotoxicity researchers and might add to unveiling the nephrotoxic mechanism of vancomycin.

Aquaporin 2 regulation: implications for water balance and polycystic kidney diseases.

Targeting the collecting duct water channel aquaporin 2 (AQP2) to the plasma membrane is essential for the maintenance of mammalian water homeostasis. The vasopressin V2 receptor (V2R), which is a GS protein-coupled receptor that increases intracellular cAMP levels, has a major role in this targeting process. Although a rise in cAMP levels and activation of protein kinase A are involved in facilitating the actions of V2R, studies in knockout mice and cell models have suggested that cAMP signalling pathways are not an absolute requirement for V2R-mediated AQP2 trafficking to the plasma membrane. In addition, although AQP2 phosphorylation is a known prerequisite for V2R-mediated plasma membrane targeting, none of the known AQP2 phosphorylation events appears to be rate-limiting in this process, which suggests the involvement of other factors; cytoskeletal remodelling has also been implicated. Notably, several regulatory processes and signalling pathways involved in AQP2 trafficking also have a role in the pathophysiology of autosomal dominant polycystic kidney disease, although the role of AQP2 in cyst progression is unknown. Here, we highlight advances in the field of AQP2 regulation that might be exploited for the treatment of water balance disorders and provide a rationale for targeting these pathways in autosomal dominant polycystic kidney disease.

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.

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 (11ßHSD2), 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.

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.