RESUMEN
Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Acuaporina 2 , Agua Corporal , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Agua Corporal/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Homeostasis , Ratones , FosforilaciónRESUMEN
SIGNIFICANCE STATEMENT: Autosomal dominant polycystic kidney disease (ADPKD) is a devastating disorder caused by mutations in polycystin 1 ( PKD1 ) and polycystin 2 ( PKD2 ). Currently, the mechanism for renal cyst formation remains unclear. Here, we provide convincing and conclusive data in mice demonstrating that Pkd2 deletion in embryonic Aqp2 + progenitor cells (AP), but not in neonate or adult Aqp2 + cells, is sufficient to cause severe polycystic kidney disease (PKD) with progressive loss of intercalated cells and complete elimination of α -intercalated cells, accurately recapitulating a newly identified cellular phenotype of patients with ADPKD. Hence, Pkd2 is a new potential regulator critical for balanced AP differentiation into, proliferation, and/or maintenance of various cell types, particularly α -intercalated cells. The Pkd2 conditional knockout mice developed in this study are valuable tools for further studies on collecting duct development and early steps in cyst formation. The finding that Pkd2 loss triggers the loss of intercalated cells is a suitable topic for further mechanistic studies. BACKGROUND: Most cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by mutations in PKD1 or PKD2. Currently, the mechanism for renal cyst formation remains unclear. Aqp2 + progenitor cells (AP) (re)generate ≥5 cell types, including principal cells and intercalated cells in the late distal convoluted tubules (DCT2), connecting tubules, and collecting ducts. METHODS: Here, we tested whether Pkd2 deletion in AP and their derivatives at different developmental stages is sufficient to induce PKD. Aqp2Cre Pkd2f/f ( Pkd2AC ) mice were generated to disrupt Pkd2 in embryonic AP. Aqp2ECE/+Pkd2f/f ( Pkd2ECE ) mice were tamoxifen-inducted at P1 or P60 to inactivate Pkd2 in neonate or adult AP and their derivatives, respectively. All induced mice were sacrificed at P300. Immunofluorescence staining was performed to categorize and quantify cyst-lining cell types. Four other PKD mouse models and patients with ADPKD were similarly analyzed. RESULTS: Pkd2 was highly expressed in all connecting tubules/collecting duct cell types and weakly in all other tubular segments. Pkd2AC mice had obvious cysts by P6 and developed severe PKD and died by P17. The kidneys had reduced intercalated cells and increased transitional cells. Transitional cells were negative for principal cell and intercalated cell markers examined. A complete loss of α -intercalated cells occurred by P12. Cysts extended from the distal renal segments to DCT1 and possibly to the loop of Henle, but not to the proximal tubules. The induced Pkd2ECE mice developed mild PKD. Cystic α -intercalated cells were found in the other PKD models. AQP2 + cells were found in cysts of only 13/27 ADPKD samples, which had the same cellular phenotype as Pkd2AC mice. CONCLUSIONS: Hence, Pkd2 deletion in embryonic AP, but unlikely in neonate or adult Aqp2 + cells (principal cells and AP), was sufficient to cause severe PKD with progressive elimination of α -intercalated cells, recapitulating a newly identified cellular phenotype of patients with ADPKD. We proposed that Pkd2 is critical for balanced AP differentiation into, proliferation, and/or maintenance of cystic intercalated cells, particularly α -intercalated cells.
Asunto(s)
Acuaporina 2 , Riñón Poliquístico Autosómico Dominante , Adulto , Animales , Humanos , Ratones , Acuaporina 2/deficiencia , Acuaporina 2/genética , Quistes , Riñón/metabolismo , Ratones Noqueados , Enfermedades Renales Poliquísticas/genética , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Insuficiencia Renal Crónica , Células Madre/metabolismo , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismoRESUMEN
Farnesoid X receptor (FXR), a ligand-activated transcription factor, plays an important role in maintaining water homeostasis by up-regulating aquaporin 2 (AQP2) expression in renal medullary collecting ducts; however, its role in the survival of renal medullary interstitial cells (RMICs) under hypertonic conditions remains unclear. We cultured primary mouse RMICs and found that the FXR was expressed constitutively in RMICs, and that its expression was significantly up-regulated at both mRNA and protein levels by hypertonic stress. Using luciferase and ChIP assays, we found a potential binding site of nuclear factor kappa-B (NF-κB) located in the FXR gene promoter which can be bound and activated by NF-κB. Moreover, hypertonic stress-induced cell death in RMICs was significantly attenuated by FXR activation but worsened by FXR inhibition. Furthermore, FXR increased the expression and nuclear translocation of hypertonicity-induced tonicity-responsive enhance-binding protein (TonEBP), the expressions of its downstream target gene sodium myo-inositol transporter (SMIT), and heat shock protein 70 (HSP70). The present study demonstrates that the NF-κB/FXR/TonEBP pathway protects RMICs against hypertonic stress.
Asunto(s)
Médula Renal , FN-kappa B , Transducción de Señal , Animales , FN-kappa B/metabolismo , Ratones , Médula Renal/metabolismo , Médula Renal/citología , Presión Osmótica , Acuaporina 2/metabolismo , Acuaporina 2/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Masculino , Ratones Endogámicos C57BL , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Regiones Promotoras Genéticas , Células Cultivadas , Regulación de la Expresión Génica , Simportadores/metabolismo , Simportadores/genética , Receptores Citoplasmáticos y NuclearesRESUMEN
High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium-Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). In line with these findings, we provide evidence that, with respect to wild-type mice, CaSR knock-in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2-targeting miRNA137 consistent with a post-transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT-305, a CaSR antagonist, increased AQP2 expression and reduced AQP2-targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short-term vasopressin response by increasing AQP2-pS261, as well as AQP2 abundance, via the p38MAPK-ATF1-miR137 pathway. KEY POINTS: Calcium-Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis. In vivo treatment with calcilytic JTT-305/MK-5442 ameliorates most of the ADH phenotypes of the CaSR knock-in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin-sensitive aquaporin-2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response. The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone-independent action through vasopressin-dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct. The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2-mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH.
Asunto(s)
Acuaporina 2 , Regulación hacia Abajo , Receptores Sensibles al Calcio , Vasopresinas , Animales , Acuaporina 2/metabolismo , Acuaporina 2/genética , Receptores Sensibles al Calcio/metabolismo , Receptores Sensibles al Calcio/genética , Ratones , Vasopresinas/metabolismo , Técnicas de Sustitución del Gen , Riñón/metabolismo , Riñón/efectos de los fármacos , Ratones Endogámicos C57BL , Masculino , Transducción de Señal , Fenotipo , Hipercalciuria/genética , Hipercalciuria/metabolismo , Hipercalciuria/tratamiento farmacológico , Calcio/metabolismo , Fosforilación , Hipocalcemia , Hipoparatiroidismo/congénitoRESUMEN
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.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteómica , Acuaporina 2/genética , Acuaporina 2/metabolismo , Cromatografía Liquida , Sistemas CRISPR-Cas , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Fosforilación , Espectrometría de Masas en Tándem , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Eliminación de Gen , RNA-Seq , Biología Computacional , Secuencias de Aminoácidos , Regulación hacia Abajo , Técnicas In VitroRESUMEN
Plant A/T-rich protein and zinc-binding protein (PLATZ) transcription factors play important roles in plant growth, development and abiotic stress responses. However, how PLATZ influences plant drought tolerance remains poorly understood. The present study showed that PLATZ4 increased drought tolerance in Arabidopsis thaliana by causing stomatal closure. Transcriptional profiling analysis revealed that PLATZ4 affected the expression of a set of genes involved in water and ion transport, antioxidant metabolism, small peptides and abscisic acid (ABA) signaling. Among these genes, the direct binding of PLATZ4 to the A/T-rich sequences in the plasma membrane intrinsic protein 2;8 (PIP2;8) promoter was identified. PIP2;8 consistently reduced drought tolerance in Arabidopsis through inhibiting stomatal closure. PIP2;8 was localized in the plasma membrane, exhibited water channel activity in Xenopus laevis oocytes and acted epistatically to PLATZ4 in regulating the drought stress response in Arabidopsis. PLATZ4 increased ABA sensitivity through upregulating the expression of ABSCISIC ACID INSENSITIVE 3 (ABI3), ABI4 and ABI5. The transcripts of PLATZ4 were induced to high levels in vegetative seedlings under drought and ABA treatments within 6 and 3 h, respectively. Collectively, these findings reveal that PLATZ4 positively influences plant drought tolerance through regulating the expression of PIP2;8 and genes involved in ABA signaling.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácido Abscísico/metabolismo , Resistencia a la Sequía , Acuaporina 2/genética , Acuaporina 2/metabolismo , Plantas Modificadas Genéticamente/genética , Sequías , Proteínas de la Membrana/metabolismo , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Estrés Fisiológico/genética , Estomas de Plantas/fisiologíaRESUMEN
Poly(ADP-ribosyl)ation (PARylation), as a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) catalyzing the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, involves a number of cellular processes. As mice lacking the PARP-1 gene (Parp1) produce more urine, we investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). In biotin-conjugated nicotinamide adenine dinucleotide (biotin-NAD+) pulldown and immunoprecipitation assays of poly(ADP)-ribose in mpkCCDc14 cells, immunoblots demonstrated that 1-deamino-8-D-arginine vasopressin (dDAVP) induced the PARylation of total proteins, associated with an increase in the cleavage of PARP-1 and cleaved caspase-3 expression. By inhibiting PARP-1 with siRNA, the abundance of dDAVP-induced AQP2 mRNA and protein was significantly diminished. In contrast, despite a substantial decrease in PARylation, the PARP-1 inhibitor (PJ34) had no effect on the dDAVP-induced regulation of AQP2 expression. The findings suggest that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. Bioinformatic analysis revealed that 408 proteins interact with PARP-1 in the collecting duct (CD) cells of the kidney. Among them, the signaling pathway of the vasopressin V2 receptor was identified for 49 proteins. In particular, ß-catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein. A significant decrease of ß-catenin phosphorylation (Ser552) in response to dDAVP was associated with siRNA-mediated PARP-1 knockdown. Taken together, PARP-1 is likely to play a role in vasopressin-induced AQP2 expression by interacting with ß-catenin in renal CD cells.NEW & NOTEWORTHY The poly(ADP-ribose) polymerase (PARP) family catalyzes poly(ADP-ribosylation) (PARylation), which is one of the posttranslational modifications of largely undetermined physiological significance. This study investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). The results demonstrated that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. ß-Catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein.
Asunto(s)
Acuaporina 2 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Ratones , Acuaporina 2/genética , beta Catenina/metabolismo , Biotina/metabolismo , Desamino Arginina Vasopresina/farmacología , Riñón/metabolismo , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño , Vasopresinas/farmacología , Vasopresinas/metabolismoRESUMEN
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 â¼70% 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 â¼63%, 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.
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Acuaporinas , Túbulos Renales Colectores , MicroARNs , Ratones , Masculino , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Cloruro de Sodio/metabolismo , Túbulos Renales Colectores/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Acuaporinas/metabolismoRESUMEN
We have previously shown that kidney collecting ducts make vasopressin. However, the physiological role of collecting duct-derived vasopressin is uncertain. We hypothesized that collecting duct-derived vasopressin is required for the appropriate concentration of urine. We developed a vasopressin conditional knockout (KO) mouse model wherein Cre recombinase expression induces deletion of arginine vasopressin (Avp) exon 1 in the distal nephron. We then used age-matched 8- to 12-wk-old Avp fl/fl;Ksp-Cre(-) [wild type (WT)] and Avp fl/fl;Ksp-Cre(+) mice for all experiments. We collected urine, serum, and kidney lysates at baseline. We then challenged both WT and knockout (KO) mice with 24-h water restriction, water loading, and administration of the vasopressin type 2 receptor agonist desmopressin (1 µg/kg ip) followed by the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). We performed immunofluorescence and immunoblot analysis at baseline and confirmed vasopressin KO in the collecting duct. We found that urinary osmolality (UOsm), plasma Na+, K+, Cl-, blood urea nitrogen, and copeptin were similar in WT vs. KO mice at baseline. Immunoblots of the vasopressin-regulated proteins Na+-K+-2Cl- cotransporter, NaCl cotransporter, and water channel aquaporin-2 showed no difference in expression or phosphorylation at baseline. Following 24-h water restriction, WT and KO mice had no differences in UOsm, plasma Na+, K+, Cl-, blood urea nitrogen, or copeptin. In addition, there were no differences in the rate of urinary concentration or dilution as in WT and KO mice UOsm was nearly identical after desmopressin and OPC-31260 administration. We conclude that collecting duct-derived vasopressin is not essential to appropriately concentrate or dilute urine.NEW & NOTEWORTHY Hypothalamic vasopressin is required for appropriate urinary concentration. However, whether collecting duct-derived vasopressin is involved remains unknown. We developed a novel transgenic mouse model to induce tissue-specific deletion of vasopressin and showed that collecting duct-derived vasopressin is not required to concentrate or dilute urine.
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Desamino Arginina Vasopresina , Túbulos Renales Colectores , Ratones Noqueados , Animales , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Desamino Arginina Vasopresina/farmacología , Capacidad de Concentración Renal/efectos de los fármacos , Arginina Vasopresina/metabolismo , Masculino , Antagonistas de los Receptores de Hormonas Antidiuréticas/farmacología , Ratones , Acuaporina 2/metabolismo , Acuaporina 2/genética , Fármacos Antidiuréticos/farmacología , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Ratones Endogámicos C57BL , Privación de Agua , Concentración Osmolar , Sodio/orina , Sodio/metabolismo , Vasopresinas/metabolismo , BenzazepinasRESUMEN
BACKGROUND: Diabetic kidney diseases (DKD) is a the most common cause of end-stage kidney disease (ESKD) around the world. Previous studies suggest that urinary podocyte stress biomarker, e.g. podocin:nephrin mRNA ratio, is a surrogate marker of podocyte injury in non-diabetic kidney diseases. METHOD: We studied 118 patients with biopsy-proved DKD and 13 non-diabetic controls. Their urinary mRNA levels of nephrin, podocin, and aquaporin-2 (AQP2) were quantified. Renal events, defined as death, dialysis, or 40% reduction in glomerular filtration rate, were determined at 12 months. RESULTS: Urinary podocin:nephrin mRNA ratio of DKD was significantly higher than the control group (p = 0.0019), while urinary nephrin:AQP2 or podocin:AQP2 ratios were not different between groups. In DKD, urinary podocin:nephrin mRNA ratio correlated with the severity of tubulointerstitial fibrosis (r = 0.254, p = 0.006). and was associated with the renal event-free survival in 12 months (unadjusted hazard ratio [HR], 1.523; 95% confidence interval [CI] 1.157-2.006; p = 0.003). After adjusting for clinical and pathological factors, urinary podocin:nephrin mRNA ratio have a trend to predict renal event-free survival (adjusted HR, 1.327; 95%CI 0.980-1.797; p = 0.067), but the result did not reach statistical significance. CONCLUSION: Urinary podocin:nephrin mRNA ratio has a marginal prognostic value in biopsy-proven DKD. Further validation is required for DKD patients without kidney biopsy.
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Diabetes Mellitus , Nefropatías Diabéticas , Podocitos , Humanos , Nefropatías Diabéticas/diagnóstico , Pronóstico , Acuaporina 2/genética , Diálisis Renal , ARN MensajeroRESUMEN
Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-ß-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear trans-Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the trans-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.NEW & NOTEWORTHY Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.
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Acuaporina 2 , Serina , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Células LLC-PK1 , Fosforilación , Serina/metabolismo , Porcinos , Vasopresinas/farmacología , Vasopresinas/metabolismo , Espacio Intracelular/metabolismoRESUMEN
Daily, we may experience mild dehydration with a rise in plasma osmolality that triggers the release of vasopressin. Although the effect of dehydration is well characterized in collecting duct principal cells (CDPCs), we hypothesized that mild dehydration (<12 h) results in many kidney cell-specific changes in transcriptomes and chromatin accessibility. Single-nucleus (sn) multiome (RNA-assay for transposase-accessible chromatin) sequencing and bulk RNA sequencing of kidneys from male and female mice that were mildly water deprived or not were compared. Water-deprived mice had a significant increase in plasma osmolality. sn-multiome-seq resulted in 19,837 nuclei that were annotated into 33 clusters. In CDPCs, aquaporin 2 (Aqp2) and aquaporin 3 (Apq3) were greater in dehydrated mice, but there were novel genes like gremlin 2 (Grem2; a cytokine) that were increased compared with ad libitum mice. The transcription factor cAMP-responsive element modulator (Crem) was greater in CDPCs of dehydrated mice, and the Crem DNA motif was more accessible. There were hundreds of sex- and dehydration-specific differentially expressed genes (DEGs) throughout the kidney, especially in the proximal tubules and thin limbs. In male mice, DEGs were enriched in pathways related to lipid metabolism, whereas female DEGs were enriched in organic acid metabolism. Many highly expressed genes had a positive correlation with increased chromatin accessibility, and mild dehydration exerted many transcriptional changes that we detected at the chromatin level. Even with a rise in plasma osmolality, male and female kidneys have distinct transcriptomes suggesting that there may be diverse mechanisms used to remain in fluid balance.NEW & NOTEWORTHY The kidney consists of >30 cell types that work collectively to maintain fluid-electrolyte balance. Kidney single-nucleus transcriptomes and chromatin accessibility profiles from male and female control (ad libitum water and food) or mildly dehydrated mice (ad libitum food, water deprivation) were determined. Mild dehydration caused hundreds of cell- and sex-specific transcriptomic changes, even though the kidney function to conserve water was the same.
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Deshidratación , Transcriptoma , Ratones , Animales , Masculino , Femenino , Deshidratación/metabolismo , Cromatina/genética , Cromatina/metabolismo , Acuaporina 2/genética , Acuaporina 2/metabolismo , Riñón/metabolismo , Agua/metabolismoRESUMEN
The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (â¼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.
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Acuaporina 2 , Insulina , Animales , Masculino , Ratones , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Acuaporina 2/genética , Acuaporina 2/metabolismo , Insulina/metabolismo , Mamíferos/metabolismo , Presión Osmótica , Vasopresinas/farmacología , Vasopresinas/metabolismo , Agua/metabolismoRESUMEN
Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5PC-KO ) had a very mild phenotype, with no clear differences in AQP2 abundance, and mild differences in renal water handling and maximal urinary concentrating capacity. In contrast, NFAT5 (NFAT5PC-KO ) mice had significantly higher water intake and their 24 h urine volume was almost 10-fold greater than controls. After challenging with dDAVP or 8 h water restriction, NFAT5PC-KO mice were unable to concentrate their urine, demonstrating that they suffer from NDI. The abundance of AQP2, other AQPs, and the urea transporter UT-A1 were greatly decreased in NFAT5PC-KO mice. In conclusion, NFAT5 is a major regulator of not only Aqp2 gene transcription, but also other genes important for water homeostasis and its absence leads to the development of NDI.
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Diabetes Insípida Nefrogénica , Diabetes Mellitus , Túbulos Renales Colectores , Factores de Transcripción/metabolismo , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Arginina Vasopresina/metabolismo , Desamino Arginina Vasopresina/metabolismo , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/metabolismo , Diabetes Mellitus/metabolismo , Factor V/metabolismo , Túbulos Renales Colectores/metabolismo , Ratones , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Linfocitos T/metabolismo , Factores de Transcripción/genética , Vasopresinas/metabolismo , Agua/metabolismoRESUMEN
The aim of this systematic review and meta-analysis was to investigate the influence of single nucleotide polymorphisms (SNPs), related to genes in salivary composition and flow, on dental caries experience. Sixteen studies were included in the systematic review and ten in the meta-analysis. Forty-four SNPS, covering four genes (CA6, AQP2, AQP5, and MUC5B) were identified. Most of the SNPs were not associated with caries in meta-analysis. Homozygous TT genotype of the SNP CA6 rs17032907(C/T) was associated with caries [OR = 3.23(1.39-7.49)]. The pool effect of the SNPs assessed in AQP5 was associated with a reduction in the likelihood of caries [OR = 0.75(0.59-0.95)]. Considering all SNPs of salivary composition and flow, the effect allele was associated with a 75% increase in the likelihood of caries [OR = 1.75(1.06-2.89)] in the homozygous genotype. The present findings showed that the genes in salivary composition and flow can play an important role in dental caries experience.
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Caries Dental , Polimorfismo de Nucleótido Simple , Humanos , Polimorfismo de Nucleótido Simple/genética , Saliva/metabolismo , Acuaporina 2/genética , Acuaporina 2/metabolismo , Caries Dental/genética , BiopelículasRESUMEN
The symptoms of diabetes insipidus may be masked by the concurrence of adrenal insufficiency and emerge after the administration of hydrocortisone, occasionally at high doses. To elucidate the mechanism underlying polyuria induced by the administration of high-dose corticosteroids in the deficiency of arginine vasopressin (AVP), we first examined the secretion of AVP in three patients in whom polyuria was observed only after the administration of high-dose corticosteroids. Next, we examined the effects of dexamethasone or aldosterone on water balance in wild-type and familial neurohypophyseal diabetes insipidus (FNDI) model mice. A hypertonic saline test showed that AVP secretion was partially impaired in all patients. In one patient, there were no apparent changes in AVP secretion before and after the administration of high-dose corticosteroids. In FNDI mice, unlike dexamethasone, the administration of aldosterone increased urine volumes and decreased urine osmolality. Immunohistochemical analyses showed that, after the administration of aldosterone in FNDI mice, aquaporin-2 expression was decreased in the apical membrane and increased in the basolateral membrane in the collecting duct. These changes were not observed in wild-type mice. The present data suggest that treatment with mineralocorticoids induces polyuria by reducing aquaporin-2 expression in the apical membrane of the kidney in partial AVP deficiency.
Asunto(s)
Diabetes Insípida Neurogénica , Diabetes Insípida , Ratones , Animales , Poliuria/genética , Acuaporina 2/genética , Mineralocorticoides , Aldosterona , Riñón/metabolismo , Arginina Vasopresina/genética , Arginina Vasopresina/metabolismo , Dexametasona/farmacologíaRESUMEN
Aquaporins (AQPs) mediate the bidirectional water flow driven by an osmotic gradient. Either gating or trafficking allows for rapid and specific AQP regulation in a tissue-dependent manner. The regulatory mechanisms of AQP2 are discussed mainly in this chapter, as the mechanisms controlling the regulation and trafficking of AQP2 have been very well studied. The targeting of AQP2 to the apical plasma membrane of collecting duct principal cells is mainly regulated by the action of arginine vasopressin (AVP) on the type 2 AVP receptor (V2R), which cause increased intracellular cAMP or elevated intracellular calcium levels. Activation of these intracellular signaling pathways results in vesicles bearing AQP2 transport, docking and fusion with the apical membrane, which increase density of AQP2 on the membrane. The removal of AQP2 from the membrane requires dynamic cytoskeletal remodeling. AQP2 is degraded through the ubiquitin proteasome pathway and lysosomal proteolysis pathway. Finally, we review updated findings in transcriptional and epigenetic regulation of AQP2.
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Acuaporinas , Túbulos Renales Colectores , Acuaporina 2/genética , Acuaporina 2/metabolismo , Epigénesis Genética , Túbulos Renales Colectores/metabolismo , Acuaporinas/genética , Acuaporinas/metabolismo , Membrana Celular/metabolismo , Transducción de SeñalRESUMEN
One of the most prevalent indications of water-electrolyte imbalance is edema. Aquaporins (AQPs) are a protein family that can function as water channels. Osmoregulation and body water homeostasis are dependent on the regulation of AQPs. Human kidneys contain nine AQPs, five of which have been demonstrated to have a role in body water balance: AQP1, AQP2, AQP3, AQP4, and AQP7. Water imbalance is connected with AQP dysfunction. Hyponatremia with elevated AQP levels can accompany edema, which can be caused by disorders with low effective circulating blood volume and systemic vasodilation, such as congestive heart failure (CHF), hepatic cirrhosis, or the syndrome of incorrect antidiuretic hormone secretion (SIADH). In CHF, upregulation of AQP2 expression and targeting is critical for water retention. AQP2 is also involved in aberrant water retention and the formation of ascites in cirrhosis of the liver. Furthermore, water retention and hyponatremia in SIADH are caused by increased expression of AQP2 in the collecting duct. Fluid restriction, demeclocycline, and vasopressin type-2 receptor antagonists are widely utilized to treat edema. The relationship between AQPs and edema is discussed in this chapter.
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Acuaporinas , Insuficiencia Cardíaca , Hiponatremia , Síndrome de Secreción Inadecuada de ADH , Humanos , Acuaporina 2/genética , Síndrome de Secreción Inadecuada de ADH/metabolismo , Hiponatremia/metabolismo , Acuaporinas/genética , Acuaporinas/metabolismo , Edema , Agua/metabolismoRESUMEN
Disruption of water and electrolyte balance is frequently encountered in clinical medicine. Regulating water metabolism is critically important. Diabetes insipidus (DI) presented with excessive water loss from the kidney is a major disorder of water metabolism. To understanding the molecular and cellular mechanisms and pathophysiology of DI and rationales of clinical management of DI is important for both research and clinical practice. This chapter will first review various forms of DI focusing on central diabetes insipidus (CDI) and nephrogenic diabetes insipidus (NDI). This is followed by a discussion of regulatory mechanisms underlying CDI and NDI, with a focus on the regulatory axis of vasopressin, vasopressin receptor 2 (V2R) and the water channel molecule, aquaporin 2 (AQP2). The clinical manifestation, diagnosis, and management of various forms of DI will also be discussed with highlights of some of the latest therapeutic strategies that are developed from in vitro experiments and animal studies.
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Acuaporinas , Diabetes Insípida Nefrogénica , Diabetes Insípida , Diabetes Mellitus , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/metabolismo , Diabetes Insípida/diagnóstico , Diabetes Insípida/genética , Acuaporinas/genética , Acuaporinas/metabolismo , Riñón/metabolismo , Agua/metabolismo , Mutación , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismoRESUMEN
BACKGROUND: Ureteral obstruction is marked by disappearance of the vasopressin-dependent water channel aquaporin-2 (AQP2) in the renal collecting duct and polyuria upon reversal. Most studies of unilateral ureteral obstruction (UUO) models have examined late time points, obscuring the early signals that trigger loss of AQP2. METHODS: We performed RNA-Seq on microdissected rat cortical collecting ducts (CCDs) to identify early signaling pathways after establishment of UUO. RESULTS: Vasopressin V2 receptor (AVPR2) mRNA was decreased 3 hours after UUO, identifying one cause of AQP2 loss. Collecting duct principal cell differentiation markers were lost, including many not regulated by vasopressin. Immediate early genes in CCDs were widely induced 3 hours after UUO, including Myc, Atf3, and Fos (confirmed at the protein level). Simultaneously, expression of NF-κB signaling response genes known to repress Aqp2 increased. RNA-Seq for CCDs at an even earlier time point (30 minutes) showed widespread mRNA loss, indicating a "stunned" profile. Immunocytochemical labeling of markers of mRNA-degrading P-bodies DDX6 and 4E-T indicated an increase in P-body formation within 30 minutes. CONCLUSIONS: Immediately after establishment of UUO, collecting ducts manifest a stunned state with broad disappearance of mRNAs. Within 3 hours, there is upregulation of immediate early and inflammatory genes and disappearance of the V2 vasopressin receptor, resulting in loss of AQP2 (confirmed by lipopolysaccharide administration). The inflammatory response seen rapidly after UUO establishment may be relevant to both UUO-induced polyuria and long-term development of fibrosis in UUO kidneys.