RESUMO
Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.
Assuntos
Infecções por Escherichia coli , Pielonefrite , Infecções Urinárias , Escherichia coli Uropatogênica , alfa-Defensinas , Animais , Variações do Número de Cópias de DNA , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Loci Gênicos , Humanos , Camundongos , Camundongos Transgênicos , Pielonefrite/genética , Pielonefrite/imunologia , Pielonefrite/microbiologia , Sistema Urinário/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologia , alfa-Defensinas/genéticaRESUMO
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.
Assuntos
Túbulos Renais Coletores , Obstrução Ureteral , Ratos , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Obstrução Ureteral/complicações , Obstrução Ureteral/metabolismo , Poliúria/metabolismo , Rim/metabolismo , Vasopressinas , RNA Mensageiro/metabolismo , Túbulos Renais Coletores/metabolismoRESUMO
BACKGROUND: Statins therapy has been primarily recommended for the prevention of cardiovascular risk in patients with chronic kidney diseases. Statins has also been proved some benefits in lipid-induced kidney diseases. The current study aims to investigate the protection and underlying mechanisms of statins on renal tubular injuries induced by cholesterol overloaded. METHODS: We used tubular suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys and mouse collecting duct cell line mpkCCD cells to investigate the effect of statins on reactive oxygen species (ROS) production induced by cholesterol. Protein and mRNA expression of NADPH oxidase 2 (NOX2) /NOX4 was examined by Western blot and RT-PCR in vitro studies and in rats with 5/6 nephrectomy and high-fat diet. Mitochondrial morphology and membrane potential was observed by Mito-tracker and JC-1. RESULTS: Statins treatment was associated with decreased NOX2 and NOX4 protein expression and mRNA levels in 5/6Nx rats with high-fat diet. Statins treatment markedly reduced the ROS production in IMCD suspensions and mpkCCD cells. Also, statins reduced NOX2 and NOX4 protein expression and mRNA levels in cholesterol overload mpkCCD cells and improved mitochondrial morphology and function. CONCLUSION: Statins prevented ROS production induced by cholesterol in the kidney, likely through inhibiting NOXs protein expression and improving mitochondrial function. Statins may be a therapeutic option in treating obesity-associated kidney diseases.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Insuficiência Renal Crônica , Animais , Colesterol/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Rim/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismoRESUMO
Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.
Assuntos
Eritropoetina/metabolismo , Glicina/análogos & derivados , Isoquinolinas/farmacologia , Inibidores de Prolil-Hidrolase/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Eritropoetina/análise , Eritropoetina/genética , Feminino , Glicina/farmacologia , Hipóxia/genética , Hipóxia/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease leading to renal failure, wherein multiple cysts form in renal tubules and collecting ducts derived from distinct precursors: the nephron progenitor and ureteric bud (UB), respectively. Recent progress in induced pluripotent stem cell (iPSC) biology has enabled cyst formation in nephron progenitor-derived human kidney organoids in which PKD1 or PKD2, the major causative genes for ADPKD, are deleted. However, cysts have not been generated in UB organoids, despite the prevalence of collecting duct cysts in patients with ADPKD. METHODS: CRISPR-Cas9 technology deleted PKD1 in human iPSCs and the cells induced to differentiate along pathways leading to formation of either nephron progenitor or UB organoids. Cyst formation was investigated in both types of kidney organoid derived from PKD1-deleted iPSCs and in UB organoids generated from iPSCs from a patient with ADPKD who had a missense mutation. RESULTS: Cysts formed in UB organoids with homozygous PKD1 mutations upon cAMP stimulation and, to a lesser extent, in heterozygous mutant organoids. Furthermore, UB organoids generated from iPSCs from a patient with ADPKD who had a heterozygous missense mutation developed cysts upon cAMP stimulation. CONCLUSIONS: Cysts form in PKD1 mutant UB organoids as well as in iPSCs derived from a patient with ADPKD. The organoids provide a robust model of the genesis of ADPKD.
Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Néfrons/patologia , Organoides/patologia , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Ureter/patologia , Técnicas de Cultura de Células , Humanos , Mutação de Sentido Incorreto/genética , Rim Policístico Autossômico Dominante/patologiaRESUMO
The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin-angiotensin-aldosterone system (RAS).
Assuntos
Angiotensina II/farmacologia , Eritropoetina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Animais , Western Blotting , Humanos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacosRESUMO
The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Epitélio/metabolismo , Furina/metabolismo , Rim/metabolismo , Subunidades Proteicas/metabolismo , Canais de Sódio/metabolismo , Aldosterona/metabolismo , Animais , Diuréticos/farmacologia , Epitélio/efeitos dos fármacos , Glicosilação , Humanos , Rim/efeitos dos fármacos , Camundongos , Proteinúria/metabolismo , Serina Endopeptidases/metabolismo , Sódio/metabolismoRESUMO
Arginine-vasopressin (AVP)-mediated translocation of aquaporin-2 (AQP2) protein-forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for the renal conservation of water. The type-1 PDZ-binding motif (PBM) in AQP2, "GTKA," is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remain obscure. We determined that synapse-associated protein-97 (SAP97), a membrane-associated guanylate kinase protein involved in establishing epithelial cell polarity, was an avid binding partner to the PBM of AQP2. The role of PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC-PK1 renal collecting cells by confocal microscopy and cell surface biotinylation techniques. These experiments indicated that distribution of AQP2 and SAP97 overlapped in the kidneys and LLC-PK1 cells and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. Binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of PKA, or knockdown of SAP97 inhibited AQP2 translocation as well as AVP- and forskolin-mediated phosphorylation of Ser256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that the targeting of PKA to the microdomain of AQP2 via SAP97-AQP2 interactions in association with cross-talk between two barcodes in AQP2, namely, the PBM and phospho-Ser256, plays an important role in the translocation of AQP2 in the kidney.
Assuntos
Aquaporina 2/metabolismo , Arginina Vasopressina/farmacologia , Proteína 1 Homóloga a Discs-Large/metabolismo , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Domínios PDZ , Animais , Aquaporina 2/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína 1 Homóloga a Discs-Large/genética , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Serina , SuínosRESUMO
microRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. The physiologic function of these noncoding RNAs in postnatal renal tubules still remains unclear. Surprisingly, they appear to be dispensable for mammalian proximal tubule (PT) function. Here, we examined the effects of miRNA suppression in collecting ducts (CDs). To conclusively evaluate the role of miRNAs, we generated three mouse models with CD-specific inactivation of key miRNA pathway genes Dicer, Dgcr8, and the entire Argonaute gene family (Ago1, 2, 3, and 4). Characterization of these three mouse models revealed that inhibition of miRNAs in CDs spontaneously evokes a renal tubule injury-like response, which culminates in progressive tubulointerstitial fibrosis (TIF) and renal failure. Global miRNA profiling of microdissected renal tubules showed that miRNAs exhibit segmental distribution along the nephron and CDs. In particular, the expression of miR-200c is nearly 70-fold higher in CDs compared with PTs. Accordingly, miR-200s are downregulated in Dicer-KO CDs, its direct target genes Zeb1, Zeb2, and Snail2 are upregulated, and miRNA-depleted CDs undergo partial epithelial-to-mesenchymal transition (EMT). Thus, miRNAs are essential for CD homeostasis. Downregulation of CD-enriched miRNAs and the subsequent induction of partial EMT may be a new mechanism for TIF progression.
Assuntos
Epitélio/metabolismo , Epitélio/patologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , MicroRNAs/genética , Animais , Proteínas Argonautas/genética , Linhagem Celular , RNA Helicases DEAD-box/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Fatores de Iniciação em Eucariotos/genética , Feminino , Fibrose , Expressão Gênica , Homeostase/genética , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , Fenótipo , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Fatores de Transcrição da Família Snail/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Background Hyperkalemia in association with metabolic acidosis that are out of proportion to changes in glomerular filtration rate defines type 4 renal tubular acidosis (RTA), the most common RTA observed, but the molecular mechanisms underlying the associated metabolic acidosis are incompletely understood. We sought to determine whether hyperkalemia directly causes metabolic acidosis and, if so, the mechanisms through which this occurs.Methods We studied a genetic model of hyperkalemia that results from early distal convoluted tubule (DCT)-specific overexpression of constitutively active Ste20/SPS1-related proline-alanine-rich kinase (DCT-CA-SPAK).Results DCT-CA-SPAK mice developed hyperkalemia in association with metabolic acidosis and suppressed ammonia excretion; however, titratable acid excretion and urine pH were unchanged compared with those in wild-type mice. Abnormal ammonia excretion in DCT-CA-SPAK mice associated with decreased proximal tubule expression of the ammonia-generating enzymes phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and overexpression of the ammonia-recycling enzyme glutamine synthetase. These mice also had decreased expression of the ammonia transporter family member Rhcg and decreased apical polarization of H+-ATPase in the inner stripe of the outer medullary collecting duct. Correcting the hyperkalemia by treatment with hydrochlorothiazide corrected the metabolic acidosis, increased ammonia excretion, and normalized ammoniagenic enzyme and Rhcg expression in DCT-CA-SPAK mice. In wild-type mice, induction of hyperkalemia by administration of the epithelial sodium channel blocker benzamil caused hyperkalemia and suppressed ammonia excretion.Conclusions Hyperkalemia decreases proximal tubule ammonia generation and collecting duct ammonia transport, leading to impaired ammonia excretion that causes metabolic acidosis.
Assuntos
Amônia/urina , Hiperpotassemia/genética , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas Serina-Treonina Quinases/genética , Acidose/etiologia , Aldosterona/urina , Amilorida/análogos & derivados , Animais , Proteínas de Transporte de Cátions/metabolismo , Diuréticos/uso terapêutico , Glutaminase/metabolismo , Hidroclorotiazida/uso terapêutico , Concentração de Íons de Hidrogênio , Hiperpotassemia/sangue , Hiperpotassemia/complicações , Hiperpotassemia/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , ATPases Translocadoras de Prótons/metabolismo , UrináliseRESUMO
The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.
Assuntos
Aquaporina 2/genética , Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/citologia , Rim/metabolismo , Receptores de Vasopressinas/genética , Fatores de Transcrição/metabolismo , Animais , Aquaporina 2/metabolismo , Contagem de Células , Linhagem Celular , Regulação para Baixo/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Integrases/metabolismo , Rim/embriologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/embriologia , Túbulos Renais Coletores/metabolismo , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Receptores Notch/metabolismo , Receptores de Vasopressinas/metabolismo , Transdução de Sinais , Regulação para Cima/genética , Ureter/embriologia , Ureter/metabolismoRESUMO
Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45â¯kDa and kidney erythropoietin at 36-40 and 42â¯kDa, both of which shifted to 22â¯kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.
Assuntos
Aldosterona/metabolismo , Eritropoetina/metabolismo , Fludrocortisona/metabolismo , Túbulos Renais Coletores/metabolismo , Néfrons/metabolismo , Animais , Hipóxia Celular , Eritropoetina/genética , Glicosilação , Túbulos Renais Coletores/citologia , Masculino , Néfrons/citologia , RNA Mensageiro/genética , Ratos Sprague-Dawley , Sistema Renina-Angiotensina , Regulação para CimaRESUMO
The vasopressin-cAMP-osmolality axis is abnormal in autosomal dominant polycystic kidney disease (ADPKD). In the Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Its Outcomes 3:4 Trial, a 3-year randomized, placebo-controlled trial in adults, the vasopressin V2 receptor antagonist tolvaptan slowed ADPKD progression in patients with preserved GFR. Here, we investigated the determinants of baseline urine osmolality (Uosm) and its value as a severity marker of ADPKD, the factors influencing the response to tolvaptan, and whether change in Uosm associated with key trial end points. At baseline, lower Uosm independently associated with female sex, presence of hypertension, lower eGFR, higher total kidney volume (TKV), and higher age. Tolvaptan consistently reduced Uosm by 200-300 mOsm/kg over 36 months. The Uosm response to tolvaptan depended on baseline eGFR and Uosm. Subjects with greater change in Uosm experienced a significant reduction in clinical progression events. Among subjects receiving tolvaptan, those with a greater suppression of Uosm had slower renal function decline. Assessment at follow-up, off medication, revealed a significant decrease in Uosm in both placebo and treated groups. Tolvaptan significantly increased plasma osmolality, which returned to baseline at follow-up. In conclusion, baseline Uosm in ADPKD reflects age, renal function, and TKV, and baseline Uosm, eGFR, and TKV influence the effect of tolvaptan on Uosm. The greatest renal benefit occurred in subjects achieving greater suppression of Uosm, that is, those with better eGFR at baseline. These results support the link between vasopressin V2 receptor signaling and ADPKD progression.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/uso terapêutico , Benzazepinas/uso terapêutico , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/urina , Adolescente , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Concentração Osmolar , Tolvaptan , Resultado do Tratamento , Adulto JovemRESUMO
The transcription factor hepatocyte nuclear factor-1ß (HNF-1ß) is essential for normal kidney development and function. Inactivation of HNF-1ß in mouse kidney tubules leads to early-onset cyst formation and postnatal lethality. Here, we used Pkhd1/Cre mice to delete HNF-1ß specifically in renal collecting ducts (CDs). CD-specific HNF-1ß mutant mice survived long term and developed slowly progressive cystic kidney disease, renal fibrosis, and hydronephrosis. Compared with wild-type littermates, HNF-1ß mutant mice exhibited polyuria and polydipsia. Before the development of significant renal structural abnormalities, mutant mice exhibited low urine osmolality at baseline and after water restriction and administration of desmopressin. However, mutant and wild-type mice had similar plasma vasopressin and solute excretion levels. HNF-1ß mutant kidneys showed increased expression of aquaporin-2 mRNA but mislocalized expression of aquaporin-2 protein in the cytoplasm of CD cells. Mutant kidneys also had decreased expression of the UT-A urea transporter and collectrin, which is involved in apical membrane vesicle trafficking. Treatment of HNF-1ß mutant mIMCD3 cells with hypertonic NaCl inhibited the induction of osmoregulated genes, including Nr1h4, which encodes the transcription factor FXR that is required for maximal urinary concentration. Chromatin immunoprecipitation and sequencing experiments revealed HNF-1ß binding to the Nr1h4 promoter in wild-type kidneys, and immunoblot analysis revealed downregulated expression of FXR in HNF-1ß mutant kidneys. These findings reveal a novel role of HNF-1ß in osmoregulation and identify multiple mechanisms, whereby mutations of HNF-1ß produce defects in urinary concentration.
Assuntos
Fator 1-beta Nuclear de Hepatócito/fisiologia , Túbulos Renais Coletores/fisiologia , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos Transgênicos , Poliúria/genética , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , UrinaRESUMO
The renal collecting duct (CD) contains two major cell types, intercalated (ICs) and principal cells (PCs). A previous report showed that deletion of ß1-integrin in the entire renal CD causes defective CD morphogenesis resulting in kidney dysfunction. However, subsequent deletion of ß1-integrin specifically in ICs and PCs, respectively, did not cause any morphological defects in the CDs. The discrepancy between these studies prompts us to reinvestigate the role of ß1-integrin in CD cells, specifically in the PCs. We conditionally deleted ß1-integrin in mouse CD PCs using a specific aquaporin-2 (AQP2) promoter Cre-LoxP system. The resulting mutant mice, ß-1f/fAQP2-Cre+, had lower body weight, failed to thrive, and died around 8-12 wk. Their CD tubules were dilated, and some of them contained cellular debris. Increased apoptosis and proliferation of PCs were observed in the dilated CDs. Trichrome staining and electron microscopy revealed the presence of peritubular and interstitial fibrosis that is associated with increased production of extracellular matrix proteins including collagen type IV and fibronectin, as detected by immunoblotting. Further analysis revealed a significantly increased expression of transforming growth factor-ß (TGF-ß)-induced protein, fibronectin, and TGF-ß receptor-1 mRNAs and concomitantly increased phosphorylation of SMAD-2 that indicates the activation of the TGF-ß signaling pathway. Therefore, our data reveal that normal expression of ß1-integrin in PCs is a critical determinant of CD structural and functional integrity and further support the previously reported critical role of ß1-integrin in the development and/or maintenance of the CD structure and function.
Assuntos
Matriz Extracelular/metabolismo , Deleção de Genes , Integrina beta1/metabolismo , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Poliúria/metabolismo , Insuficiência Renal/metabolismo , Fatores Etários , Animais , Apoptose , Aquaporina 2/genética , Proliferação de Células , Matriz Extracelular/ultraestrutura , Insuficiência de Crescimento/genética , Insuficiência de Crescimento/metabolismo , Insuficiência de Crescimento/patologia , Fibrose , Predisposição Genética para Doença , Integrases/genética , Integrina beta1/genética , Medula Renal/ultraestrutura , Túbulos Renais Coletores/ultraestrutura , Camundongos Knockout , Fenótipo , Fosforilação , Poliúria/genética , Poliúria/patologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Insuficiência Renal/genética , Insuficiência Renal/patologia , Transdução de Sinais , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Pendrin is a Cl-/HCO3- exchanger expressed in type B and non-A, non-B intercalated cells in the distal nephron, where it facilitates Cl- absorption and is involved in Na+ absorption and acid-base balance. Pendrin-knockout mice show no fluid-electrolyte abnormalities under baseline conditions, although mice with double knockout of pendrin and the Na+/Cl- cotransporter (NCC) manifest profound salt wasting. Thus, pendrin may attenuate diuretic-induced salt loss, but this function remains unconfirmed. To clarify the physiologic role of pendrin under conditions not confounded by gene knockout, and to test the potential utility of pendrin inhibitors for diuretic therapy, we tested in mice a small-molecule pendrin inhibitor identified from a high-throughput screen. In vitro, a pyrazole-thiophenesulfonamide, PDSinh-C01, inhibited Cl-/anion exchange mediated by mouse pendrin with a 50% inhibitory concentration of 1-3 µM, without affecting other major kidney tubule transporters. Administration of PDSinh-C01 to mice at predicted therapeutic doses, determined from serum and urine pharmacokinetics, did not affect urine output, osmolality, salt excretion, or acid-base balance. However, in mice treated acutely with furosemide, administration of PDSinh-C01 produced a 30% increase in urine output, with increased Na+ and Cl- excretion. In mice treated long term with furosemide, in which renal pendrin is upregulated, PDSinh-C01 produced a 60% increase in urine output. Our findings clarify the role of pendrin in kidney function and suggest pendrin inhibition as a novel approach to potentiate the action of loop diuretics. Such combination therapy might enhance diuresis and salt excretion for treatment of hypertension and edema, perhaps including diuretic-resistant edema.
Assuntos
Proteínas de Transporte de Ânions/antagonistas & inibidores , Diuréticos/farmacologia , Furosemida/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Sinergismo Farmacológico , Feminino , Camundongos , Transportadores de SulfatoRESUMO
Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling.
Assuntos
Canais de Cálcio/metabolismo , Diabetes Insípido Nefrogênico/etiologia , Diabetes Insípido Nefrogênico/metabolismo , Animais , Aquaporina 2/fisiologia , Arginina Vasopressina/fisiologia , Células Cultivadas , Masculino , Ratos , Ratos Endogâmicos SHR/genética , Molécula 1 de Interação Estromal/fisiologiaRESUMO
Tubular reabsorption of filtered sodium is tightly controlled to maintain body volume homeostasis. The rate of sodium transport by collecting duct (CD) cells varies widely in response to dietary sodium intake, GFR, circulating hormones, neural signals, and local regulatory factors. Reabsorption of filtered sodium by CD cells occurs via a two-step process. First, luminal sodium crosses the apical plasma membrane along its electrochemical gradient through epithelial sodium channels (ENaC). Intracellular sodium is then actively extruded into the interstitial space by the Na(+),K(+)-ATPase located along the basolateral membrane. Mismatch between sodium entry and exit induces variations in sodium intracellular concentration and cell volume that must be maintained within narrow ranges for control of vital cell functions. Therefore, renal epithelial cells display highly coordinated apical and basolateral sodium transport rates. We review evidence from experiments conducted in vivo and in cultured cells that indicates aldosterone and vasopressin, the two major hormones regulating sodium reabsorption by CD, generate a coordinated stimulation of apical ENaC and basolateral Na(+),K(+)-ATPase. Moreover, we discuss evidence suggesting that variations in sodium entry per se induce a coordinated change in Na(+),K(+)-ATPase activity through the signaling of protein kinases such as protein kinase A and p38 mitogen-activated protein kinase.
Assuntos
Canais Epiteliais de Sódio/fisiologia , Túbulos Renais Coletores/enzimologia , Reabsorção Renal , ATPase Trocadora de Sódio-Potássio/fisiologia , Animais , Humanos , Transporte de ÍonsRESUMO
The kidney plays a central role in the regulation of the body water balance. The process of targeting the water channel aquaporin-2 (AQP2) on the apical plasma membrane of the collecting duct (CD) principal cells is mainly regulated by the antidiuretic peptide hormone arginine vasopressin (AVP), which is responsible for the maintenance of water homeostasis. Recently, much attention has been focused on the local factors modulating renal water reabsorption by AQP2 in the collecting ducts, especially prostaglandin E2 (PGE2). PGE2 is a lipid mediator involved in a variety of physiological and pathophysiological processes in the kidney. The biological function of PGE2 is mainly mediated by four G-protein-coupled receptors, namely EP1-4, which couple to drive separate intracellular signaling pathways. Increasing evidence demonstrates that PGE2 is essential for renal water transport regulation via multiple mechanisms. Each EP receptor plays a unique role in regulating water reabsorption in renal collecting ducts. This brief review highlights the role of PGE2 in the regulation of water reabsorption and discusses the involvement of each EP receptor subtype in renal collecting duct. A better understanding of the role of PGE2 in renal water transport process may improve disease management strategies for water balance disorders, including nephrogenic diabetes insipidus.
Assuntos
Aquaporinas/metabolismo , Diabetes Insípido Nefrogênico/metabolismo , Dinoprostona/metabolismo , Túbulos Renais Coletores/metabolismo , Reabsorção Renal , Animais , HumanosRESUMO
A 1.37 Mbp region of chromosome 13 previously identified by exclusion mapping was consistently associated with a reduction of salt-induced hypertension in the Dahl salt-sensitive (SS) rat. This region contained five genes that were introgressed from the salt-insensitive Brown Norway (BN) rat. The goal of the present study was to further narrow that region to identify the gene(s) most likely to protect from salt-induced hypertension. The studies yielded a subcongenic SS rat strain containing a 0.71 Mbp insert from BN (26-P strain) in which salt-induced hypertension was reduced by 24 mmHg. The region contained two protein-coding genes (Astn1 and Pappa2) and a microRNA (miR-488). Pappa2 mRNA in the renal cortex of the protected 26-P was 6- to 10-fold greater than in SS fed a 0.4% NaCl diet but was reduced to levels observed in SS when fed 8.0% NaCl diet for 7 days. Compared with brain nuclei (NTS, RVLM, CVLM) and the adrenal gland, Pappa2 in the renal cortex was the only gene found to be differentially expressed between SS and 26-P and that responded to changes of salt diet. Immunohistochemistry studies found Pappa2 localized in the cytosol of the epithelial cells of the cortical thick ascending limbs. In more distal segments of the renal tubules, it was observed within tubular lumens and most notably bound to the apical membranes of the intercalated cells of collecting ducts. We conclude that we have identified a variant form of Pappa2 that can protect against salt-induced hypertension in the Dahl S rat.