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1.
Cell Commun Signal ; 16(1): 74, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30404645

RESUMO

BACKGROUND: We have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/ß-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe3+-free (apo-)Lcn2. AIM: To determine the effects of osmolarity/tonicity changes, Wnt/ß-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells. METHODS: Normosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. ß-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS). RESULTS: Hyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/ß-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, ß-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability. CONCLUSIONS: Lcn2-R upregulation and Lcn2 downregulation via Wnt/ß-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.


Assuntos
Infecções Bacterianas/patologia , Regulação da Expressão Gênica , Medula Renal/citologia , Medula Renal/microbiologia , Lipocalina-2/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Via de Sinalização Wnt , Animais , Infecções Bacterianas/metabolismo , Camundongos , Concentração Osmolar , Ratos
2.
Sci Rep ; 8(1): 9022, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899363

RESUMO

Nephronophthisis (NPH) is an autosomal recessive form of cystic kidney disease and the leading cause of hereditary kidney failure in children and young adults. Like other NPH proteins, the NPHP16/Anks6-interacting protein Anks3 has been identified to cause laterality defects in humans. However, the cellular functions of Anks3 remain enigmatic. We investigated the metabolic impact of Anks3 depletion in cultured murine inner medullary collecting duct cells via GC-MS profiling and LC-MS/MS analysis. Combined metabolomics successfully identified 155 metabolites; 48 metabolites were identified to be significantly altered by decreasing Anks3 levels. Especially, amino acid and purine/pyrimidine metabolism were affected by loss of Anks3. Branched-chain amino acids were identified to be significantly downregulated suggesting disrupted nutrient signalling. Tryptophan and 1-ribosyl-imidazolenicotinamide accumulated whereas NAD+ and NADP+ concentrations were diminished indicating disturbances within the tryptophan-niacin pathway. Most strikingly, nucleosides were reduced upon Anks3 depletion, while 5-methyluridine and 6-methyladenosine accumulated over time. Hence, elevated PARP1 and cleaved PARP1 levels could be detected. Furthermore, living cell number and viability was significantly declined. In combination, these results suggest that Anks3 may be involved in DNA damage responses by balancing the intracellular nucleoside pool.


Assuntos
Proteínas de Transporte/metabolismo , Doenças Renais Císticas/metabolismo , Medula Renal/citologia , Túbulos Renais Coletores/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Cromatografia Líquida , Modelos Animais de Doenças , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Túbulos Renais Coletores/citologia , Metabolômica/métodos , Camundongos , Interferência de RNA , Espectrometria de Massas em Tandem
3.
PLoS One ; 13(5): e0197078, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29734386

RESUMO

BACKGROUND: Brain natriuretic peptide (BNP) is an important biomarker for patients with cardiovascular diseases, including heart failure, hypertension and cardiac hypertrophy. It is also known that BNP levels are relatively higher in patients with chronic kidney disease and no heart disease; however, the mechanism remains unclear. METHODS AND RESULTS: We developed a BNP reporter mouse and occasionally found that this promoter was activated specifically in the papillary tip of the kidneys, and its activation was not accompanied by BNP mRNA expression. No evidence was found to support the existence of BNP isoforms or other nucleotide expression apart from BNP and tdTomato. The pBNP-tdTomato-positive cells were interstitial cells and were not proliferative. Unexpectedly, both the expression and secretion of BNP increased in primary cultured neonatal cardiomyocytes after their treatment with an extract of the renal papillary tip. Intraperitoneal injection of the extract of the papillary tips reduced blood pressure from 210 mmHg to 165 mmHg, the decrease being accompanied by an increase in serum BNP and urinary cGMP production in stroke-prone spontaneously hypertensive (SHR-SP) rats. Furthermore the induction of BNP by the papillary extract from rats with heart failure due to myocardial infarction was increased in cardiomyocytes. CONCLUSIONS: These results suggested that the papillary tip express a substance that can stimulate BNP production and secretion from cardiomyocytes.


Assuntos
Doenças Cardiovasculares/genética , GMP Cíclico/genética , Peptídeo Natriurético Encefálico/genética , Insuficiência Renal Crônica/genética , Animais , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , GMP Cíclico/metabolismo , Humanos , Medula Renal/citologia , Medula Renal/metabolismo , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Peptídeo Natriurético Encefálico/biossíntese , Cultura Primária de Células , Ratos , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia
4.
Proc Natl Acad Sci U S A ; 115(21): 5600-5605, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29739889

RESUMO

Hypertonicity in renal medulla is critical for the kidney to produce concentrated urine. Renal medullary cells have to survive high medullary osmolarity during antidiuresis. Previous study reported that farnesoid X receptor (FXR), a nuclear receptor transcription factor activated by endogenous bile acids, increases urine concentrating ability by up-regulating aquaporin 2 expression in medullary collecting duct cells (MCDs). However, whether FXR is also involved in the maintenance of cell survival of MCDs under dehydration condition and hypertonic stress remains largely unknown. In the present study, we demonstrate that 24-hours water restriction selectively up-regulated renal medullary expression of FXR with little MCD apoptosis in wild-type mice. In contrast, water deprivation caused a massive apoptosis of MCDs in both global FXR gene-deficient mice and collecting duct-specific FXR knockout mice. In vitro studies showed that hypertonicity significantly increased FXR and tonicity response enhancer binding protein (TonEBP) expression in mIMCD3 cell line and primary cultured MCDs. Activation and overexpression of FXR markedly increased cell viability and decreased cell apoptosis under hyperosmotic conditions. In addition, FXR can increase gene expression and nuclear translocation of TonEBP. We conclude that FXR protects MCDs from hypertonicity-induced cell injury very likely via increasing TonEBP expression and nuclear translocation. This study provides insights into the molecular mechanism by which FXR enhances urine concentration via maintaining cell viability of MCDs under hyperosmotic condition.


Assuntos
Capacidade de Concentração Renal/fisiologia , Medula Renal/citologia , Túbulos Renais Coletores/citologia , Pressão Osmótica/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição/genética
5.
Ann Anat ; 218: 95-104, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29660398

RESUMO

The connective stromal and epithelial compartments of the kidney have regenerative potential and phenotypic flexibility. A few studies have shown that cells appertaining to both compartments can exhibit myoid phenotypes. The purpose of our study was to investigate the myoid pattern of kidney and its association with the kidney niches containing stromal cells/telocytes (SC/TCs). We performed an immunohistochemical study using a panel of endothelial, myoid, mesenchymal and stem/progenitor markers, namely CD31, CD34, CD105 (endoglin), CD117/c-kit, nestin, desmin, α-smooth muscle actin (α-SMA) and the heavy chain of smooth muscle myosin (SMM). We used histologically normal kidney samples, obtained after nephrectomy, from nine adult patients. The capsular SC/TCs had a strong CD34 and partial nestin and CD105 immunopositivity. Subcapsular and interstitial SC/TCs expressed c-kit, nestin, CD105, but also α-SMA and SMM, therefore having a myoid phenotype. The endothelial SC/TCs phenotype was CD31+/CD34+/CD105+/nestin±/SMM±/α-SMA±. All three myoid markers were expressed in periendothelial SC/TCs. We also found a scarce expression of nestin in parietal epithelial cells of Bowman's capsule, and in podocytes. In epithelial cells, we found a positive expression for CD31, CD117/c-kit, desmin, CD34, SMM, and CD105. In epithelial tubular cells, we found a predominant basal expression of the myoid markers (SMM and desmin). In conclusion, myoepithelial tubular cells, myoid endothelial cells and myoid SC/TCs are normal constituents of the kidney.


Assuntos
Células Epiteliais/ultraestrutura , Rim/citologia , Telócitos/ultraestrutura , Idoso , Feminino , Humanos , Imuno-Histoquímica , Rim/ultraestrutura , Córtex Renal/anatomia & histologia , Córtex Renal/citologia , Medula Renal/anatomia & histologia , Medula Renal/citologia , Túbulos Renais/anatomia & histologia , Túbulos Renais/citologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Células Estromais/ultraestrutura
6.
Toxicol Pathol ; 46(3): 266-272, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29504493

RESUMO

Chronic progressive nephropathy (CPN) occurs commonly in rats, more frequently and severely in males than females. High-grade CPN is characterized by increased layers of the renal papilla lining, designated as urothelial hyperplasia in the International Harmonization of Nomenclature and Diagnostic Criteria classification. However, urothelium lining the pelvis is not equivalent to the epithelium lining the papilla. To evaluate whether the epithelium lining the renal papilla is actually urothelial in nature and whether CPN-associated multicellularity represents proliferation, kidney tissues from aged rats with CPN, from rats with multicellularity of the renal papilla epithelium of either low-grade or marked severity, and from young rats with normal kidneys were analyzed and compared. Immunohistochemical staining for uroplakins (urothelial specific proteins) was negative in the papilla epithelium in all rats with multicellularity or not, indicating these cells are not urothelial. Mitotic figures were rarely observed in this epithelium, even with multicellularity. Immunohistochemical staining for Ki-67 was negative. Papilla lining cells and true urothelium differed by scanning electron microscopy. Based on these findings, we recommend that the epithelium lining the papilla not be classified as urothelial, and the CPN-associated lesion be designated as vesicular alteration of renal papilla instead of hyperplasia and distinguished in diagnostic systems from kidney pelvis urothelial hyperplasia.


Assuntos
Epitélio/anatomia & histologia , Medula Renal/citologia , Insuficiência Renal Crônica/patologia , Animais , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Urotélio/citologia
7.
Int J Mol Sci ; 18(12)2017 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-29236055

RESUMO

The content of hyaluronan (HA) in the interstitium of the renal medulla changes in relation to body hydration status. We investigated if hormones of central importance for body fluid homeostasis affect HA production by renomedullary interstitial cells in culture (RMICs). Simultaneous treatment with vasopressin and angiotensin II (Ang II) reduced HA by 69%. No change occurred in the mRNA expressions of hyaluronan synthase 2 (HAS2) or hyaluronidases (Hyals), while Hyal activity in the supernatant increased by 67% and CD44 expression reduced by 42%. The autocoid endothelin (ET-1) at low concentrations (10-10 and 10-8 M) increased HA 3-fold. On the contrary, at a high concentration (10-6 M) ET-1 reduced HA by 47%. The ET-A receptor antagonist BQ123 not only reversed the reducing effect of high ET-1 on HA, but elevated it to the same level as low concentration ET-1, suggesting separate regulating roles for ET-A and ET-B receptors. This was corroborated by the addition of ET-B receptor antagonist BQ788 to low concentration ET-1, which abolished the HA increase. HAS2 and Hyal2 mRNA did not alter, while Hyal1 mRNA was increased at all ET-1 concentrations tested. Hyal activity was elevated the most by high ET-1 concentration, and blockade of ET-A receptors by BQ123 prevented about 30% of this response. The present study demonstrates an important regulatory influence of hormones involved in body fluid balance on HA handling by RMICs, thereby supporting the concept of a dynamic involvement of interstitial HA in renal fluid handling.


Assuntos
Angiotensina II/farmacologia , Endotelinas/farmacologia , Ácido Hialurônico/metabolismo , Medula Renal/efeitos dos fármacos , Vasopressinas/farmacologia , Animais , Células Cultivadas , Endotelinas/metabolismo , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/análise , Hialuronoglucosaminidase/antagonistas & inibidores , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Himecromona/farmacologia , Medula Renal/citologia , Medula Renal/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Transcriptoma/efeitos dos fármacos
8.
PLoS One ; 12(11): e0188006, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29155857

RESUMO

The final adjustment of urine volume occurs in the inner medullary collecting duct (IMCD), chiefly mediated by the water channel aquaporin 2 (AQP2). With vasopressin stimulation, AQP2 accumulation in the apical plasma membrane of principal cells allows water reabsorption from the lumen. We report that FXYD1 (phospholemman), better known as a regulator of Na,K-ATPase, has a role in AQP2 trafficking. Daytime urine of Fxyd1 knockout mice was more dilute than WT despite similar serum vasopressin, but both genotypes could concentrate urine during water deprivation. FXYD1 was found in IMCD. In WT mice, phosphorylated FXYD1 was detected intracellularly, and vasopressin induced its dephosphorylation. We tested the hypothesis that the dilute urine in knockouts was caused by alteration of AQP2 trafficking. In WT mice at baseline, FXYD1 and AQP2 were not strongly co-localized, but elevation of vasopressin produced translocation of both FXYD1 and AQP2 to the apical plasma membrane. In kidney slices, baseline AQP2 distribution was more scattered in the Fxyd1 knockout than in WT. Apical recruitment of AQP2 occurred in vasopressin-treated Fxyd1 knockout slices, but upon vasopressin washout, there was more rapid reversal of apical AQP2 localization and more heterogeneous cytoplasmic distribution of AQP2. Notably, in sucrose gradients, AQP2 was present in a detergent-resistant membrane domain that had lower sedimentation density in the knockout than in WT, and vasopressin treatment normalized its density. We propose that FXYD1 plays a role in regulating AQP2 retention in apical membrane, and that this involves transfers between raft-like membrane domains in endosomes and plasma membranes.


Assuntos
Aquaporina 2/metabolismo , Endossomos/metabolismo , Túbulos Renais Coletores/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Fosfoproteínas/genética , Vesículas Transportadoras/metabolismo , Animais , Aquaporina 2/genética , Centrifugação com Gradiente de Concentração , Endossomos/química , Endossomos/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Medula Renal/citologia , Medula Renal/efeitos dos fármacos , Medula Renal/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Masculino , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos dos fármacos , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Microtomia , Fosfoproteínas/deficiência , Fosforilação , Transporte Proteico , Sacarose , Técnicas de Cultura de Tecidos , Vesículas Transportadoras/química , Vesículas Transportadoras/efeitos dos fármacos , Vasopressinas/genética , Vasopressinas/metabolismo , Vasopressinas/farmacologia
9.
Cell Physiol Biochem ; 42(3): 1264-1273, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28693025

RESUMO

BACKGROUND: Our previous study has detected a stem cell deficiency in the renal medulla in Dahl salt-sensitive (S) rats. This study determined whether infusion of valproic acid (VA), an agent known to stimulate the stem cell function, attenuated salt-sensitive hypertension in Dahl S rats. METHODS: Uninephrectomized Dahl S rats were infused with vehicle or VA (50mg/kg/d) into the renal medulla and fed with a low (LS) or high salt diet (HS). Stem cell marker and number were analyzed by immunohistochemistry, Real-time RT-PCR and Western blot. Sodium excretion and blood pressure were measured. RESULTS: VA significantly increased the mRNA and protein levels of FGF2, a stem cell niche factor, and CD133, a stem cell marker. The number of CD133+ cells was significantly increased in the renal medulla in VA-treated rats. Meanwhile, high salt-induced increases in the mRNA level of proinflammatory factors interleukin-1ß and interleukin-6 were blocked in VA-treated rats. Functionally, sodium excretion in response to the blood pressure increase and acute sodium loading was significantly enhanced, sodium retention attenuated, high salt-induced increase of blood pressure reduced in VA-treated rats. CONCLUSION: Activation of stem cell function by VA inhibits the activation of proinflammatory factors and attenuates salt-sensitive hypertension in Dahl S rats.


Assuntos
Anti-Hipertensivos/farmacologia , Inibidores Enzimáticos/farmacologia , Hipertensão/tratamento farmacológico , Medula Renal/citologia , Medula Renal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Ácido Valproico/farmacologia , Antígeno AC133/análise , Antígeno AC133/metabolismo , Animais , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hipertensão/metabolismo , Masculino , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta/metabolismo , Células-Tronco/citologia , Ácido Valproico/administração & dosagem
10.
BMC Nephrol ; 18(1): 209, 2017 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-28673338

RESUMO

BACKGROUND: Although TGF-ß and the transcription factor Egr-1 play an important role in both kidney fibrosis and in response to acute changes of renal medullary osmolarity, their role under sustained hypo- or hyperosmolar conditions has not been elucidated. We investigated the effects of chronic hypertonicity and hypotonicity on the renal medullary TGF-ß and Egr-1 expression. METHODS: Male adult Sprague Dawley rats (n = 6/group) were treated with 15 mg/day furosemide, or the rats were water restricted to 15 ml/200 g body weight per day. Control rats had free access to water and rodent chow. Kidneys were harvested after 5 days of treament. In cultured inner medullary collecting duct (IMCD) cells, osmolarity was increased from 330 mOsm to 900 mOsm over 6 days. Analyses were performed at 330, 600 and 900 mOsm. RESULTS: Urine osmolarity has not changed due to furosemide treatment but increased 2-fold after water restriction (p < 0.05). Gene expression of TGF-ß and Egr-1 increased by 1.9-fold and 7-fold in the hypertonic medulla, respectively (p < 0.05), accompanied by 6-fold and 2-fold increased c-Fos and TIMP-1 expression, respectively (p < 0.05) and positive immunostaining for TGF-ß and Egr-1 (p < 0.05). Similarly, hyperosmolarity led to overexpression of TGF-ß and Egr-1 mRNA in IMCD cells (2.5-fold and 3.5-fold increase from 330 to 900 mOsm, respectively (p < 0.05)) accompanied by significant c-Fos and c-Jun overexpressions (p < 0.01), and increased Col3a1 and Col4a1 mRNA expression. CONCLUSION: We conclude that both TGF-ß and Egr-1 are upregulated by sustained hyperosmolarity in the rat renal medulla, and it favors the expression of extracellular matrix components.


Assuntos
Ingestão de Líquidos/fisiologia , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Medula Renal/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/genética , Expressão Gênica , Medula Renal/citologia , Masculino , Concentração Osmolar , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/genética
11.
Physiol Rep ; 5(12)2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28646097

RESUMO

To learn more about controlling renal interstitial hydrostatic pressure (RIHP), we assessed its response to renal medullary direct interstitial volume expansion (rmDIVE = 100 µL bolus infusion/30 sec). Three experimental series (S) were performed in hydropenic, anesthetized, right-nephrectomized, acute left renal-denervated and renal perfusion pressure-controlled rats randomly assigned to groups in each S. S1: Rats without hormonal clamp were contrasted before and after rmDIVE induced via 0.9% saline solution bolus (SS group) or 2% albumin in SS bolus (2% ALB + SS group). Subcapsular ΔRIHP rose slowly, progressively and similarly in both groups by ~3 mmHg. S2: Rats under hormonal clamp were contrasted before and after sham rmDIVE (time CTR group) and real rmDIVE induced via either SS bolus (SS group) or SS bolus containing the subcutaneous tissue fibroblast relaxant dibutyryl-cAMP (SS + db-cAMP group). ΔRIHP showed time, group, and time*group interaction effects with a biphasic response (early: ~1 mmHg; late: ~4 mmHg) in the SS group that was absent in the SS + db-cAMP group. S3: Two groups of rats (SS and SS + db-cAMP) under hormonal clamp were contrasted as in S2, producing similar ΔRIHP results to those of S2 but showing a slow, progressive, and indistinct decrease in renal outer medullary blood flow in both groups. These results provide highly suggestive preliminary evidence that the renal interstitium is capable of contracting reactively in vivo in response to rmDIVE with SS and demonstrate that such a response is abolished when db-cAMP is interstitially and concomitantly infused.


Assuntos
Pressão Hidrostática , Medula Renal/fisiologia , Animais , Bucladesina/farmacologia , Fibroblastos/efeitos dos fármacos , Medula Renal/citologia , Medula Renal/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Cloreto de Sódio/farmacologia
12.
Pflugers Arch ; 469(7-8): 869-876, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28624952

RESUMO

This review aims to summarize the knowledge about the sensor and endocrine response functions of resident interstitial cells of the kidney. By the production of renin, erythropoietin and arachidonate metabolites (medullipin) subsets of renal interstitial fibroblasts and pericytes in different kidney zones play a central role in salt, blood pressure and oxygen homeostasis of the body. Common to these endocrine functions is that their regulation mainly occurs by (de)recruitment of active cells.


Assuntos
Medula Renal/metabolismo , Animais , Ácido Araquidônico/metabolismo , Eritropoetina/metabolismo , Fibroblastos/metabolismo , Humanos , Medula Renal/citologia , Pericitos/metabolismo , Renina/metabolismo
13.
Physiol Rep ; 5(5)2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28270594

RESUMO

Recent studies suggest that aldosterone-mediated sulfenic acid modification of the endothelin B receptor (ETB) promotes renal injury in an ischemia/reperfusion model through reduced ETB-stimulated nitric oxide production. Similarly, aldosterone inactivation of ETB signaling promotes pulmonary artery hypertension. Consequently, we asked whether aldosterone inhibits collecting duct ETB signaling; this could promote fluid retention since CD ETB exerts natriuretic and diuretic effects. A mouse inner medullary collecting duct cell line (IMCD3) was treated with aldosterone for 48 h followed by sarafotoxin-6c, an ETB-selective agonist, and extracellular signal-related kinase 1/2 (ERK) phosphorylation assessed. S6c increased the phospho/total-ERK ratio similarly in control and aldosterone-treated cells (aldosterone alone increased phospho/total-ERK). Since cultured IMCD cell lines lack ETB inhibited AVP signaling, the effect of S6c on AVP-stimulated cAMP in acutely isolated IMCD was assessed. Rats (have much higher CD ETB expression than mice) were exposed to 3 days of a normal or low Na+ diet, or low Na+ diet + desoxycorticosterone acetate. S6c inhibited AVP-stimulated cAMP in rat IMCD by the same degree in the high mineralocorticoid groups compared to controls. Finally, S6c-stimulated cGMP accumulation in cultured IMCD, or S6c-stimulated nitric oxide or cGMP in acutely isolated IMCD, was not affected by prior aldosterone exposure. These findings provide evidence that aldosterone does not modify ETB effects on ERK phosphorylation, AVP-dependent cAMP inhibition, or NO/cGMP accumulation in the IMCD Thus, while aldosterone can inhibit endothelial cell ETB activity to promote hypertension and injury, this response does not appear to occur in the IMCD.


Assuntos
Aldosterona/farmacologia , Medula Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor de Endotelina B/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Medula Renal/citologia , Medula Renal/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Fosforilação/efeitos dos fármacos , Venenos de Víboras/farmacologia
14.
Cell Physiol Biochem ; 41(1): 124-136, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28214900

RESUMO

BACKGROUND/AIMS: Recent studies have indicated that microRNA-21 (miR-21) is involved in the inflammatory response in relation to renal disease. Sirtuin1 (SIRT1) exerts renoprotective properties by counteracting inflammation. The activation of CD40 triggers inflammation that participates in renal inflammation and injury. The relationship between miR-21, SIRT1 and CD40, however, remains elusive. METHODS: Immunohistochemistry, small-interfering RNA (siRNA) transfection, quantitative real-time PCR and western blotting were applied to assess the morphological, functional and molecular mechanisms in primary cultured renal inner medullary collecting duct (IMCD) cells. RESULTS: TNF-α induced miR-21, CD40 and acetylated-NF-κBp65 (Ac-p65) expressions and reduced SIRT1 expression in IMCD cells. miR-21 mimics increased SIRT1 expression and attenuated Ac-p65 and CD40 expressions in TNF-α-induced IMCD cells, and the corresponding changes were observed with a miR-21 inhibitor. SIRT1 overexpression or activation by SRT1720 diminished TNF-α-induced CD40 and Ac-p65 expressions, which was reversed by SIRT1 siRNA or the inhibitors Ex527 and sirtinol and augmented by pretreatment with NF-κB siRNA. Further study found that the inhibitory effect of miR-21 on Ac-p65 and CD40 expressions was impeded by pretreatment with SIRT1 siRNA. CONCLUSION: The present study indicates that miR-21 inhibits TNF-α-induced CD40 expression in IMCD cells via the SIRT1-NF-κB signalling pathway, which provides new insight in understanding the anti-inflammatory effect of miR-21.


Assuntos
Antígenos CD40/metabolismo , Medula Renal/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antagomirs/metabolismo , Antígenos CD40/genética , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Medula Renal/citologia , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Microscopia de Fluorescência , NF-kappa B/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
15.
Clin Exp Pharmacol Physiol ; 43(12): 1225-1233, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27593225

RESUMO

Vasopressin (AVP) regulates the body salt-water balance. Brattleboro rats carry an AVP gene mutation resulting in a recessive form of central diabetes insipidus, being ideal for AVP deficiency studies. Herein, we studied the water permeability of the apical and basolateral sides of outer medullary collecting duct (OMCD) principal cells in response to dDAVP (a V2 receptor agonist) administration in Wistar and Brattleboro rats. Biophysical measurements of the water permeability (Pf ) of isolated OMCD principal cells were performed with the calcein quenching method with/without dDAVP (10-8  mol/L). mRNA transcripts and protein levels of AQP2, AQP3 and AQP4 were assessed by RT-PCR and western blot respectively. dDAVP increased the apical and basolateral Pf of OMCD principal cells in Wistar rats, while in Brattleboro rats this effect was present basolaterally. Long-term dDAVP administration in both strains resulted in a significant increase in mRNA expression of all assessed AQP's while only the protein levels of AQP2 and AQP3 were significantly increased. Short-term (20 minutes) dDAVP treatment of isolated OMCD fragments resulted in significantly increased plasma membrane expression of AQP2 in Wistar rats and of AQP2 and AQP3 in Brattleboro rats. In summary, dDAVP induces different expression of AQP2, AQP3 and AQP4 in Wistar and Brattleboro rats during short- and long-term treatment. In Wistar rats dDAVP mainly increased AQP2 expression while in Brattleboro rats it increased functional water permeability mainly by AQP3 expression.


Assuntos
Medula Renal/citologia , Medula Renal/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Água/metabolismo , Animais , Aquaporinas/biossíntese , Desamino Arginina Vasopressina/farmacologia , Medula Renal/efeitos dos fármacos , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Concentração Osmolar , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Brattleboro , Ratos Wistar , Água/administração & dosagem
16.
Nephron ; 133(1): 71-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27161213

RESUMO

AIM: Glycogen synthase kinase 3 (GSK3) regulates urine concentration by mediating the vasopressin-induced aquaporin 2 expression and water permeability, although it is unknown whether GSK3 also mediates the accumulation of the urea transporter A1 (UT-A1). The aim of this study is to investigate the effect of GSK3 on UT-A1 distribution. METHODS: Mouse inner medullary collecting duct 3 cells were transfected with UT-A1-GFP construct. The stable transfected cells were cultured under hypertonic conditions, treated with GSK3 inhibitor lithium chloride, GSK3 activator, lysosome or proteasome inhibitor. The expression levels of UT-A1, GSK3, and phospho-GSK3 were analyzed using western blot. The interaction between UT-A1 and the Golgi apparatus was examined using confocal immunofluorescence microscope. The UT-A1 trafficking was examined using the biotinylation of surface membranes. RESULTS: UT-A1 dissociated away from the Golgi apparatus and translocated to the plasma membrane under hypertonic-NaCl and NaCl plus urea stimulation. This movement was accompanied by the increased phosphorylation of GSK3 and its localization on the cellular membrane. Moreover, these results were duplicated by treating the cells with the GSK3 inhibitor, and by contrast, were partially reversed by the GSK3 activator. Treating cells with a lysosome or proteasome inhibitor failed to attenuate the effects of hypertonic stimulus, indicating that the loss of UT-A1 from the Golgi was not due to degradation. CONCLUSION: Our results suggest that GSK3 may in part modulate the hypertonic-induced intracellular UT-A1 redistribution and its accumulation on the plasma membrane, which may constitute another mechanism by which GSK3 modulates urine concentration.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Linhagem Celular Transformada , Medula Renal/citologia , Túbulos Renais Coletores/citologia , Camundongos , Concentração Osmolar , Transporte Proteico
17.
PLoS One ; 11(1): e0147831, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26824839

RESUMO

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H(+)-ATPases and alternative functions in H(+)-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H(+)-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H(+)-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H(+)-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H(+)-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H(+)-ATPases. In mice treated with NH4Cl, NaHCO3, KHCO3, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H(+)-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH4Cl or NaHCO3 treated mice demonstrated always colocalization of PRR/Atp6ap2 with H(+)-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H(+)-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H(+)-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H(+)-ATPase activity in intercalated cells.


Assuntos
Córtex Renal/efeitos dos fármacos , Medula Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , ATPases Translocadoras de Prótons/genética , Receptores de Superfície Celular/genética , Renina/farmacologia , Cloreto de Amônio/farmacologia , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Aquaporina 2/genética , Aquaporina 2/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cães , Regulação da Expressão Gênica , Córtex Renal/citologia , Córtex Renal/metabolismo , Medula Renal/citologia , Medula Renal/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Células Madin Darby de Rim Canino , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , ATPases Translocadoras de Prótons/metabolismo , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais , Bicarbonato de Sódio/farmacologia , Cloreto de Sódio/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato
18.
J Am Soc Nephrol ; 27(3): 835-46, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26293821

RESUMO

The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc-/-) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc-/- mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.


Assuntos
Anti-Infecciosos/farmacologia , Infecções por Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Hepcidinas/metabolismo , Hepcidinas/farmacologia , Infecções Urinárias/metabolismo , Animais , Anti-Infecciosos/metabolismo , Carga Bacteriana/genética , Células Cultivadas , Contagem de Colônia Microbiana , Citocinas/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Hepcidinas/genética , Ferro/metabolismo , Medula Renal/citologia , Medula Renal/metabolismo , Medula Renal/microbiologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Nefrite/metabolismo , Nefrite/microbiologia , Nefrite/patologia , Neutrófilos , Fosforilação , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Infecções Urinárias/microbiologia
19.
J Biol Chem ; 290(9): 5582-91, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25533472

RESUMO

The kidney is an important organ for arterial blood pressure (BP) maintenance. Reduced NO generation in the kidney is associated with hypertension in insulin resistance. NO is a critical regulator of vascular tone; however, whether insulin regulates NO production in the renal inner medullary collecting duct (IMCD), the segment with the greatest enzymatic activity for NO production in kidney, is not clear. Using an NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescent dye, we found that insulin increased NO production in mouse IMCD cells (mIMCD) in a time- and dose-dependent manner. A concomitant dose-dependent increase in the NO metabolite (NOx) was also observed in the medium from insulin-stimulated cells. NO production peaked in mIMCD cells at a dose of 100 nm insulin with simultaneously increased NOx levels in the medium. At this dose, insulin significantly increased p-eNOS(Ser1177) levels in mIMCD cells. Pretreatment of cells with a PI 3-kinase inhibitor or insulin receptor silencing with RNA interference abolished these effects of insulin, whereas insulin-like growth factor-1 receptor (IGF-1R) silencing had no effect. We also showed that chronic insulin infusion to normal C57BL/6J mice resulted in increased endothelial NOS (eNOS) protein levels and NO production in the inner medulla. However, insulin-infused IRKO mice, with targeted deletion of insulin receptor from tubule epithelial cells of the kidney, had ∼50% reduced eNOS protein levels in their inner medulla along with a significant rise in BP relative to WT littermates. We have previously reported increased baseline BP and reduced urine NOx in IRKO mice. Thus, reduced insulin receptor signaling in IMCD could contribute to hypertension in the insulin-resistant state.


Assuntos
Insulina/farmacologia , Medula Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Óxido Nítrico/biossíntese , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Fluoresceínas/metabolismo , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Immunoblotting , Insulina/administração & dosagem , Medula Renal/citologia , Medula Renal/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Fatores de Tempo
20.
Am J Physiol Renal Physiol ; 306(2): F259-70, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24226522

RESUMO

Oxidative stress resulting from unilateral ureteral obstruction (UUO) may be aggravated by increased production of ROS. Previous studies have demonstrated increased cyclooxygenase (COX)-2 expression in renal medullary interstitial cells (RMICs) in response to UUO. We investigated, both in vivo and in vitro, the role of ROS in the induction of COX-2 in rats subjected to UUO and in RMICs exposed to oxidative and mechanical stress. Rats subjected to 3-day UUO were treated with two mechanistically distinct antioxidants, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) and the complex I inhibitor rotenone (ROT), to interfere with ROS production. We found that UUO-mediated induction of COX-2 in the inner medulla was attenuated by both antioxidants. In addition, DPI and ROT reduced tubular damage and oxidative stress after UUO. Moreover, mechanical stretch induced COX-2 and oxidative stress in RMICs. Likewise, RMICs exposed to H2O2 as an inducer of oxidative stress showed increased COX-2 expression and activity, both of which were reduced by DPI and ROT. Similarly, ROS production, which was increased after exposure of RMICs to H2O2, was also reduced by DPI and ROT. Furthermore, oxidative stress-induced phosphorylation of ERK1/2 and p38 was blocked by both antioxidants, and inhibition of ERK1/2 and p38 attenuated the induction of COX-2 in RMICs. Notably, COX-2 inhibitors further exacerbated the oxidative stress level in H2O2-exposed RMICs. We conclude that oxidative stress as a consequence of UUO stimulates COX-2 expression through the activation of multiple MAPKs and that the induction of COX-2 may exert a cytoprotective function in RMICs.


Assuntos
Ciclo-Oxigenase 2/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Obstrução Ureteral/enzimologia , Animais , Antioxidantes/farmacologia , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Dinoprostona/metabolismo , Eletroforese em Gel de Poliacrilamida , Indução Enzimática/fisiologia , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Medula Renal/citologia , Medula Renal/metabolismo , Masculino , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Inclusão em Parafina , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Estresse Mecânico
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