RESUMO
Nuclear factor of activated T cells 5 (NFAT5; also called TonEBP/OREBP) is a transcription factor that is activated by hypertonicity and induces osmoprotective genes to protect cells against hypertonic conditions. In the kidney, renal tubular NFAT5 is known to be involved in the urine concentration mechanism. Previous studies have suggested that NFAT5 modulates the immune system and exerts various effects on organ damage, depending on organ and disease states. Pathophysiological roles of NFAT5 in renal tubular cells, however, still remain obscure. We conducted comprehensive analysis by performing transcription start site (TSS) sequencing on the kidney of inducible and renal tubular cell-specific NFAT5 knockout (KO) mice. Mice were subjected to unilateral ureteral obstruction to examine the relevance of renal tubular NFAT5 in renal fibrosis. TSS sequencing analysis identified 722 downregulated TSSs and 1,360 upregulated TSSs, which were differentially regulated ≤-1.0 and ≥1.0 in log2 fold, respectively. Those TSSs were annotated to 532 downregulated genes and 944 upregulated genes, respectively. Motif analysis showed that sequences that possibly bind to NFAT5 were enriched in TSSs of downregulated genes. Gene Ontology analysis with the upregulated genes suggested disorder of innate and adaptive immune systems in the kidney. Unilateral ureteral obstruction significantly exacerbated renal fibrosis in the renal medulla in KO mice compared with wild-type mice, accompanied by enhanced activation of immune responses. In conclusion, NFAT5 in renal tubules could have pathophysiological roles in renal fibrosis through modulating innate and adaptive immune systems in the kidney.NEW & NOTEWORTHY TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.
Assuntos
Obstrução Ureteral , Animais , Camundongos , Fibrose , Expressão Gênica , Rim , Camundongos KnockoutRESUMO
Many large-scale studies show that exogenous erythropoietin, erythropoiesis-stimulating agents, lack any renoprotective effects. We investigated the effects of endogenous erythropoietin on renal function in kidney ischemic reperfusion injury (IRI) using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat (ROX). Four h of hypoxia (7% O2) and 4 h treatment by ROX prior to IRI did not improve renal function. In contrast, 24-72 h pretreatment by ROX significantly improved the decline of renal function caused by IRI. Hypoxia and 4 h ROX increased interstitial cells-derived Epo production by 75- and 6-fold, respectively, before IRI, and worked similarly to exogenous Epo. ROX treatment for 24-72 h increased Epo production during IRI by 9-fold. Immunohistochemistry revealed that 24 h ROX treatment induced Epo production in proximal and distal tubules and worked similarly to endogenous Epo. Our data show that tubular endogenous Epo production induced by 24-72 h ROX treatment results in renoprotection but peritubular exogenous Epo production by interstitial cells induced by hypoxia and 4 h ROX treatment did not. Stimulation of tubular, but not peritubular, Epo production may link to renoprotection.
Assuntos
Eritropoetina , Inibidores de Prolil-Hidrolase , Traumatismo por Reperfusão , Humanos , Eritropoetina/farmacologia , Rim , Epoetina alfa/farmacologia , Inibidores de Prolil-Hidrolase/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , HipóxiaRESUMO
Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin ß pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin ß pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.
Assuntos
Líquidos Corporais , Eritropoetina , Reprodutibilidade dos Testes , Focalização Isoelétrica/métodos , Western Blotting , Anticorpos , Eletroforese em Gel de Poliacrilamida , Detecção do Abuso de Substâncias/métodos , Proteínas RecombinantesRESUMO
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
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
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
BACKGROUND: The localization and role of the calcium-sensing receptor (CaSR) along the nephron including the collecting ducts is still open to debate. METHODS: Using the quantitative, highly sensitive in situ hybridization technique and a double-staining immunohistochemistry technique, we investigated the axial distribution and expression of CaSR along the nephron in mice (C57B/6J) treated for 6 days with acid or alkali diets. RESULTS: Under control condition, CaSR was specifically localized in the cortical and medullary thick ascending limb of Henle's loop (CTAL and MTAL), macula densa (MD), distal convoluted tubule (DCT), and CCD (TALs, MD > DCT, CCD). Along the CCD, CaSR was co-localized with an anion exchanger type 4 (AE4), a marker of the basolateral membrane of type-B intercalated cell (IC-B) in mice. On the contrary, CaSR was not detected either in principal cells (PC) or in type-A intercalated cell (IC-A). CaSR expression levels in IC-B significantly (P < 0.005) decreased when mice were fed NH4Cl (acid) diets and increased when animals were given NaHCO3 (alkali) diets. As expected, cell heights of IC-A and IC-B significantly (P < 0.005) increased in the above experimental conditions. Surprisingly, single infusion (ip) of neomycin, an agonist of CaSR, significantly (P < 0.005) increased urinary Ca excretion without further increasing the hourly urine volume and significantly (P < 0.05) decreased urine pH. CONCLUSION: CaSR, cloned from rat kidney, was localized in the basolateral membrane of IC-B and was more expressed during alkali-loading. Its alkali-sensitive expression may promote urinary alkali secretion for body acid-base balance.
Assuntos
Túbulos Renais Coletores/metabolismo , Rim/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Animais , Cálcio/urina , Tamanho Celular , Diuréticos/farmacologia , Concentração de Íons de Hidrogênio , Hibridização In Situ , Rim/citologia , Túbulos Renais Coletores/citologia , Camundongos , Camundongos Endogâmicos C57BL , Néfrons/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Detecção de Cálcio , Receptores Acoplados a Proteínas G/genéticaRESUMO
Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in ß-intercalated or non α/non ß-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.
Assuntos
Eritropoetina/biossíntese , Néfrons/metabolismo , Animais , Hipóxia Celular , Linhagem Celular , Eritropoetina/genética , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Rim/citologia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b.
Assuntos
Desidratação/metabolismo , Rim/metabolismo , Isoformas de Proteínas/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Animais , Sequência de Bases , Bradicinina/farmacologia , Primers do DNA , Expressão Gênica/efeitos dos fármacos , Masculino , Reação em Cadeia da Polimerase , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Membro 1 da Família 12 de Carreador de Soluto/genéticaRESUMO
BACKGROUND: Vasopressin V1a receptor null (V1aR(-/-)) mice recently showed incomplete urinary concentration due to higher urine volume during control and water diuresis (euhydration), but showed normal response during dehydration (Aoyagi et al., Am J Physiol 295: F100-7, 2008). METHODS: Water balance, plasma vasopressin, plasma and urine osmolality, and aquaporin 2 (AQP2) expression in the kidney of wild-type (WT) and V1aR(-/-) mice were therefore further examined using improved methods of urine collection (urinary bladder urine). RESULTS: V1aR(-/-) mice demonstrated a lower urine osmolality (3,360 ± 138 vs. 3,610 ± 47 mOsm/kgH2O) and a higher plasma osmolality (354.3 ± 1.3 vs. 342.5 ± 1.5 mOsm/kgH2O) after dehydration for 24 h compared to WT mice (P < 0.05). In contrast, the plasma vasopressin concentration was significantly (P < 0.001) higher in the V1aR(-/-) mice (48.8 ± 4.8 vs. 22.1 ± 2.4 pg/ml). On the other hand, although the AQP2 protein expression in the kidney was increased after dehydration, the basal (control) and dehydration-induced AQP2 protein levels were significantly lower in V1aR(-/-) mice compared to WT mice (by Western blotting). Staining by an anti-AQP2 antibody in the luminal membrane of the collecting ducts was increased in both V1aR(-/-) and WT mice after dehydration, but was relatively weaker in the V1aR(-/-) mice (by immunohistochemistry). Moreover, urinary excretion of AQP2 protein, an index of the luminal AQP2 expression, was significantly (P < 0.05) lower in the V1aR(-/-) mice. CONCLUSION: V1aR signaling may be fundamentally important for the expression of AQP2 in the collecting ducts during control conditions and dehydration.
Assuntos
Aquaporina 2/biossíntese , Túbulos Renais Coletores/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/fisiologia , Animais , Análise Química do Sangue , Western Blotting , Desidratação/genética , Desidratação/metabolismo , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas/isolamento & purificação , Proteínas/metabolismo , Frações Subcelulares/metabolismo , UrináliseRESUMO
BACKGROUND: Vasopressin V1a receptor (V1aR) null mice have insufficient acid-base balance, but the target cell for V1aR signaling which results in the urinary acidification has not been identified. METHODS: By using a quantitative in situ hybridization technique and a double-staining technique with an anti-AQP3 antibody in mice, we investigated the axial distribution and acidosis-induced expression of V1aR mRNA along the nephron. We also investigated the acidosis-induced morphological change in the tubule cells from wild-type and V1aR-null (V1aR(-/-)) mice. RESULTS: In the normal condition, V1aR mRNA was moderately expressed in the medullary thick ascending limb (MTAL) and highly expressed in the intercalated cell (IC) throughout the collecting duct (CD). However, no expression was observed in the proximal tubule, thin limbs of Henle's loop, and the principal cell of the CD. Importantly, V1aR mRNA was upregulated significantly both in the TAL and the IC of the CD in the inner stripe of the outer medulla (MTALis and IC of OMCDis, respectively) when mice were treated with NH4Cl (0.28 mol/L) for 6 days. Acidosis-induced hypertrophy, which was completely attenuated in V1aR(-/-) mice, was observed only in the IC of OMCDis (P < 0.005). In addition, urinary excretion of ammonia (NH3/NH4 (+)) was significantly decreased on day 3 (P < 0.05) and day 6 (P < 0.005) in the V1aR(-/-) mice treated with NH4Cl. CONCLUSION: In conclusion, the IC of OMCDis may be the target cell stimulated by the vasopressin V1aR axis and contribute to urinary acidification, at least during metabolic acidosis.
Assuntos
Túbulos Renais Coletores/citologia , Receptores de Vasopressinas/fisiologia , Acidose/fisiopatologia , Acidose/urina , Amônia/urina , Cloreto de Amônio/farmacologia , Animais , Células Epiteliais/metabolismo , Concentração de Íons de Hidrogênio , Hibridização In Situ , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores de Vasopressinas/biossíntese , Receptores de Vasopressinas/genéticaRESUMO
In gastrointestinal (GI) physiology, anion and fluid secretion is an important function for host defense and is induced by changes in the luminal environment. The transient receptor potential A1 (TRPA1) channel is considered to be a chemosensor in several sensory tissues. Although the function of TRPA1 has been studied in GI motility, its contribution to the transepithelial ion transport system has rarely been discussed. In the present study, we investigated the secretory effect of the potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colon using an Ussing chamber. The mucosal application of AITC (10(-6)-10(-3) M) induced Cl(-) and HCO(3)(-) secretion in a concentration-dependent manner, whereas the serosal application induced a significantly weaker effect. AITC-evoked anion secretion was attenuated by tissue pretreatment with piroxicam and prostaglandin (PG) E(2); however, this secretion was not affected by TTX, atropine, or extracellular Ca(2+) depletion. These experiments indicate that TRPA1 activation induces anion secretion through PG synthesis, independent of neural pathways in the colon. Further analysis also indicates that AITC-evoked anion secretion is mediated mainly by the EP(4) receptor subtype. The magnitude of the secretory response exhibited segmental heterogeneity in rat colon. Real-time PCR analysis showed the segmental difference was corresponding to the differential expression of EP(4) receptor and cyclooxygenase-1 and -2. In addition, RT-PCR, in situ hybridization, and immunohistochemical studies showed TRPA1 expression in the colonic epithelia. Therefore, we conclude that the activation of TRPA1 in colonic epithelial cells is likely involved in the host defense mechanism through rapid anion secretion.
Assuntos
Canais de Cálcio/metabolismo , Colo/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Idoso , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Canais de Cálcio/genética , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Piroxicam/farmacologia , Transporte Proteico , Ratos , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP4/genética , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , Canais de Potencial de Receptor Transitório/genéticaRESUMO
BACKGROUND: The calcium (Ca)-activated potassium (K) channel is an alternative K-secretory pathway in the apical membranes of the distal nephrons of adrenalectomized (ADX) animals. As a potential approach for estimating intracellular Ca(2+) increase, we investigated normal and ADX mice to determine whether dietary K intake would stimulate the expression of the calbindin D28k protein, a cytosolic Ca(2+)-binding protein, along the distal nephron consisting of the early and late portions of the distal convoluted tubule (DCT1 and DCT2, respectively), the CNT, and CCD. METHODS: ADX mice received a control diet plus either 0.3% NaCl solution (C) or a 0.3% NaCl plus 3% KCl solution (HK) for 7 days before the experiment. RESULTS: The mean plasma K concentration and pH were significantly (P < 0.001) higher (7.9 ± 0.3 mEq/l) and lower (7.28 ± 0.02) in the K-loaded ADX mice than in the control ADX mice. The mean urinary K excretion (mEq/day) and urine flow (ml/day) increased significantly (P < 0.0001) from 0.47 ± 0.07 (C) to 4.80 ± 0.57 (HK) and from 1.1 ± 0.2 (C) to 8.8 ± 1.0 (HK). Urinary Ca excretion significantly (P < 0.005 and P < 0.05, respectively) increased in K-loaded normal and ADX mice compared with control normal and ADX mice. Immunofluorescence studies revealed that the relative staining of calbindin was 167.0 ± 15.4%, 291.3 ± 13.8%, and 206.3 ± 11.3% for DCT1, DCT2/CNT, and CCD of normal control mice, respectively. These values increased significantly (P < 0.0001) only in DCT2/CNT (574.8 ± 42%) of the K-loaded ADX mice. CONCLUSION: Upregulation of calbindin in the late distal tubule suggests that Ca(2+)-dependent K transport may function as an alternative mechanism for urinary K excretion in ADX mice.
Assuntos
Glândulas Suprarrenais/fisiologia , Túbulos Renais Distais/fisiologia , Potássio/farmacologia , Proteína G de Ligação ao Cálcio S100/biossíntese , Adrenalectomia , Aldosterona/sangue , Animais , Calbindina 1 , Calbindinas , Cálcio/urina , Eletrólitos/sangue , Eletrólitos/urina , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Masculino , Camundongos , Potássio/metabolismo , Potássio/urina , Regulação para Cima , Vasopressinas/urinaRESUMO
Rhesus C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14 M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spironolactone and GÓ§6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level.
Assuntos
Aldosterona/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/metabolismo , Glicoproteínas de Membrana/metabolismo , Equilíbrio Ácido-Base , Aldosterona/administração & dosagem , Cloreto de Amônio/administração & dosagem , Compostos de Amônio/urina , Animais , Proteínas de Transporte de Cátions/genética , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Infusões Subcutâneas , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Potássio/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The pronuclear injection (PI)-based targeted transgenesis (PITT) method allows the generation of targeted transgenic (Tg) mice wherein a single copy of a transgene is integrated into the Rosa26 locus following PI. The Rosa26 locus allows unbiased ubiquitous expression of integrated transgenes; however, it remains little known whether tissue-specific promoters retain their functional properties when placed at the Rosa26 locus. We evaluated tissue-specific activity and reproducibility of exogenous tissue-specific promoters targeted to the Rosa26 locus by generating Thy1-Dre/Dre reporter mice using PITT and assessed spatial expression patterns of the transgenes. The Thy1 promoter targeted to the Rosa26 locus appeared active in virtually all Purkinje cells in the cerebellum and hippocampus. However, mosaic expression of the transgene under the Thy1 promoter was observed in many other organs. This phenomenon was consistent in all the Tg lines generated by PITT, indicating a high degree of reproducibility for this experiment.
Assuntos
Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , RNA não Traduzido/genética , Animais , Encéfalo/metabolismo , Genótipo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , RNA não Traduzido/metabolismo , Transgenes/genéticaRESUMO
Doping tests for the illegal use of erythropoiesis-stimulating agents (ESAs) have been developed. We developed a new Western blotting method to detect and distinguish endogenous erythropoietin (Epo, 35-38 kDa) and exogenous ESAs (epoetin α and ß, 38-42 kDa; darbepoetin α, 47-50 kDa; epoetin ß pegol, 93-110 kDa). Epo and ESAs are glycoproteins and deglycosylation using peptide-N-glycosidase F shifted all Epo and ESA bands except epoetin ß pegol to 22 kDa. We cut the bands of Epo and ESAs from SDS-PAGE gels and analyzed them by Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS detected all endogenous Epo and exogenous ESAs as deglycosylated 22 kDa Epo, indicating that LC/MS analysis could confirm the presence of Epo or ESA, but could not distinguish between endogenous Epo and exogenous ESAs. We propose the following Epo doping tests: 1) detect Epo or ESAs by Western blotting of the glycosylated form; 2) increase the reliability by the band shift following deglycosylation; and 3) complete confirmation of Epo or ESA by LC/MS analysis using cut gels. One of the advantages of our method is that pre-purification of samples for Epo is not required in our Western blotting.
RESUMO
The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin ß pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.
Assuntos
Eritropoetina/biossíntese , Hipóxia/metabolismo , Rim/metabolismo , Fígado/metabolismo , Anemia/sangue , Anemia/urina , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Eritropoetina/sangue , Eritropoetina/urina , Glicosilação , Humanos , Hipóxia/sangue , Hipóxia/urina , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
ATP-sensitive K+ (K(ATP)) channels in the kidney are considered to play roles in regulating membrane potential according to changes in the intracellular ATP concentration. They are composed of two types of subunits; the pore subunits (Kir6.1, Kir6.2), which are members of the inwardly rectifying K+ channel family, and the regulatory subunits, the sulphonylurea receptors, which belong to the ATP-binding cassette (ABC) superfamily. The sulphonylurea receptors (SURs) are receptors of sulphonylureas widely used for the treatment of type 2 diabetes mellitus. The SURs are divided into two isoforms, SUR1 and SUR2, the latter was further divided into SUR2A and SUR2B. In the present study, we have investigated the mRNA expression by RT-PCR assay, and protein expression profiles by immunoblotting, immunohistochemistry, and immunoelectron microscopy with anti SUR2A and anti SUR2B antibodies. RT-PCR detected the presence of mRNA transcripts of the SUR2A and SUR2B, while SUR1 mRNA was barely detected. In immunoblotting, SUR2A protein was detected distinctly in the microsomal fraction, weakly in the mitochondrial fraction and at negligible level in the cell membrane fraction. In contrast, the SUR2B protein was detected intensely in the microsomal fraction, with a low level in the mitochondrial fraction and scarcely in the cell membrane fraction. In immunohistochemistry SUR2A and SUR2B proteins were widely distributed in renal tubular epithelial cells, glomerular mesangial cells, and the endothelium and the smooth muscle of blood vessels. In immunoelectron microscopy, the immunoreactivity was localized in the endoplasmic reticulum and mitochondria throughout the epithelial cells for SUR2A, and dominantly in the apical cytoplasm of the cells for SUR2B. In conclusion, the regulatory subunits of the K(ATP) channel in the rat kidney are SUR2A and SUR2B; they also are candidate regulatory subunits for the mitochondrial K(ATP) channel.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Túbulos Renais/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Western Blotting , Epitélio/metabolismo , Epitélio/ultraestrutura , Regulação da Expressão Gênica , Imuno-Histoquímica , Túbulos Renais/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Canais de Potássio/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores de Droga/genética , Receptores de SulfonilureiasRESUMO
Metabolic acidosis often results from chronic kidney disease; in turn, metabolic acidosis accelerates the progression of kidney injury. The mechanisms for how acidosis facilitates kidney injury are not fully understood. To investigate whether low pH directly affects the expression of genes controlling local homeostasis in renal tubules, we performed transcription start site sequencing (TSS-Seq) using IN-IC cells, a cell line derived from rat renal collecting duct intercalated cells, with acid loading for 24 h. Peak calling identified 651 up-regulated and 128 down-regulated TSSs at pH 7.0 compared with those at pH 7.4. Among them, 424 and 38 TSSs were ≥ 1.0 and ≤ -1.0 in Log2 fold change, which were annotated to 193 up-regulated and 34 down-regulated genes, respectively. We used gene ontology analysis and manual curation to profile the up-regulated genes. The analysis revealed that many up-regulated genes are involved in renal fibrosis, implying potential molecular mechanisms induced by metabolic acidosis. To verify the activity of the ubiquitin-proteasome system (UPS), a candidate pathway activated by acidosis, we examined the expression of proteins from cells treated with a proteasome inhibitor, MG132. The expression of ubiquitinated proteins was greater at pH 7.0 than at pH 7.4, suggesting that low pH activates the UPS. The in vivo study demonstrated that acid loading increased the expression of ubiquitin proteins in the collecting duct cells in mouse kidneys. Motif analysis revealed Egr1, the mRNA expression of which was increased at low pH, as a candidate factor that possibly stimulates gene expression in response to low pH. In conclusion, metabolic acidosis can facilitate renal injury and fibrosis during kidney disease by locally activating various pathways in the renal tubules.
Assuntos
Acidose/genética , Injúria Renal Aguda/genética , Insuficiência Renal Crônica/genética , Sítio de Iniciação de Transcrição , Acidose/complicações , Acidose/patologia , Injúria Renal Aguda/complicações , Injúria Renal Aguda/patologia , Animais , Fibrose/genética , Fibrose/patologia , Regulação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Rim/metabolismo , Rim/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Leupeptinas/administração & dosagem , Camundongos , Ratos , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/patologia , Transdução de Sinais/genéticaRESUMO
Background and Aim: To examine the effects of intratesticular transplantation on the growth and maturation of young murine ovaries. Methods: Two-week-old ovaries from transgenic mice with enhanced green fluorescent protein expression were transplanted under the testicular capsule of 4-week-old non-transgenic mice. Results: Two months after transplantation all successfully grafted ovaries had survived, based on the presence of bright green fluorescence. The grafts showed various stages of folliculogenesis, including expanded follicles. The neighboring seminiferous tubules had a normal structure and mature sperm in their lumens, indicating active spermatogenesis, and all the recipient males were fertile. There was no evidence of extensive cell migration from the grafted ovaries into the testis. Similar findings were obtained for the grafted ovaries 6 months after surgery, although cell death (as evidenced by yellowish or pale fluorescence) was more frequent. Conclusion: Young murine ovaries can grow and mature autonomously for at least 6 months unaffected by the male hormonal environment. (Reprod Med Biol 2006; 5: 71-77).