RESUMEN
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.
Asunto(s)
Eritropoyetina , Inhibidores de Prolil-Hidroxilasa , Daño por Reperfusión , Humanos , Eritropoyetina/farmacología , Riñón , Epoetina alfa/farmacología , Inhibidores de Prolil-Hidroxilasa/farmacología , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/prevención & control , HipoxiaRESUMEN
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.
Asunto(s)
Líquidos Corporales , Eritropoyetina , Reproducibilidad de los Resultados , Focalización Isoeléctrica/métodos , Western Blotting , Anticuerpos , Electroforesis en Gel de Poliacrilamida , Detección de Abuso de Sustancias/métodos , Proteínas RecombinantesRESUMEN
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.
Asunto(s)
Eritropoyetina/metabolismo , Glicina/análogos & derivados , Isoquinolinas/farmacología , Inhibidores de Prolil-Hidroxilasa/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Animales , Eritropoyetina/análisis , Eritropoyetina/genética , Femenino , Glicina/farmacología , Hipoxia/genética , Hipoxia/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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).
Asunto(s)
Angiotensina II/farmacología , Eritropoyetina/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Animales , Western Blotting , Humanos , Riñón/efectos de los fármacos , Hígado/efectos de los fármacosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Eritropoyetina/biosíntesis , Hipoxia/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Anemia/sangre , Anemia/orina , Animales , Western Blotting/métodos , Modelos Animales de Enfermedad , Eritropoyetina/sangre , Eritropoyetina/orina , Glicosilación , Humanos , Hipoxia/sangre , Hipoxia/orina , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. METHODS: We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H+ secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. RESULTS: Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H+-ATPase in A-ICs. CONCLUSIONS: Our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.
Asunto(s)
Equilibrio Ácido-Base/efectos de los fármacos , Receptores de Vasopresinas/efectos de los fármacos , Vasopresinas/farmacología , Equilibrio Ácido-Base/genética , Animales , Células Cultivadas/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Inmunohistoquímica , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratas Brattleboro , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de Vasopresinas/genética , Sensibilidad y Especificidad , Urinálisis/métodosRESUMEN
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.
Asunto(s)
Aldosterona/metabolismo , Eritropoyetina/metabolismo , Fludrocortisona/metabolismo , Túbulos Renales Colectores/metabolismo , Nefronas/metabolismo , Animales , Hipoxia de la Célula , Eritropoyetina/genética , Glicosilación , Túbulos Renales Colectores/citología , Masculino , Nefronas/citología , ARN Mensajero/genética , Ratas Sprague-Dawley , Sistema Renina-Angiotensina , Regulación hacia ArribaRESUMEN
We have previously demonstrated that the nestin-expressing cells from the upper part of the hair follicle can differentiate to neurons and other cell types. We have termed these cells as hair-associated-pluripotent (HAP) stem cells. In the present chapter, we describe methods for HAP stem cells to differentiate to beating cardiac muscle cells. The mouse vibrissa hair follicle was divided into three parts (upper, middle, and lower), and each part was suspended separately in DMEM containing 10 % fetal bovine serum (FBS). All three parts of hair follicle differentiate to neurons, glial cells, keratinocytes, smooth muscle cells, and cardiac muscle cells. The differentiation potential to cardiac muscle is greatest in the upper part of the follicle. Hair spheres comprised of HAP stem cells formed from the upper part of vibrissa hair follicle can differentiate to cardiac muscle cells.
Asunto(s)
Diferenciación Celular , Folículo Piloso/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Células Madre Pluripotentes/citología , Animales , Biomarcadores , Técnica del Anticuerpo Fluorescente , Inmunofenotipificación , Ratones , Células Madre Pluripotentes/metabolismo , VibrisasRESUMEN
Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair-follicle-associated pluripotent (HAP) stem cells. Cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells are described in this chapter. Intact hair follicles from green fluorescent protein (GFP) transgenic mice were cryopreserved by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in DMEM with fetal bovine serum (FBS). After 4 weeks culture, cells from the upper part of the hair follicles grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week culture, the growing cells formed hair spheres, each containing approximately 1 × 10(2) HAP stem cells. The hair spheres contained cells which could differentiate to neurons, glial cells, and other cell types. The formation of hair spheres by the thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. These results suggest that the cryopreservation of the whole hair follicle is an effective way to store HAP stem cells for personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use.
Asunto(s)
Criopreservación , Expresión Génica , Folículo Piloso/citología , Nestina/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Autorrenovación de las Células , Células Cultivadas , Criopreservación/métodos , Genes Reporteros , Inmunohistoquímica , Ratones Transgénicos , Nestina/metabolismo , VibrisasRESUMEN
We have previously discovered nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells and have shown that they can differentiate to neurons, glia, and many other cell types. HAP stem cells can be used for nerve and spinal cord repair. We have recently shown the HAP stem cells can differentiate to beating heart-muscle cells and tissue sheets of beating heart-muscle cells. In the present study, we determined the efficiency of HAP stem cells from mouse vibrissa hair follicles of various ages to differentiate to beating heart-muscle cells. We observed that the whiskers located near the ear were more efficient to differentiate to cardiac-muscle cells compared to whiskers located near the nose. Differentiation to cardiac-muscle cells from HAP stem cells in cultured whiskers in 4-week-old mice was significantly greater than in 10-, 20-, and 40-week-old mice. There was a strong decrease in differentiation potential of HAP stem cells to cardiac-muscle cells by 10 weeks of age. In contrast, the differentiation potential of HAP stem cells to other cell types did not decrease with age. The possibility of rejuvenation of HAP stem cells to differentiate at high efficiency to cardiac-muscle cells is discussed.
Asunto(s)
Envejecimiento/fisiología , Diferenciación Celular , Folículo Piloso/citología , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Animales , Biomarcadores/metabolismo , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vibrisas/citologíaRESUMEN
Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of the follicle. Previous studies have shown that HAP stem cells can differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. HAP stem cells effected nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. The differentiation potential to cardiac muscle cells was greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol. In the present study, we observed that isoproterenol directs HAP stem cells to differentiate to cardiac muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. These results demonstrate that HAP stem cells have great potential to form beating cardiac muscle cells in tissue sheets.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Isoproterenol/farmacología , Células Madre Pluripotentes/fisiología , Activinas/fisiología , Animales , Proteína Morfogenética Ósea 4/fisiología , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/fisiología , Folículo Piloso/citología , Ratones Endogámicos C57BL , Ratones Transgénicos , Contracción Miocárdica , Miocardio/citología , Miocitos Cardíacos/fisiología , Células Madre Pluripotentes/efectos de los fármacosRESUMEN
We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. In the present study, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We separated the mouse vibrissa hair follicle into 3 parts (upper, middle, and lower), and suspended each part separately in DMEM containing 10% FBS. All three parts of hair follicle differentiated to beating cardiac muscle cells as well as neurons, glial cells, keratinocytes and smooth muscle cells. The differentiation potential to cardiac muscle is greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol and inhibited by propanolol. HAP stem cells have potential for regenerative medicine for heart disease as well as nerve and spinal cord repair.
Asunto(s)
Folículo Piloso/citología , Miocitos Cardíacos/metabolismo , Nestina/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Antígenos CD34/metabolismo , Diferenciación Celular/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Queratina-15/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos Animales , Miocitos Cardíacos/citología , Neuronas/fisiología , Células Madre Pluripotentes/citología , Regeneración , Regeneración de la Medula Espinal/fisiologíaRESUMEN
Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. In the present study, we established efficient cryopreservation methods of the hair follicle that maintained the pluripotency of HAP stem cells. We cryopreserved the whole hair follicle from green fluorescent protein transgenic mice by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) with fetal bovine serum (FBS). After 4 weeks of culture, cells from the upper part of the hair follicle grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week of culture, the growing cells formed hair spheres, each containing â¼1×10(2) HAP stem cells. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. In contrast, rapid-cooling (vitrification) cryopreservation poorly preserved the pluripotency of the hair follicle stem cells. Stem cell marker genes (nestin, Sox2, and SSEA-1) were as highly expressed in slow-rate cooled cryopreserved follicles, after thawing, as in fresh follicles. However, in the vitrification cryopreserved follicles, the expression of the stem cell marker genes was greatly reduced. Direct cryopreservation of hair spheres by either the rapid-cooling, or slow-cooling method, resulted in loss of pluripotency. These results suggest that the slow-rate cooling cryopreservation of the whole hair follicle is effective to store HAP stem cells. Stored HAP stem cells would be very useful in personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Criopreservación , Folículo Piloso , Nestina/biosíntesis , Células Madre Pluripotentes , Animales , Bovinos , Folículo Piloso/citología , Folículo Piloso/metabolismo , Ratones , Ratones Transgénicos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
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.
Asunto(s)
Túbulos Renales Colectores/metabolismo , Riñón/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Animales , Calcio/orina , Tamaño de la Célula , Diuréticos/farmacología , Concentración de Iones de Hidrógeno , Hibridación in Situ , Riñón/citología , Túbulos Renales Colectores/citología , Ratones , Ratones Endogámicos C57BL , Nefronas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Sensibles al Calcio , Receptores Acoplados a Proteínas G/genéticaRESUMEN
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.
Asunto(s)
Deshidratación/metabolismo , Riñón/metabolismo , Isoformas de Proteínas/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , Secuencia de Bases , Bradiquinina/farmacología , Cartilla de ADN , Expresión Génica/efectos de los fármacos , Masculino , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Miembro 1 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
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.
Asunto(s)
Eritropoyetina/biosíntesis , Nefronas/metabolismo , Animales , Hipoxia de la Célula , Línea Celular , Eritropoyetina/genética , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Inmunohistoquímica , Hibridación in Situ , Riñón/citología , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The Kir4.1/Kir5.1 channel mediates basolateral K(+) recycling in renal distal tubules; this process is critical for Na(+) reabsorption at the tubules. Mutations in Kir4.1 are associated with EAST/SeSAME syndrome, a genetic disorder characterized by renal salt wasting. In this study, we found that MAGI-1 anchors Kir4.1 channels (Kir4.1 homomer and Kir4.1/Kir5.1 heteromer) and contributes to basolateral K(+) recycling. The Kir4.1 A167V mutation associated with EAST/SeSAME syndrome caused mistrafficking of the mutant channels and inhibited their expression on the basolateral surface of tubular cells. These findings suggest mislocalization of the Kir4.1 channels contributes to renal salt wasting.
Asunto(s)
Pérdida Auditiva Sensorineural/metabolismo , Discapacidad Intelectual/metabolismo , Túbulos Renales Distales/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Convulsiones/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Moléculas de Adhesión Celular , Moléculas de Adhesión Celular Neuronal/metabolismo , Polaridad Celular , Perros , Guanilato-Quinasas , Células HEK293 , Pérdida Auditiva Sensorineural/genética , Humanos , Discapacidad Intelectual/genética , Células de Riñón Canino Madin Darby , Mutación Missense , Canales de Potasio de Rectificación Interna/genética , Transporte de Proteínas , Convulsiones/genéticaRESUMEN
OBJECTIVE: This study was conducted to evaluate the relationship between hearing and cochlear histopathology after arginine vasopressin administration in rats. METHODS: A total of 30 Wistar rats were injected with either 0.02 unit/g of arginine vasopressin or the same amount of isotonic saline solution. The initial auditory brain stem response threshold was recorded and additional measurements were made at 10, 30, 60, and 90 min after injection of arginine vasopressin or isotonic saline solution. The threshold for each timepoint was compared with the initial threshold. Histological quantitative assessment of endolymphatic hydrops in the cochlea was performed using light microscopy and assessment of the basal, intermediate, and marginal cells of the stria vascularis was performed with electron microscopy. RESULTS: The auditory brain stem threshold 60 min after arginine vasopressin injection increased significantly in comparison with the initial threshold (P<0.05). Although the index for endolymphatic hydrops in rats administered arginine vasopressin was not different from that in controls (P>0.05), vacuoles in the intermediate cells were increased significantly in the treated rats (P<0.01). CONCLUSION: Hearing impairment was detected without endolymphatic hydrops in rats administered arginine vasopressin. An increase of vacuoles in the intermediate cells may account for the hearing impairment induced by arginine vasopressin injection.