RESUMO
In the collecting duct (CD), the interactions of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) modulate Na+ reabsorption, volume homeostasis, and blood pressure. In this study, we used a mouse kidney cortical CD cell line (M-1 cells) to test the hypothesis that in the CD, the activation of bradykinin B2 receptor (B2R) increases renin synthesis and release. Physiological concentrations of bradykinin (BK) treatment of M-1 cells increased renin mRNA and prorenin and renin protein contents in a dose-dependent manner and increased threefold renin content in the cell culture media. These effects were mediated by protein kinase C (PKC) independently of protein kinase A (PKA) because B2R antagonism with Icatibant and PKC inhibition with calphostin C, prevented these responses, but PKA inhibition with H89 did not modify the effects elicited by the B2R activation. BK-dependent stimulation of renin gene expression in CD cells also involved nitric oxide (NO) pathway because increased cGMP levels and inhibition of NO synthase with L-NAME prevented it. Complementary renin immunohistochemical studies performed in kidneys from mice with conventional B2R knockout and conditional B2R knockout in the CD, showed marked decreased renin immunoreactivity in CD, regardless of the renin presence in juxtaglomerular cells in the knockout mice. These results indicate that the activation of B2R increases renin synthesis and release by the CD cells through PKC stimulation and NO release, which support further the interactions between the RAS and KKS.
Assuntos
Bradicinina/farmacologia , Córtex Renal/metabolismo , Óxido Nítrico/metabolismo , Receptor B2 da Bradicinina/metabolismo , Renina/metabolismo , Animais , Bradicinina/análogos & derivados , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Linhagem Celular , Isoquinolinas/farmacologia , Córtex Renal/citologia , Córtex Renal/efeitos dos fármacos , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Naftalenos/farmacologia , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologiaRESUMO
We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G2 /M phases of the cell cycle. This acceleration is due, at least in part, to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to increase cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na+ /H+ exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Three cortical collecting duct cell lines were used: WT-RCCD1 (not expressing AQPs), AQP2-RCCD1 (transfected with AQP2), and mpkCCDc14 (with inducible AQP2 expression). We here demonstrate, for the first time, that both NHE2 protein activity and expression were increased in AQP2-expressing cells. NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G2 /M phases only if AQP2 was expressed. Finally, we observed that only in AQP2-expressing cells a NHE2-dependent RVI response was activated in the S phase. These observations suggest that the AQP2-increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2-expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. J. Cell. Biochem. 118: 967-978, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Aquaporina 2/metabolismo , Córtex Renal/citologia , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Aquaporina 2/genética , Ciclo Celular , Linhagem Celular , Proliferação de Células , Tamanho Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Córtex Renal/metabolismo , RatosRESUMO
The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Rim/citologia , Rim/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Feminino , Rim/embriologia , Rim/enzimologia , Córtex Renal/citologia , Córtex Renal/enzimologia , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Sphingosine-1-phosphate, the product of sphingosine kinase (SK) activity, is a sphingolipid metabolite that regulates cell growth, survival and migration. It is also known to affect diuresis, natriuresis and renovascular contraction in rats, although the mechanisms through which it affects these processes are not known. No previous report has addressed the differences among the kidney zones regarding endogenous SK expression and activity. Therefore, we examined SK1 distribution and activity in the various kidney zones: cortex, medulla and papilla. We found that SK1 expression does not correlate with enzyme activity. Study of the expression showed that the enzyme is highly expressed in cortex, followed by medulla and papilla. However, medulla had the highest enzyme activity. In all kidney zones, SK1 expression was mainly cytosolic. Regarding enzyme activity, whereas we found no difference between cytosol, membrane and nucleus in renal medulla, the membrane-bound enzyme presented the highest activity in cortex and papilla. SK1 distribution observed by immunohistochemical staining showed higher expression in cortical proximal convoluted epithelial cells. In medulla, immunostaining was observed as patches of staining, whereas in papilla, positive immunostaining was exclusively restricted to collecting duct cells. We also evaluated the effects of bradykinin and angiotensin II on SK1 activity.
Assuntos
Rim/enzimologia , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Angiotensina II/farmacologia , Animais , Bradicinina/farmacologia , Rim/citologia , Córtex Renal/citologia , Córtex Renal/enzimologia , Medula Renal/citologia , Medula Renal/enzimologia , Masculino , Especificidade de Órgãos , Ratos , Ratos Wistar , Esfingosina/metabolismoRESUMO
The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 microg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 microg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.
Assuntos
Técnicas de Cultura de Células/economia , Venenos de Crotalídeos/toxicidade , Crotalus , Córtex Renal/citologia , Testes de Toxicidade/economia , Animais , Técnicas de Cultura de Células/métodos , Chlorocebus aethiops , Cromatografia em Gel , Córtex Renal/efeitos dos fármacos , Dose Letal Mediana , Masculino , Camundongos , Testes de Toxicidade/métodos , Células VeroRESUMO
La patogénesis de la lesion renal ha sido relacionada a la miolisis, hemólisis, hipotensión y/o el efecto directo del veneno. Tanto el cultivo primario o el cultivo celular continuo proveen una alternativa in vitro para la evaluación cuantitativa de la toxicidad de venenos de serpiente. El veneno crudo de Crotalus vegrandis fue fraccionado por una cromatografía de exclusión molecular. La toxicidad del veneno crudo de C. vegrandis, sus fracciones hemorrágicas y neurotóxicas fueron evaluadas en células renales primarias de ratón y una línea continua de células Vero mantenidas in vitro. Las células fueron aisladas de la corteza renal murina y se cultivaron en placas de 96 pozos con medio Dulbecco (DMEM). Allí fueron tratadas con el veneno crudo y sus fracciones. Las células de la corteza renal murina tuvieron una morfología de células epiteliales y la mayoría se tiñeron con un anticuerpo anti-músculo actina. La citotoxicidad fue evaluada por el método colorimétrico del tetrazolium. La viabilidad de las células fue menor en las células tratadas con el veneno crudo, seguida por la fracción hemorrágica y neurotóxica, con un CT50 de 4.93, 18.41 y 50.22 µg/mL, respectivamente. Los cultivos de células Vero parecieron ser más sensibles con un CT50 de 2.9 y 1.4 µg/mL para el veneno crudo y el pico hemorrágico, respectivamente. Los resultados de este estudio muestran la potencialidad de usar sistemas de cultivo celular para evaluar la toxicidad de los venenos.
Assuntos
Animais , Masculino , Camundongos , Técnicas de Cultura de Células , Crotalus , Venenos de Crotalídeos/toxicidade , Córtex Renal/citologia , Testes de Toxicidade/economia , Técnicas de Cultura de Células , Chlorocebus aethiops , Cromatografia em Gel , Córtex Renal/efeitos dos fármacos , Testes de Toxicidade/métodos , Células VeroRESUMO
We have investigated the temporal maturation of the rat kidney during the postnatal developmental period. As a result, we observed the following: an active process of cortical cell proliferation and differentiation occurs as late as day 20. The medulla is the most immature zone at birth and displays the greatest morphological changes during this period. At birth, no distinction exists between inner and outer medulla, and the outer and inner strip of the outer medulla can be distinguished as late as day 30. Remodeling of the ECM surrounding collecting ducts occurs in the medulla twice, stopping at day 11 and it occurs in the papilla three times, stopping at day 20. The increase of kidney size is temporally different for each kidney zone. The cortex and the papilla acquire the morphological appearance of the adult kidney before the medulla does. Consequently, the medulla remains at the highest degree of immaturation among the kidney zones for a relatively long postnatal period.
Assuntos
Córtex Renal/citologia , Córtex Renal/crescimento & desenvolvimento , Medula Renal/citologia , Medula Renal/crescimento & desenvolvimento , Animais , Divisão Celular/fisiologia , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Glicosilação , Lectinas/metabolismo , Lectinas/farmacologia , Masculino , Antígeno Nuclear de Célula em Proliferação/análise , Ratos , Ratos WistarRESUMO
To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Córtex Renal/fisiologia , Túbulos Renais Coletores/fisiologia , Nifedipino/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Animais Recém-Nascidos , Transporte Biológico/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Citosol/metabolismo , Di-Hidropiridinas/farmacologia , Diltiazem/farmacologia , Ácido Egtázico/farmacologia , Gadolínio/farmacologia , Peróxido de Hidrogênio/farmacologia , Isradipino/farmacologia , Córtex Renal/citologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Cinética , Lantânio/farmacologia , Protaminas/farmacologia , Ratos , Ratos Sprague-Dawley , Tapsigargina/farmacologia , Verapamil/farmacologiaRESUMO
Rats submitted to chronic inhibition of nitric oxide synthase (NOS) have developed systemic hypertension and consequent renal injury. The present study aims to determine glomerular quantitative changes due to NOS inhibition in rats. Adults and normotensive Wistar rats were separated into control and L-NAME groups (each group n=10). The animals received water and food ad libitum, while L-NAME rats received NG-Nitro-L-Arginine methyl Ester hydrochloride to inhibit NOS (50mg/kg/day) in drinking water during 40 days. After that period the rats were sacrificed, the kidneys were removed, measured, and prepared for histological and stereological analyses. The glomerular density per area [NA(glom)] and the mean glomerular volume [v] were determined per animal in 15 random fields. In L-NAME rat the blood pressure was 76% higher than the respective control group with the same age. Glomeruli had global or segmental glomerular sclerosis; some glomeruli only presented an atrophic structure. The renal volume was not different between control and L-NAME rats (p>0.05). However, L-NAME rats had the NA(glom) 33% smaller than the control rats (p=0.0001) and, concomitantly, L-NAME rats had the v (glom) 33% higher than the control ones (p=0.004). These results demonstrate morphological renal alterations caused by NOS inhibition and hypertension.