RESUMO
Group V (GV) phospholipase A2 (PLA2) is a member of the family of secreted PLA2 (sPLA2) enzymes. This enzyme has been identified in several organs, including the kidney. However, the physiologic role of GV sPLA2 in the maintenance of renal function remains unclear. We used mice lacking the gene encoding GV sPLA2 (Pla2g5-/-) and wild-type breeding pairs in the experiments. Mice were individually housed in metabolic cages and 48-h urine was collected for biochemical assays. Kidney samples were evaluated for glomerular morphology, renal fibrosis, and expression/activity of the (Na+ + K+)-ATPase α1 subunit. We observed that plasma creatinine levels were increased in Pla2g5-/- mice following by a decrease in creatinine clearance. The levels of urinary protein were higher in Pla2g5-/- mice than in the control group. Markers of tubular integrity and function such as γ-glutamyl transpeptidase, lactate dehydrogenase, and sodium excretion fraction (FENa+) were also increased in Pla2g5-/- mice. The increased FENa+ observed in Pla2g5-/- mice was correlated to alterations in cortical (Na+ + K+) ATPase activity/ expression. In addition, the kidney from Pla2g5-/- mice showed accumulation of matrix in corticomedullary glomeruli and tubulointerstitial fibrosis. These data suggest GV sPLA2 is involved in the maintenance of tubular cell function and integrity, promoting sodium retention through increased cortical (Na+ + K+)-ATPase expression and activity.
Assuntos
Fosfolipases A2 do Grupo V/fisiologia , Túbulos Renais Distais/enzimologia , Rim/enzimologia , Sódio/metabolismo , Animais , Homeostase , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.
Assuntos
Aldosterona/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Túbulos Renais Distais/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Animais , Regulação para Baixo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação , Transdução de Sinais , Ubiquitinação , Proteínas de Xenopus , Xenopus laevisRESUMO
In the present paper the effect of Ang-(1-7) on the distal tubule (Na(+)+K+)ATPase activity was evaluated by using MDCK cells as a model. Confluent cell monolayers were incubated with increasing concentrations of Ang-(1-7) for 30 min. Thereafter, the (Na(+)+K+)ATPase activity was evaluated and a dose-dependent (from 10(-12) to 10(-7) M) inhibition was observed. The maximal inhibitory effect (54%) was reached at the concentration of 10(-8) M. The inhibitory effect of Ang-(1-7) was not affected by the AT2 receptor selective antagonist PD123319 (from 10(-10) to 10(-7) M) but was blocked in a dose-dependent manner by the AT1 receptor selective antagonists losartan (10(-10) M), candesartan (10(-17) M), irbesartan (2 x 10(-12) M) and telmisartan (2 x 10(-16) M). The signaling pathway triggered by stimulation of the AT(1) receptor was also investigated. The PI-phospholipase C (PI-PLC) inhibitor U73122 (5 x 10(-8) M) blocked the inhibitory effect elicited by Ang-(1-7). Involvement of the protein kinase C (PKC) was evidenced by the sensitivity of the inhibitory effect of Ang-(1-7) to calphostin C (6.32 x 10(-7) M) and the lack of additive effects when the cells were co-incubated with Ang-(1-7) and 3.2 x 10(-8) M PMA. Altogether, these results demonstrate that Ang-(1-7) inhibits the (Na(+)+K+)ATPase activity of the prototypic distal tubule cell MDCK through the AT1 receptor-mediated stimulation of PI-PLC/PKC signaling pathway.
Assuntos
Angiotensina I/farmacologia , Anti-Hipertensivos/farmacologia , Túbulos Renais Distais/enzimologia , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Túbulos Renais Distais/citologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
Bicarbonate reabsorption was evaluated by stationary microperfusion of in vivo early distal (ED) and late distal (LD) segments of rat kidney. Intratubular pH was recorded by double-barreled H ion-exchange resin/reference (1 M KCl) microelectrodes for the determination of HCO3- reabsorption. In the presence of luminal arginine vasopressin (AVP, 10(-9) M), a significant increase in HCO3- reabsorption was observed both in ED (from 0.931 +/- 0.061 to 2.12 +/- 0.171 nmol.cm-2.s-1] and LD segments [from 0.542 +/- 0.086 to 1.67 +/- 0.111 nmol.cm-2.s-1]. The addition of the V1-receptor antagonist [(d (CH2)5, Tyr (Et)2) arginine vasopressin] (10(-5) M) to luminal perfusion blocked luminal AVP mediated stimulation in ED and LD segments. 5-(N, N-hexamethylene) amiloride (10(-4) M) added to luminal perfusion inhibited luminal AVP-mediated stimulation in ED (by 63.7%) and LD (by 34.1%) segments. The addition of Bafilomycin A1 (2 x 10(-7) M) to the luminal perfusion did not affect luminal AVP-mediated stimulation in ED segments, but reduced it (by 31.7%) in LD segments. Our results indicate that luminal AVP acts to stimulate the Na(+)-H+ exchange in ED and LD segments via activation of V1 receptors, as well as the vacuolar H(+)-ATPase in LD segments.