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Albumin endocytosis in proximal tubule cells is modulated by angiotensin II through an AT2 receptor-mediated protein kinase B activation.

ABSTRACT: Albumin endocytosis in renal proximal tubule cells is a clathrin- and receptor-mediated mechanism that, in several pathophysiological conditions, is involved in initiating or promoting tubule-interstitial disease. Although much work has been done on this pathway, the regulation of albumin endocytosis in proximal tubule cells is not well understood. Here, we study the modulation by angiotensin II (Ang II) of albumin endocytosis in LLC-PK1, a model of proximal tubule cells. We observed that Ang II increases albumin endocytosis by approximately 100% at 10(-9) M. This effect is completely reversed by 10(-9) M PD123319, a specific AT(2) receptor antagonist, but not by losartan, a specific AT(1) receptor antagonist, at concentrations up to 10(-7) M. The Ang II effect on albumin endocytosis is also reversed by: phosphoinositide 3-kinase inhibitors LY294002 (2.5 x 10(-6) M) or wortmannin (10(-7) M), the protein kinase B inhibitor (2 x 10(-5) M), and staurosporine (2 x 10(-6) M), an inhibitor of 3'-phosphoinositide-dependent kinase 1. Ang II induced the selective phosphorylation of protein kinase B (PKB) at the Thr-308 residue without a change in Ser-473 phosphorylation, a combination that leads to an increase in PKB activity. These effects were completely abolished by 3 x 10(-6) M staurosporine or 10(-8) M PD123319. Our experiments also showed that PKB is present in the membrane fraction in overnight-starved LLC-PK1 cells. Taken together, these data show that Ang II increases albumin endocytosis through an AT(2) receptor mediated by activation of PKB in the plasma membrane, which depends on the basal activity of the phosphatidyl-inositol 3-kinase.

SUBMITTER: Caruso-Neves C

PROVIDER: S-EPMC1297674 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Albumin endocytosis in proximal tubule cells is modulated by angiotensin II through an AT2 receptor-mediated protein kinase B activation.

Caruso-Neves Celso C Kwon Sang-Ho SH Guggino William B WB

Proceedings of the National Academy of Sciences of the United States of America 20051117 48

Albumin endocytosis in renal proximal tubule cells is a clathrin- and receptor-mediated mechanism that, in several pathophysiological conditions, is involved in initiating or promoting tubule-interstitial disease. Although much work has been done on this pathway, the regulation of albumin endocytosis in proximal tubule cells is not well understood. Here, we study the modulation by angiotensin II (Ang II) of albumin endocytosis in LLC-PK1, a model of proximal tubule cells. We observed that Ang II ...[more]

PMID: 16293694

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Project description:BackgroundChronic exposure to low cadmium (Cd) levels produces urinary excretion of low molecular weight proteins, which is considered the critical effect of Cd exposure. However, the mechanisms involved in Cd-induced proteinuria are not entirely clear. Therefore, the present study was designed to evaluate the possible role of megalin and cubilin (important endocytic receptors in proximal tubule cells) and angiotensin II type 1 (AT1) receptor on Cd-induced microalbuminuria.MethodsFour groups of female Wistar rats were studied. Control (CT) group, vehicle-treated rats; LOS group, rats treated with losartan (an AT1 antagonist) from weeks 5 to 8 (10 mg/kg/day by gavage); Cd group, rats subchronically exposed to Cd (3 mg/kg/day by gavage) during 8 weeks, and Cd + LOS group, rats treated with Cd for 8 weeks and LOS from weeks 5-8. Kidney Cd content, glomerular function (evaluated by creatinine clearance and plasma creatinine), kidney injury and tubular function (evaluated by Kim-1 expression, urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and glucose, and microalbuminuria), oxidative stress (measured by lipid peroxidation and NAD(P)H oxidase activity), mRNA levels of megalin, expressions of megalin and cubilin (by confocal microscopy) and AT1 receptor (by Western blot), were measured in the different experimental groups. Data were analyzed by one-way ANOVA or Kruskal-Wallis test using GraphPad Prism 5 software (Version 5.00). P < 0.05 was considered statistically significant.ResultsAdministration of Cd (Cd and Cd + LOS groups) increased renal Cd content. LOS-treatment decreased Cd-induced microalbuminuria without changes in: plasma creatinine, creatinine clearance, urinary NAG and glucose, oxidative stress, mRNA levels of megalin and cubilin, neither protein expression of megalin nor AT1 receptor, in the different experimental groups studied. However, Cd exposure did induce the expression of the tubular injury marker Kim-1 and decreased cubilin protein levels in proximal tubule cells whereas LOS-treatment restored cubilin levels and suppressed Kim-1 expression.ConclusionLOS treatment decreased microalbuminuria induced by Cd apparently through a cubilin receptor-dependent mechanism but independent of megalin.

Project description:Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na+/K+-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.

Dataset Information

Albumin endocytosis in proximal tubule cells is modulated by angiotensin II through an AT2 receptor-mediated protein kinase B activation.

Publications

Albumin endocytosis in proximal tubule cells is modulated by angiotensin II through an AT2 receptor-mediated protein kinase B activation.

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