Sirolimus and everolimus reduce albumin endocytosis in proximal tubule cells via an angiotensin II-dependent pathway.

BACKGROUND: The proliferation signal inhibitors (PSIs) sirolimus (SRL) and everolimus (ERL) often induce proteinuria due to glomerular but also tubular dysfunction in transplant patients. The beneficial effect of angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II (Ang II) type 1 receptor blockers (ARB) has been reported. AIM: This study aimed to investigate: (i) the role of an Ang II-dependent mechanism and Ang II type 1 receptor (AT(1)R) in the regulation of receptor-mediated albumin endocytosis on proximal tubular epithelial cells (PTEC) following PSI treatment; (ii) the specific roles of the albumin receptors cubilin and megalin in albumin binding and uptake in PTEC. METHODS: A human renal PTEC line (HK-2) was used in the study. The binding of Alexa 488 conjugated albumin was measured by flow cytometry. Albumin uptake and the expression of cubilin and megalin were determined by cellular ELISA. RESULTS: The administration of PSIs resulted in decreased albumin binding and uptake and downregulation of cubilin and megalin expression in PTEC. These effects were significantly reversed by the administration of an ACE-I (ramipril) or an ARB (losartan). Combined use of ramipril and losartan demonstrated additive effects on cubilin expression, but not on megalin expression or albumin binding and uptake. CONCLUSIONS: Our findings suggest that decreased albumin endocytosis in PTECs following PSI treatment involve an Ang II-dependent pathway via AT(1)R. The albumin receptor megalin may play a more crucial role than cubulin, since its expression correlated directly with albumin uptake.

Anti-HLA I antibodies induce VEGF production by endothelial cells, which increases proliferation and paracellular permeability.

Anti-human leukocyte antigen class I (HLA I) antibodies were shown to activate several protein kinases in endothelial cells (ECs), which induces proliferation and cell survival. An important phenomenon in antibody-mediated rejection is the occurrence of interstitial edema. We investigated the effect of anti-HLA I antibodies on endothelial proliferation and permeability, as one possible underlying mechanism of edema formation. HLA I antibodies increased the permeability of cultured ECs isolated from umbilical veins. Anti-HLA I antibodies induced the production of vascular endothelial growth factor (VEGF) by ECs, which activated VEGF receptor 2 (VEGFR2) in an autocrine manner. Activated VEGFR2 led to a c-Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and its degradation. Aberrant VE-cadherin expression resulted in impaired adherens junctions, which might lead to increased endothelial permeability. This effect was only observed after cross-linking of HLA I molecules by intact antibodies. Furthermore, our results suggest that increased endothelial proliferation following anti-HLA I treatment occurs via autocrine VEGFR2 activation. Our data indicate the ability of anti-HLA I to induce VEGF production in ECs. Transactivation of VEGFR2 leads to increased EC proliferation and paracellular permeability. The autocrine effect of VEGF on endothelial permeability might be an explanation for the formation of interstitial edema after transplantation.

Proliferation signal inhibitor-induced decrease of vascular endothelial cadherin expression and increase of endothelial permeability in vitro are prevented by an anti-oxidant.

BACKGROUND: Rapamycines, sirolimus (SRL) and everolimus (ERL), are proliferation signal inhibitors (PSIs). PSI therapy often leads to edema. We hypothesized that increased oxidative stress in response to PSIs may modulate the expression of vascular endothelial (VE)-cadherin on endothelial cells (ECs) and, subsequently, vascular permeability, which in turn may be involved in the development of edema. METHODS: Experiments were performed on human umbilical vein ECs (HUVECs). Oxidative stress was measured by dichlorofluorescein-diacetate. Expression of VE-cadherin was evaluated by immunofluorescent staining and western blot analysis. Endothelial "permeability" was assessed using a transwell model. RESULTS: SRL and ERL, at concentrations of 1, 10 and 100 nmol/liter, enhanced oxidative stress (SRL: 24 +/- 12%, 29 +/- 9%, 41 +/- 13% [p < 0.05, in all three cases]; ERL: 13 +/- 10%, 27 +/- 2%, 40 +/- 12% [p < 0.05, in the latter two cases], respectively) on HUVECs, which was inhibited by the anti-oxidant, N-acetyl-cysteine (NAC) and, to a lesser extent, by the specific inhibitor of nitric oxide synthase, N-Omega-nitro-L-arginine methylester. By the use of NAC, VE-cadherin expression remained comparable with control, according to both immunocytochemistry and western blot analysis. Permeability was significantly increased by SRL and ERL at 100 nmol/liter (29.5 +/- 6.4% and 33.8 +/- 4.2%, respectively); however, co-treatment with NAC abrogated the increased permeability. CONCLUSIONS: EC homeostasis, as indicated by VE-cadherin expression, may be damaged by SRL and ERL, but resolved by the anti-oxidant NAC.