AT₁receptor-mediated accumulation of extracellular angiotensin II in proximal tubule cells: role of cytoskeleton microtubules and tyrosine phosphatases

Abstract

Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG II is taken up by proximal tubule cells (PTCs) through AT(1) receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase-dependent mechanisms, and 3) AT(1) receptor-mediated endocytosis of ANG II has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [(125)I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the AT(1) receptor blocker losartan (16.5 +/- 4.6%, P < 0.01 vs. ANG II, 78.3 +/- 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 +/- 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by approximately 58% (basal, 229.8 +/- 11.4 vs. ANG II, 361.3 +/- 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan (P < 0.01), the cytoskeleton microtubule inhibitor colchicine (P < 0.05), and PAO (P < 0.01), whereas depletion of clathrin-coated pits with hyperosmotic sucrose had no effect (356.1 +/- 25.5 pg ANG II/mg protein, not significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 +/- 2.8 vs. ANG II, 9.1 +/- 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 +/- 8.6 vs. forskolin + ANG II, 42.8 +/- 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through AT(1) receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling.

Review: Novel roles of intracrine angiotensin II and signalling mechanisms in kidney cells

“…36 Ang II has also been shown to stimulate AT 1a -receptor internalisation 95,96 we showed that intracellular Ang II levels were increased more than two-fold when they were incubated with exogenous Ang II, and this response was inhibited by the ARB losartan and the cytoskeleton microtubule inhibitor colchicine or the tyrosine phosphatase inhibitor phenylarsine oxide (PAO). 46 These results support the concept that proximal tubule cells take up extracellular Ang II via AT 1 -receptor-mediated endocytosis and the process requires interactions with cytoskeleton microtubules and tyrosine phosphatases.…”

“…[42][43][44] Whether internalised or intracellular Ang II plays a physiological and/or pathological role in proximal tubule cells remains to be further studied. 45,46 Since proximal tubule cells reabsorb more than 65-70% of glomerular filtered sodium load, and increased sodium reabsorption in this nephron segment by extracellular and intracellular Ang II will promote sodium and fluid retention and consequently the development of hypertension. Acting as a powerful intracellular cytokine and growth factor, intracellular Ang II may also play an important role in the pathogenesis of Ang IIinduced hypertensive renal injury, especially tubulo-interstitial fibrosis.…”

Redox Control of Renal Function and Hypertension

“…This is a major drawback of this compound, as we now know that nonphagocytic NAD(P)H oxidase is active in the cytosol itself. Some studies have found utility for this compound in modulating Ang II signaling through its anti-tyrosine phosphatase activity (138,186). Tyrosine phosphatases play an important role in the internalization of AT 1 R and Ang II into PTCs (186).…”

“…Some studies have found utility for this compound in modulating Ang II signaling through its anti-tyrosine phosphatase activity (138,186). Tyrosine phosphatases play an important role in the internalization of AT 1 R and Ang II into PTCs (186). Others have demonstrated activation of eNOS, leading to improvement in endothelial dysfunction (68).…”

Renin-angiotensin system in the kidney: What is new?

“…The canonical clathrin-dependent endocytosis pathway for Ang Ⅱ occurs in different cell types, such as vascular smooth muscle and human embryonic kidney (HEK-293) cells through the AT1 receptor, c-Src and clathrin Adapter Protein 2 [112] . In rabbit proximal tubule cells, the alternative microtubule-associated endocytic pathway rather than the clathrin-dependent pathway participates in the AT1 receptor-mediated uptake of Ang Ⅱ [113] . Another alternative endocytic pathway for Ang Ⅱ internalization in proximal tubule cells has been described by Gonzalez-Villalobos et al [114] , where anti-megalin antisera interferes with Ang Ⅱ binding in cell brush-border membrane vesicles extracted from mice, indicating that Ang Ⅱ internalization is a megalin-dependent process.…”