Novel signaling mechanisms of intracellular angiotensin II-induced NHE3 expression and activation in mouse proximal tubule cells

Expression of a cytosolic cyan fluorescent fusion protein of angiotensin II (ECFP/ANG II) in proximal tubules increases blood pressure in rodents. To determine cellular signaling pathways responsible for this response, we expressed ECFP/ANG II in transport-competent proximal convoluted tubule cells (mPCT) from wild type (WT) and type 1a ANG II receptor-deficient (AT1a-KO) mice and measured its effects on intracellular ANG II levels, surrogates of NHE3-dependent Na+ absorption as well as MAP kinases and NF-¿B signaling. In WT mPCT cells, ECFP/ANG II expression doubled ANG II levels, increased NHE3 expression and membrane phospho-NHE3 proteins 3-fold, and intracellular [Na+]i by 65%. These responses were associated with 3-fold increases in phospho-ERK 1/2 and phospho-p38 MAPK, 5-fold increases in p65 subunit of NF-¿B, and 3-fold increases in phospho-IKKa/ß (Ser 176/180) proteins. These signaling responses to ECFP/ANG II were inhibited by losartan (AT1 blocker), PD123319 (AT2 blocker), U0126 (MEK1/MEK2 inhibitor), and RO 106-9920 (NF-¿B inhibitor). In mPCT cells of AT1a-KO mice, ECFP/ANG II also increased the levels of NHE3, p-ERK1/2, and p65 proteins above their controls, but considerably less so than in WT cells. In WT mice, selective expression of ECFP/ANG II in vivo in proximal tubules significantly increased blood pressure and indices of sodium reabsorption, in particular levels of phosphorylated NHE3 protein in the membrane fraction and proton gradient-stimulated 22Na+ uptake by proximal tubules. We conclude that intracellular ANG II may induce NHE3 expression and activation in mPCTs via AT1a and AT2 receptor-mediated activation of MAP kinases, ERK 1/2, and NF-¿B signaling pathways.