p66Shc regulates renal vascular tone in hypertension-induced nephropathy
Bradley Miller, … , John D. Imig, Andrey Sorokin
Bradley Miller, … , John D. Imig, Andrey Sorokin
Published June 6, 2016
Citation Information: J Clin Invest. 2016;126(7):2533-2546. https://doi.org/10.1172/JCI75079.
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Research Article Nephrology Vascular biology

p66Shc regulates renal vascular tone in hypertension-induced nephropathy

Abstract

Renal preglomerular arterioles regulate vascular tone to ensure a large pressure gradient over short distances, a function that is extremely important for maintaining renal microcirculation. Regulation of renal microvascular tone is impaired in salt-sensitive (SS) hypertension–induced nephropathy, but the molecular mechanisms contributing to this impairment remain elusive. Here, we assessed the contribution of the SH2 adaptor protein p66Shc (encoded by Shc1) in regulating renal vascular tone and the development of renal vascular dysfunction associated with hypertension-induced nephropathy. We generated a panel of mutant rat strains in which specific modifications of Shc1 were introduced into the Dahl SS rats. In SS rats, overexpression of p66Shc was linked to increased renal damage. Conversely, deletion of p66Shc from these rats restored the myogenic responsiveness of renal preglomerular arterioles ex vivo and promoted cellular contraction in primary vascular smooth muscle cells (SMCs) that were isolated from renal vessels. In primary SMCs, p66Shc restricted the activation of transient receptor potential cation channels to attenuate cytosolic Ca2+ influx, implicating a mechanism by which overexpression of p66Shc impairs renal vascular reactivity. These results establish the adaptor protein p66Shc as a regulator of renal vascular tone and a driver of impaired renal vascular function in hypertension-induced nephropathy.

Authors

Bradley Miller, Oleg Palygin, Victoriya A. Rufanova, Andrew Chong, Jozef Lazar, Howard J. Jacob, David Mattson, Richard J. Roman, Jan M. Williams, Allen W. Cowley Jr., Aron M. Geurts, Alexander Staruschenko, John D. Imig, Andrey Sorokin

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Figure 4

Generation of a panel of mutant rat strains on the genetic background of Dahl SS rats.

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Generation of a panel of mutant rat strains on the genetic background of...
(A) Schematic diagram of p66Shc protein showing positions of p66Shc domains and regulatory phosphorylation site Ser36 along with the changes in amino acid sequence caused by ZFNs. The newly introduced stop codons are indicated. CH1, collagen homologue 1 region; PTB, phosphotyrosine binding domain. (B) WB revealed deficiency in p66Shc isoform in renal medulla from rats with modified Shc1 gene (M1 and M4), but not in their WT littermates. Blot was probed with antibodies that recognized all 3 Shc isoforms. Equal loading was verified by blotting with anti–β-actin antibodies. Lanes were run on the same blot, but were noncontiguous. (C) Knockin strategy to generate single amino acid substitution in endogenous rat p66Shc. Ser36Ala substitution strategy used the double-strand break caused by microinjection of ZFNs targeting Shc1 gene to stimulate homology-dependent repair (HDR) and knock in of a Ser36Ala substitution in 1-cell rat embryo. (D) Sanger sequencing confirming introduced Ser36Ala substitution. Raw sequencing data are shown along with sequences of WT and mutated Shc1 alleles. Additional changes were introduced to knock in BamH1 restriction site and to destroy ZFN target site in the edited rat genome. Due to degeneracy of genetic code, these additional changes did not cause amino acid substitutions. (E) WB analysis with phosphospecific antibodies against Ser36 revealed the absence of ET-1–induced Ser36 phosphorylation in SMCs derived from renal vessels of Ser36Ala rats when compared with SMCs derived from their WT littermates. Treatment for 5 minutes with 100 nM ET-1 is indicated by plus signs. Equal expression of p66Shc in these cells was verified by antibodies that recognize p66Shc regardless of its phosphorylation status.