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Hydrogen sulfide targets EGFR Cys797/Cys798 residues to induce Na(+)/K(+)-ATPase endocytosis and inhibition in renal tubular epithelial cells and increase sodium excretion in chronic salt-loaded rats.

ABSTRACT: The role of hydrogen sulfide (H2S) in renal sodium and water homeostasis is unknown. We investigated whether H2S promoted Na(+)/K(+)-ATPase endocytosis via the H2S/EGFR/gab1/PI3K/Akt pathway in renal tubular epithelial cells.H2S decreased Na(+)/K(+)-ATPase activity and induced its endocytosis in renal tubular epithelial cells, which was abrogated by small interfering RNA (siRNA) knockdown of epidermal growth factor receptor (EGFR) and gab1, a dominant-negative mutant of Akt and PI3K inhibitors. H2S increased EGFR, gab1, PI3K, and Akt phosphorylation in both renal tubular epithelial cells and kidneys of chronic salt-loaded rats. These increases were abrogated by siRNA knockdown of EGFR, but not of c-Src. Radiolabeled H2S exhibited transient, direct binding to EGFR and directly activated EGFR. Some disulfide bonds in EGFR intracellular kinase domain were susceptible to H2S-induced cleavage. Mutations of EGFR Cys797 (human) or Cys798 (rat) residues increased EGFR activity and prevented H2S-induced Na(+)/K(+)-ATPase endocytosis. H2S also inhibited sodium hydrogen exchanger-3 (NHE3) activity in renal tubular epithelial cells. H2S treatment increased sodium excretion in chronic and acute salt-loaded rats and decreased blood pressure in chronic salt-loaded rats.H2S directly targets some disulfide bonds in EGFR, which activates the EGFR/gab1/PI3K/Akt pathway and subsequent Na(+)/K(+)-ATPase endocytosis and inhibition in renal tubular epithelial cells. EGFR Cys797/Cys798 residues are essential for an intrinsic inhibitory mechanism and for H2S actions in renal tubular epithelial cells. Other pathways, including NHE3, may be involved in mediating the renal effects of H2S. Our results reveal a new renal sodium homeostasis mechanism, which may provide for novel treatment approaches for diseases related to renal sodium homeostasis dysfunction.

SUBMITTER: Ge SN

PROVIDER: S-EPMC4215382 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Hydrogen sulfide targets EGFR Cys797/Cys798 residues to induce Na(+)/K(+)-ATPase endocytosis and inhibition in renal tubular epithelial cells and increase sodium excretion in chronic salt-loaded rats.

Ge Shun-Na SN Zhao Man-Man MM Wu Dong-Dong DD Chen Ying Y Wang Yi Y Zhu Jian-Hua JH Cai Wen-Jie WJ Zhu Yi-Zhun YZ Zhu Yi-Chun YC

Antioxidants & redox signaling 20140508 15

<h4>Aims</h4>The role of hydrogen sulfide (H2S) in renal sodium and water homeostasis is unknown. We investigated whether H2S promoted Na(+)/K(+)-ATPase endocytosis via the H2S/EGFR/gab1/PI3K/Akt pathway in renal tubular epithelial cells.<h4>Results</h4>H2S decreased Na(+)/K(+)-ATPase activity and induced its endocytosis in renal tubular epithelial cells, which was abrogated by small interfering RNA (siRNA) knockdown of epidermal growth factor receptor (EGFR) and gab1, a dominant-negative mutant ...[more]

PMID: 24684506

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Project description:A potent angiotensin II type-1 receptor blocker, azilsartan, has been reported to reduce blood pressure more effectively than candesartan. Interestingly, azilsartan can also restore the circadian rhythm of blood pressure. We hypothesized that azilsartan could also improve salt sensitivity; thus, we examined the effect of azilsartan on sodium handling in renal tubules. Subtotal nephrectomized C57BL/6 mice received azilsartan (1.0 mg/kg/day), candesartan (0.3 mg/kg/day), or vehicle via the oral route in conjunction with a normal- (0.3%) or high-salt (8.0%) diet. Two weeks later, the azilsartan group showed significantly lower blood pressure during the light period than the candesartan and vehicle groups (azilsartan: 103.1 ± 1.0; candesartan: 111.7 ± 2.7; vehicle: 125.5 ± 2.5 mmHg; P < 0.05; azilsartan or candesartan vs. vehicle). The azilsartan group also showed higher urinary fractional excretion of sodium during the dark period than the candesartan and vehicle groups (azilsartan: 21.37 ± 3.69%; candesartan: 14.17 ± 1.42%; vehicle: 13.85 ± 5.30%; P < 0.05 azilsartan vs. candesartan or vehicle). A pressure-natriuresis curve demonstrated that azilsartan treatment restored salt sensitivity. Immunofluorescence and western blotting showed lower levels of Na+-H+ exchanger-3 (NHE3) protein (the major sodium transporter in renal proximal tubules) in the azilsartan group, but not in the candesartan or vehicle groups. However, azilsartan did not affect NHE3 transcription levels. Interestingly, we did not observe increased expression of downstream sodium transporters, which would have compensated for the increased flow of sodium and water due to non-absorption by NHE3. We also confirmed the mechanism stated above using cultured opossum kidney proximal tubular cells. Results revealed that a proteasomal inhibitor (but not a lysosomal inhibitor) blocked the azilsartan-induced decrease in NHE3 protein expression, suggesting that azilsartan increases NHE3 ubiquitination. In conclusion, azilsartan (but not candesartan) improved salt sensitivity possibly by decreasing NHE3 expression via ubiquitin-proteasomal degradation.

Project description:The giant mudskipper, Periophthalmodon schlosseri, is an amphibious fish that builds burrows in the mudflats. It can actively excrete ammonia through its gills, and tolerate high environmental ammonia. This study aimed to examine the effects of seawater (salinity 30; SW) acclimation and/or environmental ammonia exposure on the kinetic properties of Na(+)/K(+)-ATPase (Nka) from, and mRNA expression and protein abundance of nka/Nka α-subunit isoforms in, the gills of P. schlosseri pre-acclimated to slightly brackish water (salinity 3; SBW). Our results revealed that the Nka from the gills of P. schlosseri pre-acclimated to SBW for 2 weeks had substantially higher affinity to (or lower K m for) K(+) than NH(+) 4, and its affinity to NH(+) 4 decreased significantly after 6-days exposure to 75 mmol l(-1) NH4Cl in SBW. Hence, Nka transported K(+) selectively to maintain intracellular K(+) homeostasis, instead of transporting NH(+) 4 from the blood into ionocytes during active NH(+) 4 excretion as previously suggested. Two nkaα isoforms, nkaα1 and nkaα3, were cloned and sequenced from the gills of P. schlosseri. Their deduced amino acid sequences had K(+) binding sites identical to that of Nkaα1c from Anabas testudineus, indicating that they could effectively differentiate K(+) from NH(+) 4. Six days of exposure to 75 mmol l(-1) NH4Cl in SBW, or to SW with or without 50 mmol l(-1) NH4Cl led to significant increases in Nka activities in the gills of P. schlosseri. However, a significant increase in the comprehensive Nkaα protein abundance was observed only in the gills of fish exposed to 50 mmol l(-1) NH4Cl in SW. Hence, post-translational modification could be an important activity modulator of branchial Nka in P. schlosseri. The fast modulation of Nka activity and concurrent expressions of two branchial nkaα isoforms could in part contribute to the ability of P. schlosseri to survive abrupt transfer between SBW and SW or abrupt exposure to ammonia.

Dataset Information

Hydrogen sulfide targets EGFR Cys797/Cys798 residues to induce Na(+)/K(+)-ATPase endocytosis and inhibition in renal tubular epithelial cells and increase sodium excretion in chronic salt-loaded rats.

Publications

Hydrogen sulfide targets EGFR Cys797/Cys798 residues to induce Na(+)/K(+)-ATPase endocytosis and inhibition in renal tubular epithelial cells and increase sodium excretion in chronic salt-loaded rats.

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