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Knockdown of the Expression of Gamma‐Adducin in the Renal Afferent Arteriole Enhances BK Channel Activity
Author(s) -
Fan Fan,
Ge Ying,
Pabbidi Mallikarjuna R,
Harder David R,
Roman Richard J
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1224.18
Subject(s) - gene knockdown , afferent arterioles , transfection , arteriole , myogenic contraction , vascular smooth muscle , kidney , gene isoform , small interfering rna , microbiology and biotechnology , medicine , biology , chemistry , endocrinology , pathology , cell culture , blood pressure , gene , microcirculation , renin–angiotensin system , biochemistry , genetics , smooth muscle
We recently identified a region on chromosome 1 containing 15 genes including Gamma‐Adducin (Add3) that causes the impaired myogenic response in the renal afferent arteriole (Af‐Art) and the development of renal vascular injury in Fawn Hood Hypertensive (FHH) rats. To determine which isoform of Add3 is expressed in the vasculature and whether Add3 is the viable candidate gene that plays a role in the modulation of myogenic response in renal microvessels, we knocked down the expression of Add3 with newly designed 27‐mer Dicer‐Substrate RNAi (DsiRNA) in rat Af‐art cultured for 36 hours. We then identified positively transfected vascular smooth muscle cells (VSMCs) using a red fluorescent indicator, and examined the large conductance calcium sensitive potassium (BK) current using patch clamp techniques. Both Add3 isoforms were detected in brain, kidney and liver, however, only isoform 2 was detected in the vasculature. The expression of Add3 was reduced in the renal microvessels and in the membrane fraction of the brain of FHH vs. FHH.1 BN rats. Add3 DsiRNA completely blocked the expression of Add3 protein in 293 cells and reduced the expression of mRNA in a dose‐dependent manner up to 50% in the cultured Af‐Art. The inner diameters of the Af‐Art transfected with Add3 DsiRNA dilated by 4 ± 3 % in response to an elevation in transmural pressure from 60 to 120 mmHg. In contrast, these vessels still constricted normally by 10 ± 1 % in vessels transfected with scrambled siRNA. The myogenic response of Af‐Art isolated from our novel Add3 KO rats is impaired versus controls. BK current was 3‐fold higher in VSMC freshly isolated from Af‐Art that were transfected with Add3 DsiRNA as indicated by the appearance of siGLO red fluorescence compared with that seen in non‐transfected cells. Similarly, BK current was higher in 293BKα cells transfected with Add3 DsiRNA or in VSMC isolated from Af‐Art of FHH relative to control rats and was blocked by IBTX. BKα protein expression was greater in the membrane fraction in FHH vs. FHH.1 BN rats. It appeared a stronger florescence at the membrane of 293BKα cells transfected with Add3 DsiRNA when staining with BKα implying an inhibition of endocytosis of BKα from the cell membrane. These results indicate that vasculature specifically expressed Add3 isoform 2, knockdown of the expression of Add3 impairs the myogenic response of Af‐Art that is similar as seen in FHH rats and associated with an elevation in BK channel activity. It supports the hypothesis that Add3 is a viable candidate gene that may play a causal role in the impaired myogenic response of the renal circulation in FHH rats and contributes to the development of renal injury. Support or Funding Information This study was supported by grants HL36279 and DK104184 (RJR) from the National Institutes of Health.