Renal proximal tubules from old Fischer 344 rats grow into epithelial cells in cultures and exhibit increased oxidative stress and reduced D1 receptor function

Abstract

Earlier we reported defects in D1 receptor function in renal proximal tubules (RPTs) of aged Fischer 344 (F344) and obese Zucker rats. However, the defects in the receptor function in RPTs of obese Zucker rats do not pass onto primary cultures of RPTs from these animals. Here, we determined whether the defects in D1 receptor function in RPTs of aged F344 rats pass onto the primary cultures. RPTs from aged (24-mo) and adult (6-mo) F344 rats were grown into primary cultures. The microscopic studies showed that cells in cultures from adult and old rats were healthy as determined by the shape and size of the cells and nuclei. D1 receptor agonist SKF-38393 produced inhibition of (86)Rb (rubidium) uptake, index of Na-K-ATPase activity, in cells from adult rats, but this was reduced in old rats. Also, SKF-38393 increased the [(35)S]GTPgammaS binding, index of receptor activation, in the membranes of cells from adult rats but to a lesser extent from old rats. Furthermore, there was a downward trend in the levels of D1 receptor numbers and in the receptor proteins in old rats. Interestingly, gp(91phox) subunit of NADPH oxidase and cellular protein carbonyl levels (oxidative stress marker) were higher in cultures from old rats. These results show that RPTs from adult and old F344 rats grow into epithelial cells in cultures. Furthermore, cells in cultures from old rats are at a higher level of oxidative stress, which may be contributing to the reduced D1 receptor function in the cells from old compared with adult rats.

Fig. 1.

Renal proximal tubules (RPTs) from adult (6 mo) and old (24 mo) Fischer 344 (F344) rats grow into epithelial cells in primary cultures. Epithelial cells grown on glass cover-slips (details in methods and materials) were scanned under microscope using ×20 phase (A) and ×60 fluorescence (B) objectives. Arrows in A show cell boundaries. Arrowheads in A and B show cell nuclei.

Fig. 2.

D1 receptor agonist SKF-38393 (10⁻⁸ to 10⁻⁵ M) inhibits Na-K-ATPase to a lesser extent in the proximal tubular epithelial cell cultures of old F344 rats. Na-K-ATPase activity was measured as rubidium (⁸⁶Rb) uptake, an index of Na-K-ATPase activity. Results are means ± SE from proximal tubular epithelial cells in cultures from 4 rats in each group. *Significantly different from SKF-38393 (10⁻⁸ M) in culture from adult rats. #Significantly different from SKF-38393 (10⁻⁸ M) in culture from old rats. ANOVA and post hoc Newman-Keuls test was applied to determine the significance at P < 0.05.

Fig. 3.

D1 receptor agonist SKF-38393 (10⁻¹⁰ to 10⁻⁷ M) stimulates D1 receptor to a lesser extent in the proximal tubular epithelial cell cultures of old F344 rats. D1 receptor stimulation was measured as binding of [³⁵S]GTPγS to G proteins, an index of receptor G protein coupling. Results are means ± SE from proximal tubular epithelial cells in cultures from 5 rats in each group. *Significantly different from SKF-38393 (10⁻¹⁰ M) in culture from adult rats. #Significantly different from SKF-38393 (10⁻¹⁰ M) in culture from old rats. ANOVA and post hoc Newman-Keuls test was applied to determine the significance at P < 0.05.

Fig. 4.

D1 receptor numbers and D1 receptor proteins in the membranes of proximal tubular epithelial cells of adult and old F344 rats. A: D1 receptor numbers were determined using 20 nM of radioactive D1 receptor-specific antagonist [³H]SCH-23390. B: D1 receptor proteins were measured by Western blot analysis using specific D1 receptor antibody. Bars are results as means ± SE (n = 4 cultures from 4 different rats). B: top right, immunoblot of D1 receptor proteins (lanes 1, 2, 5, 6: cultures from adult F344 rats; lanes 3, 4, 7, 8: cultures from old F344 rats); left, a positive control for D1 receptors in brain striatum of Sprague-Dawley rats (lane 1, 5 μg protein; lane 2, 15 μg protein).

Fig. 5.

NADPH gp^91phox over expresses in the membranes of proximal tubular epithelial cell cultures from old F344. A: top, gp^91phox in the membranes of cell cultures of adult (lanes 1, 3, 5) and old (lanes 2, 4, 6) F344 rats. B: top, rac1 in the membranes of cell cultures of adult (lanes 1, 3, 5) and old (lanes 2, 4, 6) F344 rats. Bars are results as means ± SE (n = 3 cultures from 3 different rats). *Significantly different from cultures from adult rats in A (t-test, P < 0.01).

Fig. 6.

Proximal tubular epithelial cells in cultures from old F344 rats are endowed with higher protein carbonyls. Protein carbonyl, a marker of oxidative stress, was determined by Western blot analysis using a kit (details in methods and materials). A: lane M (markers of protein carbonyl); lanes 1, 3, 5 (primary cell cultures from adult F344 rats); lanes 2, 4, 6 (primary cell cultures from old F344 rats). Arrows point to protein bands with carbonyls. B: densities of protein carbonyls in bands A, B, C and D are means ± SE (n = 3 cultures from 3 different animals) and represented as bars. *Significantly different from respective cultures from adult rats (t-test, P < 0.05).