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

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 86Rb (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 [35S]GTPγS 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.

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Role of oxidative stress in defective renal dopamine D1 receptor-G protein coupling and function in old Fischer 344 rats

AJP Renal Physiology ◽

10.1152/ajprenal.00111.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. F945-F951 ◽

Cited By ~ 33

Author(s):

Riham Zein Fardoun ◽

Mohammad Asghar ◽

Mustafa Lokhandwala

Keyword(s):

Oxidative Stress ◽

G Protein ◽

D1 Receptor ◽

Serine Phosphorylation ◽

D1 Receptors ◽

Adult Rats ◽

G Protein Coupling ◽

Protein Coupling ◽

Age Related ◽

Old Rats

Aging is associated with an increase in oxidative stress. Previously, we have reported that dopamine failed to inhibit proximal tubular Na-K-ATPase and to promote sodium excretion in old rats (Beheray S, Kansra V, Hussain T, and Lokhandwala MF. Kidney Int 58: 712–720, 2000). This was due to uncoupling of dopamine D1 receptors from G proteins resulting from hyperphosphorylation of D1 receptors. The present study was designed to test the role of oxidative stress in the age-related decline in renal dopamine D1 receptor function. We observed that old animals had increased malondialdehyde (MDA) levels, a biomarker of oxidative stress, and decreased D1 receptor number and protein in the proximal tubules (PT) compared with adult rats. In old rats, there was increased G protein-coupled receptor kinase-2 (GRK-2) abundance, increased basal serine phosphorylation of D1 receptors, and defective D1 receptor-G protein coupling in PT membranes. Interestingly, supplementation with an antioxidant, tempol (1 mmol/l in drinking water for 15 days), lowered MDA levels and normalized D1 receptor number and protein in old rats to the level seen in adult rats. Furthermore, tempol decreased GRK-2 abundance and D1 receptor serine phosphorylation and restored D1 receptor-G protein coupling in PT of old rats. The functional consequence of these changes was the restoration of the natriuretic response to D1 receptor activation in tempol-supplemented old rats. Therefore, in old rats, tempol reduces oxidative stress and prevents GRK-2 membranous abundance and hyperphosphorylation of D1 receptors, resulting in restoration of D1 receptor-G protein coupling and the natriuretic response to SKF-38393. Thus tempol, by lowering oxidative stress, normalizes the age-related decline in dopamine receptor function.

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Tempol Reduces Oxidative Stress, Improves Insulin Sensitivity, Decreases Renal Dopamine D1 Receptor Hyperphosphorylation, and Restores D1 Receptor-G-Protein Coupling and Function in Obese Zucker Rats

Diabetes ◽

10.2337/diabetes.54.7.2219 ◽

2005 ◽

Vol 54 (7) ◽

pp. 2219-2226 ◽

Cited By ~ 83

Author(s):

A. A. Banday ◽

A. Marwaha ◽

L. S. Tallam ◽

M. F. Lokhandwala

Keyword(s):

Oxidative Stress ◽

Insulin Sensitivity ◽

G Protein ◽

Dopamine D1 Receptor ◽

D1 Receptor ◽

Zucker Rats ◽

G Protein Coupling ◽

Obese Zucker Rats ◽

Renal Dopamine ◽

And Function

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Exercise decreases oxidative stress and inflammation and restores renal dopamine D1 receptor function in old rats

AJP Renal Physiology ◽

10.1152/ajprenal.00272.2007 ◽

2007 ◽

Vol 293 (3) ◽

pp. F914-F919 ◽

Cited By ~ 75

Author(s):

Mohammad Asghar ◽

Liza George ◽

Mustafa F. Lokhandwala

Keyword(s):

Oxidative Stress ◽

G Protein ◽

Sch 23390 ◽

D1 Receptor ◽

Skf 38393 ◽

Receptor Function ◽

Protein Activation ◽

G Protein Activation ◽

Anti Oxidant ◽

Old Rats

Recently, we reported that oxidative stress decreases D1 receptor numbers and G protein activation in renal proximal tubules (RPT), resulting in diminished natriuretic response to dopamine in old rats. We tested the hypothesis that exercise in old rats will decrease oxidative stress and restore natriuretic response to D1 receptor agonist, SKF 38393. Old (23 mo) rats were subjected to rest (sedentary) or to treadmill exercise followed by measurement of oxidative stress [malondialdehyde (MDA)], inflammation [C-reactive protein (CRP)], anti-inflammation (IL-10), antioxidant enzyme [superoxide dismutase (SOD)], and natriuretic response to SKF 38393. We found that MDA levels decreased and total SOD activity and Cu/ZnSOD protein increased in RPT of exercised rats. Exercise increased the nuclear levels of Nrf2 transcription factor (which binds to anti-oxidant response elements) in RPT. The levels of CRP decreased and IL-10 increased in RPT of exercised rats. Furthermore, exercise increased the basal bindings of [3H]SCH 23390 and [35S]GTPγS (indexes of D1 receptor number and G protein activation, respectively) together with D1 receptor and Gαq proteins in RPT membranes. Moreover, SKF 38393 increased sodium excretion in exercised rats. Also, exercise decreased the levels of proteins in the urine of old rats. These results demonstrate that exercise decreases oxidative stress, inflammation, and proteinuria and increases anti-oxidant defense and D1 receptor function in old rats. Therefore, exercise may prove beneficial in preventing age-associated increases in oxidative stress, inflammation, and preserving kidney function, in general, and renal D1 receptor responsiveness, in particular.

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Renal dopamine D1 receptor dysfunction is acquired and not inherited in obese Zucker rats

AJP Renal Physiology ◽

10.1152/ajprenal.00396.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. F109-F116 ◽

Cited By ~ 17

Author(s):

Anees Ahmad Banday ◽

Tahir Hussain ◽

Mustafa F. Lokhandwala

Keyword(s):

G Protein ◽

D1 Receptor ◽

Receptor Expression ◽

Receptor Protein ◽

Zucker Rats ◽

Receptor Function ◽

Rat Serum ◽

Obese Rats ◽

Obese Zucker Rats ◽

Renal Dopamine

In essential hypertension, the defect in renal dopamine (DA) D1 receptor function is intrinsic to proximal tubules as this phenomenon is also seen in primary proximal tubule cultures from spontaneously hypertensive rats (SHR) and essential hypertensive patients. Previously, a defect was reported in renal D1 receptor function in obese Zucker rats. In the present study, we sought to determine whether this D1 receptor dysfunction is intrinsic in these animals. In primary proximal tubular epithelial cells (PTECs) from lean and obese rats, DA inhibited Na-K-ATPase (NKA) activity in PTECs from both groups of rats. Basal NKA activity, D1 receptor protein expression, and their coupling to G proteins were similar in cells from both groups. However, when PTECs from lean and obese rats were cultured in 20% serum from obese rats, DA failed to inhibit NKA activity, which was accompanied by a reduction in D1 receptor expression and a defect in D1 receptor-G protein coupling. No such defects in the inhibitory effect of DA on NKA activity, D1 receptor numbers, or coupling were seen when PTECs from both lean and obese rats were grown in 20% serum from lean or rosiglitazone-treated obese (RTO) rats. RTO rat serum had normal blood glucose and reduced plasma levels of insulin compared with serum from obese rats. Furthermore, chronic insulin treatment of PTECs from lean and obese rats caused an attenuation in DA-induced NKA inhibition, a decrease in D1 receptor expression, and D1 receptor-G protein uncoupling. These results suggest that defective D1 receptor function in obese Zucker rats is not inherited but contributed to by hyperinsulinemia and/or other circulating factors associated with obesity.

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Exercise as an antioxidant decreases age‐associated oxidative stress and protein kinase C activity and restores D1 receptor function in renal proximal tubules of old rats

The FASEB Journal ◽

10.1096/fasebj.23.1_supplement.lb152 ◽

2009 ◽

Vol 23 (S1) ◽

Author(s):

Liza E. George ◽

Mustafa F. Lokhandwala ◽

Mohammad Asghar

Keyword(s):

Oxidative Stress ◽

Protein Kinase C ◽

Protein Kinase ◽

D1 Receptor ◽

Proximal Tubules ◽

Receptor Function ◽

Renal Proximal Tubules ◽

Kinase C ◽

Protein Kinase C Activity ◽

Old Rats

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Tempol reduces oxidative stress, improves insulin sensitivity and restores renal dopamine D1 receptor-G protein coupling and function in obese Zucker rats

American Journal of Hypertension ◽

10.1016/j.amjhyper.2004.03.572 ◽

2004 ◽

Vol 17 (5) ◽

pp. S214-S215

Author(s):

A BANDAY

Keyword(s):

Oxidative Stress ◽

Insulin Sensitivity ◽

G Protein ◽

Dopamine D1 Receptor ◽

D1 Receptor ◽

Zucker Rats ◽

G Protein Coupling ◽

Obese Zucker Rats ◽

Renal Dopamine ◽

And Function

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Cold-induced thermogenesis in younger and older Fischer 344 rats following exercise training

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1988.254.6.r908 ◽

1988 ◽

Vol 254 (6) ◽

pp. R908-R916 ◽

Cited By ~ 1

Author(s):

R. B. McDonald ◽

B. A. Horwitz ◽

J. S. Stern

Keyword(s):

Exercise Training ◽

Body Mass ◽

Cold Exposure ◽

O2 Consumption ◽

Fischer 344 ◽

Before And After ◽

Old Rats ◽

F344 Rats ◽

Maintain Body Temperature ◽

Brown Fat Mitochondria

The inability of old rats to maintain body temperature during cold exposure has been well documented. This study evaluated the effect of exercise on the rates of cold-induced O2 consumption and the contribution of nonshivering thermogenesis (NST) to these rates. Younger (12 mo) and older (24 mo) male Fischer 344 (F344) rats were divided into exercised and sedentary groups. Exercised rats were run on a motor-driven treadmill 60 min/day, at 19-24 m/min, 5 days/wk for 6 mo. At the conclusion of the 6-mo training period, O2 consumption of all four groups was measured at thermoneutrality (26 degrees C) and during 6 h of exposure to 6 degrees C. Rectal temperatures were recorded before and after cold exposure. NST was estimated from the ability of isolated brown fat mitochondria to bind guanosine 5'-diphosphate (GDP). Core temperature of older sedentary rats fell 5.1 +/- 0.4 degrees C after cold exposure (36.3 +/- 0.3 vs. 31.2 +/- 0.8 degrees C). Exercise training in older animals prevented this fall from occurring (36.4 +/- 0.2 vs. 35.3 +/- 0.3 degrees C). Core temperatures of cold-exposed younger exercised and sedentary rats did not differ from thermoneutral values. Exercise did not alter the rates of resting body mass-independent (ml.min-1.kg body mass-0.67) O2 consumption in younger or older rats. However, body mass-independent and lean body mass (LBM)-independent (ml.min-1.g LBM-0.67) cold-induced O2 consumptions of older exercised rats were significantly elevated relative to those of older sedentary animals. This effect of exercise was not seen in younger rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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Overexpression of PKC-βI and -δ contributes to higher PKC activity in the proximal tubules of old Fischer 344 rats

AJP Renal Physiology ◽

10.1152/ajprenal.00198.2003 ◽

2003 ◽

Vol 285 (6) ◽

pp. F1100-F1107 ◽

Cited By ~ 15

Author(s):

Mohammad Asghar ◽

Tahir Hussain ◽

Mustafa F. Lokhandwala

Keyword(s):

Skf 38393 ◽

Proximal Tubules ◽

Fischer 344 Rats ◽

Adult Rats ◽

Altered Expression ◽

Fischer 344 ◽

Pkc Isoforms ◽

Diuretic Response ◽

Old Rats ◽

Pkc Ζ

Previously, we reported that natriuretic and diuretic response to dopamine is diminished in old Fischer 344 rats, which is due to higher basal protein kinase C (PKC) activity and hyperphosphorylation of Na-K-ATPase in the proximal tubules (PTs) of old rats. The present study was conducted to determine whether higher PKC activity could be due to altered expression of some of the PKC isoforms in the superficial cortex (rich in PTs) of old rats. Fluorimetric measurement showed almost twofold increase in the PKC activities in homogenates and membranes of old (24 mo) compared with adult (6 mo) rats. Interestingly, in the basal state PKC-βI was overexpressed in the membranes, whereas PKC-δ expression was increased in the cytosol of old compared with adult rats. Treatment of the cortical slices with either SKF-38393, a D1-like agonist, or PDBu, a direct activator of PKC, caused translocation of PKC-βI from cytosol to membranes in adult but not in old rats. Both of these drugs caused translocation of PKC-δ from membranes to cytosol in adult but not in old rats. These drugs had no effect on translocation of PKC-ζ in both adult and old rats. Both PKC-βI and -δ coimmunoprecipiated with α1-subunit of Na-K-ATPase in adult and old rats. These observations suggest that both SKF-38393 and PDBu differentially regulate PKC-βI and -δ in adult but not in old rats. Also, PKC-βI and -δ seem to interact with Na-K-ATPase in these animals. The overexpression of both PKC-βI and -δ in old rats could be responsible for a higher basal PKC activity, which causes the hyperphosphorylation of Na-K-ATPase and contributes to the diminished inhibition of Na-K-ATPase activity by dopamine in old rats.

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The age-related increase in renal clusterin mRNA is accelerated in obese Zucker rats.

Journal of the American Society of Nephrology ◽

10.1681/asn.v9138 ◽

1998 ◽

Vol 9 (1) ◽

pp. 38-45 ◽

Cited By ~ 2

Author(s):

N J Laping ◽

B A Olson ◽

J R Day ◽

B M Brickson ◽

L C Contino ◽

...

Keyword(s):

Mrna Expression ◽

Tissue Injury ◽

Rat Kidney ◽

Zucker Rats ◽

Mrna Levels ◽

Zucker Rat ◽

Age Related ◽

Obese Zucker Rat ◽

Obese Zucker Rats ◽

F344 Rats

Clusterin is a multifunctional glycoprotein associated with development and tissue injury. Because renal function decreases with advancing age in the obese Zucker rat, clusterin mRNA expression was examined in the kidney of young adult Zucker rats and compared with age-related changes in renal clusterin mRNA expression in Fischer 344 (F344) rats. Renal clusterin mRNA levels in the obese Zucker rat were 2.5-fold higher by 3 mo of age and fourfold higher at 5 mo of age compared with the lean strain. In comparison, renal clusterin mRNA in 12-mo-old F344 rats was twofold higher than in 3-mo-old animals and was tenfold higher at 24 mo of age. Clusterin mRNA was positively correlated with urinary protein excretion and negatively correlated with creatinine clearance in Zucker rats. Clusterin was increased in select nephrons of the obese Zucker rat kidney and in 24-mo-old F344 rat kidney as assessed by in situ hybridization. Increased expression of clusterin mRNA occurred mostly in the tubular epithelium of dilated, convoluted proximal tubules. These data indicate that renal clusterin mRNA levels increase as a function of age and that age-related increases in renal clusterin and the associated tubular abnormalities are accelerated in obese Zucker rats.

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