Date of Award

Spring 2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Anca D. Dobrian

Committee Member

Stephen J. Beebe

Committee Member

Frank A. Lattanzio

Committee Member

Russell L. Prewitt

Abstract

Hypertension is one of the common complications of obesity. Using a rat model of diet induced obesity and hypertension we investigated some of the mechanisms that are involved in regulation of blood pressure in obesity. The first aim of this study was to determine the role of proximal tubule transporters on the renal sodium handling in obese hypertensive (OP) and lean normotensive (OR) rats. An acute increase in renal perfusion pressure resulted in a blunted natriuretic response in OP vs. OR rats and indicated that increased sodium reabsorption in the proximal tubule is casual, at least in part, for hypertension in OP rats. Subsequently, protein expression and activity of Na,K-ATPase and NHE3 were increased in obese rats compared to lean rats. Moreover, in OP rats more NHE3 was associated with its active pool located in the microvillus region. Together, these results suggest that hypertension in obese rats is characterized by the impaired pressure-natriuresis and diuresis that can be explained by the increased activity of proximal tubule sodium transporters.

Previous results from our lab determined that peroxisome proliferator activated receptor γ (PPARγ) has reduced expression and activity in the kidney of OP vs. OR rats. Therefore, in the second aim we investigated the effect of PPARγ ligand activation on expression and activity of proximal tubule Na+ transporters in OP and OR rats. In addition, by employing in vitro studies using proximal tubule epithelial cells, we determined whether pioglitazone exerts its effect via direct PPARγ activation. Pioglitazone reduced systolic blood pressure in obese rats while having no effect in lean rats. However, it increased sodium retention in the lean group. Pioglitazone increased Na,K-ATPase activity in OP rats, while its protein expression was increased in both groups. In contrast, NHE3 activity was reduced in obese rats treated with pioglitazone and protein expression was decreased in both groups. Pioglitazone did not have an effect on NHE3 localization in obese rats, but in lean rats, it had tendency to redistribute NHE3 towards the more active membrane pool. In cells transiently transfected to overexpress or silence PPARγ, we demonstrated that pioglitazone reduced Na,K-ATPase and NHE3 abundance via PPARγ activation. Collectively, the results indicated that pioglitazone reduced blood pressure in the obese group most likely by decreasing activity of NHE3. However, other factors besides trafficking are involved in the transporter regulation. Pioglitazone did not reduce blood pressure in lean rats, suggesting that the metabolic milieu is an important determinant of the pioglitazone differential effect on the blood pressure and on the proximal tubule transporters.

Nitric oxide (NO) plays an important role in regulating pressure natriuresis and diuresis and its availability seems to be altered in obese animals and humans. The third aim was designed to examine the role of NO on blood pressure, pressure natriuresis and expression of sodium transporters NHE3 and Na,K-ATPase in OP and OR rats. To determine the role of NO, we performed in vivo study using L-NAME for chronic NO inhibition. The NO inhibition did not change glomerular filtration rate in either of the groups. Natriuresis and diuresis was significantly decreased only in treated OR rats. Also, NHE3 protein expression and activity were significantly elevated in treated vs. non-treated OR rats, with no significant changes in OP rats. Moreover, L-NAME caused a shift of NHE3 to the active pool located in microvillus region in OR group only. In conclusion, normotensive OR rats are more susceptible to NO deficiency and the mechanism involves an increase in activity of NHE3 with the transporter redistribution playing a significant role. In addition, we investigated in cell culture whether hormones elevated in obesity can modulate Na,K-ATPase and NHE3 via cGMP production. In vitro experiments provided some evidence that angiotensin II and insulin interact with the NO signaling pathway at the level of cGMP. cGMP could affect transporter activity by phosphorylation which could account for the effects determined in vivo.

Comments

Dissertation Submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/hvs7-v927

ISBN

9781109705966

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