The effect of exercise on endothelin and endothelin receptor levels in SHR and WKY male and female rats

The role of exercise as a means to modulate renal function in hypertension is unclear. However, prior studies in the laboratory of Dr. Penny Knoblich indicate that exercised female spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats excrete significantly more sodium in response to a rise in blood pressure relative to exercised male and sedentary female SHR and WKY rats. The purpose of this study was to evaluate the effects of exercise on endothelin and endothelin receptor levels in the urine and kidneys of male and female SHR and WKY rats, to elucidate potential mechanisms to explain an increase in urinary sodium excretion in exercised female SHR and WKY rats as previously described by Dr. Penny Knoblich. Eighty male and female SHR and WKY rats were randomly assigned to an exercise or a sedentary control group. Each rat assigned to the exercise group ran voluntarily on a wire wheel for ten to twelve weeks. After ten to twelve weeks, the urine and kidneys of each exercised and sedentary rat was collected. The urine from each group of rats was pooled and analyzed using an ELISA assay to determine the concentration of endothelin-1 (ET-1). The excised kidneys from each rat was examined using Western blot analysis and immunofluorescence microscopy to determine the amount and location of ET-1, endothelin-A (ET-A) receptors, and endothelin-B (ET-B) receptors within the tissue. The amount of urinary ET-1 excretion was elevated in female rats relative to male rats of the same strain and activity level, with exercised female SHR rats being the only exception. Male and female WKY rats were found to excrete more ET-1 than male and female SHR rats. Exercise had a variable effect on urinary ET-1 excretion levels. ET-A receptor levels were increased in kidney lysate of SHR rats when compared to WKY rats and in male rats relative to female rats of the same strain and activity level, with exercised male SHR rats being the only exception. Exercise had a variable effect on renal ET-A receptor expression. ET-B receptors were detectable in the kidney lysate of exercised female SHR rats, but not observed in any other group of rats. Immunofluorescence microscopy revealed that ET-A and ET-B receptor staining was very broad and localized in most components of the nephron. However, while ET-A receptor staining was observed in each group of rats, ET-B receptors were only detected in renal tissue extracted from exercised female SHR rats. ET-1 receptors were undetectable in the kidney tissue of each group of rats using Western blot analysis and immunofluorescence microscopy. An upregulation of ET-B receptors in the kidney tissue of exercised female SHR rats may explain previous reports that these rats excrete more sodium in response to a rise in blood pressure relative to other experimental groups of rats. An increase in ET-B receptor expression was not observed in the renal tissue of exercised female WKY rats. However, exercised female WKY rats excreted the greatest amount of ET-1 in their urine, which may be responsible for their previously reported increase in urinary sodium excretion in response to a rise in blood pressure. TABLE OF CONTENTS INTRODUCTION.1 Cardiovascular Physiology and Hypertension.1 Cardiovascular Regulation of Mean Arterial Pressure.1 The Effect of Hypertension on the Cardiovascular System.4 Renal Physiology and Hypertension.6 Renal Regulation of Blood Volume.8 Pressure Natriuresis Relationship and Regulation.11 Hormonal Control of Mean Arterial Pressure.14 The Effect of Hypertension on the Renal System.16 Types of Hypertension.17 Mechanisms of Hypertension.17 Sodium Consumption and Hypertension.18 Kidney Structure and Function and Hypertension.18 Endothelin System and Hypertension.22 Endothelin-1 Synthesis.22 Endothelin-1 and the Regulation of Mean Arterial Pressure.23 The Physiological Effects of Endothelin-A Receptor Binding.26 The Physiological Effects of Endothelin-B Receptor Binding.27 The Pathophysiology of the Endothelin System.28 Exercise and Hypertension.29 Physiological Effects of Exercise.30 Exercise and the Endothelin System.31 Spontaneously Hypertensive Rats.32 The Effects of Exercise on Spontaneously Hypertensive Rats.32 The Endothelin System of Spontaneously Hypertensive Rats.33 Purpose of Current Study.34 MATERIALS AND METHODS.37 Animal Husbandry and Exercise Regime.37 Surgical Procedure.37 Enzyme-Linked Immunosorbent Assay.38 Reagent Preparation.38 Assay Procedure.39 Western Blot Analysis.40 Solubilization of the Kidneys.40 SDS-PAGE and Transfer to Nitrocellulose Membrane.41 Western Blotting.42 Image Analysis.44 Immunohistochemistry.45 Cryosectioning.45 TABLE OF CONTENTS Immunostaining.45 Immunoflourescence and Image Capturing Software.46 RESULTS.48 Exercise Distance.48 Kidney and Body Mass.49 Levels of Endothelin in Urine.49 Endothelin and Endothelin Receptor Levels in Solubilzed Kidney Tissue.50 Endothelin and Endothelin Receptor Location within Kidney Tissue.51 DISCUSSION.91 Exercise Behavior.91 Kidney and Body Mass.92 Levels of Renal Endothelin-A Receptor.93 Levels of Renal Endothelin-B Receptor.95 Renal Localization of Endothelin-A and Endothelin-B Receptors.98 Levels of Renal and Urinary Endothelin-1.100 Conclusion.103 Further Studies.105 LITERATURE CITED.108