Nephrotic syndrome is a common pediatric disease. Its treatment relies heavily on the use of glucocorticoids to induce remission, as well as symptomatic treatment with loop diuretics furosemide and bumetanide. While the diuretic effect of these drugs is well studied, vascular effects that they are claimed to possess remain poorly investigated and understood. One characteristic of nephrotic syndrome, revealed by studying experimental models, is an impaired ultrafiltration barrier function of the glomerulus, attributed to mesangial cell contraction under the action of angiotensin II. We have recently established the presence in mesangial cells of a loop diuretic-inhibitable Na/K/Cl cotransport, which is sensitive to vasoconstrictors, including angiotensin II. We would like to characterize the role of this transport system in mesangial cells, and examine the regulation and functional responses to angiotensin II. We will employ well established transport studies, as well as a powerful experimental tool unique to this transporter, binding of 3H bumetanide, which has been shown to directly reflect transporter number. We will also examine the signal transduction mechanisms involved in transporter regulation. Mesangial cell contraction will be directed and quantitatively assessed by myosin light- chain phosphorylation, a technique recently developed in our laboratory. These in vitro studies will be supplemented by in vivo studies in the rat model of puromycin aminonucleoside nephrosis, which closely resembles human minimal change disease. The use of unilateral nephrosis will allow us to dissociate direct glomerular pathology from hemodynamic effects secondary to systemic hypoproteinemia or intravascular volume contraction. Taken together, these studies will provide valuable data regarding the Na/K/Cl transporter activity, the mechanism of action of loop diuretics and its relation the functional responses of mesangial cells to vasoconstrictors; the findings can potentially be expanded to vascular smooth muscle cells. Furthermore, they will increase our understanding of the pathophysiological mechanisms at work in nephrotic syndrome.
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