The immunosuppressive effect of rapamycin is mediated by inhibition of interleukin-2-stimulated T cell proliferation. We report for the first time that rapamycin also inhibits growth factor-induced proliferation of cultured mouse proximal tubular (MPT; IC(50) ~1 ng/ml) cells and promotes apoptosis of these cells by impairing the survival effects of the same growth factors. On the basis of these in vitro data, we tested the hypothesis that rapamycin would impair recovery of renal function after ischemic acute renal failure induced in vivo by renal artery occlusion (RAO). Rats given daily injections of rapamycin or vehicle were subjected to RAO or sham surgery. Rapamycin had no effect on the glomerular filtration rate (GFR) of sham-operated animals. In rats subjected to RAO, GFR fell to comparable levels 1 day later in vehicle- and rapamycin-treated rats (0.25 +/- 0.08 and 0.12 +/- 0.05 ml. min(-1). 300 g(-1), respectively) (P = not significant). In vehicle-treated rats subjected to RAO, GFR increased to 0.61 +/- 0.08 ml. min(-1). 300 g(-1) on day 3 (P < 0.02 vs. day 1) and then rose further to 0.99 +/- 0.09 ml. min(-1). 300 g(-1) on day 4 (P < 0.02 vs. day 3). By contrast, GFR did not improve in rapamycin-treated rats subjected to RAO over the same time period. Rapamycin also increased apoptosis of tubular cells while markedly reducing their proliferative response after RAO. Furthermore, rapamycin inhibited activation of 70-kDa S6 protein kinase (p70(S6k)) in cultured MPT cells as well as in the renal tissue of rats subjected to RAO. We conclude that rapamycin severely impairs the recovery of renal function after ischemia-reperfusion injury. This effect appears to be due to the combined effects of increased tubular cell loss (via apoptosis) and profound inhibition of the regenerative response of tubular cells. These effects are likely mediated by inhibition of p70(S6k).

Proteinuria is a risk factor for progression of chronic renal failure. A model of proteinuria-associated tubulointerstitial injury was developed and was used to examine the therapeutic effect of rapamycin. Two studies were performed. In study A, proteinuric rats were given sheep anti-Fx1A to induce experimental membranous nephropathy; control rats received normal sheep serum. Four weeks later, groups were subdivided and underwent laparotomy alone (two kidneys), nephrectomy alone (one kidney), or nephrectomy with polectomy (0.6 kidney). Renal function and morphology were evaluated 4 wk later. Whereas control rats never developed proteinuria, anti-Fx1A induced severe proteinuria. Proteinuria was unaffected by renal mass reduction. Proteinuric rats developed tubulointerstitial disease that was most severe in rats with 0.6 kidneys. Renal function (GFR) was reduced by loss of renal mass and was reduced further in proteinuric rats with 0.6 kidneys. In study B, the effect of rapamycin on the expression of candidate proinflammatory and profibrotic genes and the progression of proteinuria-associated renal disease were examined. All rats received an injection of anti-Fx1A and were nephrectomized and then divided into groups to receive rapamycin or vehicle. Gene expression, renal morphology, and GFR were evaluated after 4, 8, and 12 wk. Rapamycin reduced expression of the proinflammatory and profibrotic genes (monocyte chemotactic protein-1, vascular endothelial growth factor, PDGF, TGF-␤ 1 , and type 1 collagen). Tubulointerstitial inflammation and progression of interstitial fibrosis that were present in vehicle-treated rats were ameliorated by rapamycin. Rapamycin also completely inhibited compensatory renal hypertrophy. In summary, rapamycin ameliorates the tubulointerstitial disease associated with chronic proteinuria and loss of renal mass.

Age is a risk factor in the development of atherosclerosis. In this study we investigated the hypothesis that proliferation ofvascular smooth muscle cells (SMCs), an integral part of atherosclerotic plaque formation, changes with age. SMC growth kinetics ofold rats (21-24 months) were compared to those ofyoung adult rats (3-4 months). Rat aortas were denuded oftheir endothelium and the animals were killed after [3H]thymidine and Evans blue injections at 0-28 days after denudation. Incorporation of [3H]thymidine into SMC peaked in the young animals by day 2, whereas the older animals responded to endothelial removal with greater incorporation at day 2 and a more sustained rate of incorporation peaking at day 4. The [3H]thymidine incorporation curves decreased sharply from their peaks at 2 and 4 days, respectively, and paralleled each other after day 7.[3H]Thymidine uptake reflected the subsequent SMC intimal growth as measured morphometrically, with old animals showing greater numbers of intimal SMC than did the younger animals. The difference in response of SMC to injury with age suggests that aging produces a change in the vascular SMC that enhances proliferation. This change in response implies that the more pronounced atherosclerotic plaque growth seen with aging may be a result of an agerelated increase in response to injury rather than merely the accumulation of time-related intimal change.Advancing age is a major risk factor for the development ofatherosclerosis in a variety of vascular beds (1). Despite the increasing prevalence and severity of atherosclerosis in senescence, little is known regarding the relationship of the aging process itself to the biology of the arterial wall or to atherosclerosis. One basic question which has attracted little experimental attention relates to whether atherosclerosis is more notable in the elderly due to accumulation of intimal changes or because the aging process alters responsiveness of the arterial wall to injury, thus enhancing plaque formation in older animals. In this study we have compared smooth muscle cell growth kinetics in the aortas of young and old rats after de-endothelialization. Because the major cell of the progressive phase of atherosclerosis is the vascular smooth muscle cell (SMC) (2), which is the most likely source of extracellular connective tissue in the plaque, change in the growth kinetics ofthese cells as a function of age is of importance to a fuller understanding of the pathogenesis of atherosclerosis. Our studies indicate that the proliferative response ofSMCs to endothelial injury is enhanced with advancing age. METHODS Animals. Male Sprague-Dawley rats 4-5 months and 21-24 months of age were studied. Animals 21-24 months of age (referred to hereafter as old animals) were obtained from Charles River Breeding Laboratories and weighed 634 ± 86 (SD) g. The animals were barrier-reared and were housed in barrier conditions providing 12 hr of light and 12 hr ofdarkness. They were fed rat chow (Charles River) and water ad lib and were ...

Rapamycin delays but does not prevent renal recovery after ARF. MPT cells become resistant to rapamycin after prolonged exposure. We speculate that the ultimate recovery of renal function after ARF is due to the development of acquired tubular cell resistance to rapamycin.

A new quantitative assay for studying the kinetics of vascular smooth muscle cells in vivo is reported. The assay was used to determine the specific activity of DNA from rabbit aortic smooth muscle cells stimulated to grow by removal of the endothelial layer. The specific activity of the DNA was correlated with the rate of tritiated thymidine incorporation as measured by autoradiography and with the rate of DNA synthesis as estimated by direct measurement of cellular proliferation. Smooth muscle cells exhibit a 24-hour latent period in vivo prior to DNA synthesis; the synthesis peaks at 48 hours and then rapidly declines. The decline in DNA synthesis is not related to endothelial regrowth, and may be of homeostatic significance in limiting luminal stenosis. The assay offers a rapid and reliable alternative to autoradiographic and morphometric techniques for evaluating growth kinetics and growth regulation in vivo.