Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells.

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).

Effects of hemoglobin-based oxygen-carrying solutions in anesthetized rats with acute ischemic renal failure.

The effects of three hemoglobin solutions were compared with those of iso-oncotic human serum albumin in rats with ischemic renal failure and sham-operated controls. Unmodified and alpha-alpha cross-linked hemoglobins both increase mean arterial pressure and systemic vascular resistance and reduce cardiac output substantially and to a comparable extent. In contrast, omicron-raffinose cross-linked hemoglobin has no deleterious effect on any of these parameters. In sham-operated rats unmodified hemoglobin reduces the glomerular filtration rate (GFR) by approximately 30%, whereas neither of the two cross-linked hemoglobins has any adverse effect on GFR in this group. None of the three hemoglobin solutions exacerbated the degree to which GFR was reduced by ischemia-reperfusion injury. Also, the degree of tubular necrosis induced by ischemia-reperfusion injury was also comparable in all groups. We conclude the following: (1) omicron-raffinose cross-linking, but not alpha-alpha cross-linking, ameliorates the effects of unmodified hemoglobin on vascular resistance and cardiac output; (2) both forms of cross-linking reduce the nephrotoxicity exhibited by unmodified hemoglobin in sham-operated rats; and (3) none of the hemoglobin solutions exacerbate renal injury induced by ischemia-reperfusion.

O-raffinose cross-linking markedly reduces systemic and renal vasoconstrictor effects of unmodified human hemoglobin.

The hemodynamic effects of a 20% exchange-transfusion with different solutions of highly purified human hemoglobin A-zero (A0) were evaluated. We compared unmodified hemoglobin with hemoglobin cross-linked with O-raffinose. Unmodified hemoglobin increased systemic vascular resistance and mean arterial pressure more than the O-raffinose cross-linked hemoglobin solution (by approximately 45% and approximately 14%, respectively). Unmodified hemoglobin markedly reduced cardiac output (CO) by approximately 21%, whereas CO was unaffected by the O-raffinose cross-linked hemoglobin solution. Unmodified and O-raffinose cross-linked hemoglobin solutions increased mean arterial pressure to comparable extents ( approximately 14% and approximately 9%, respectively). Unmodified hemoglobin increased renal vascular resistance 2-fold and reduced the glomerular filtration rate by 58%. In marked contrast, the O-raffinose cross-linked hemoglobin had no deleterious effect on the glomerular filtration rate, renal blood flow, or renal vascular resistance. The extents to which unmodified and O-raffinose cross-linked hemoglobin solutions inactivated nitric oxide also were compared using three separate in vitro assays: platelet nitric oxide release, nitric oxide-stimulated platelet cGMP production, and endothelium-derived relaxing factor-mediated inhibition of platelet aggregation. Unmodified hemoglobin inactivated or oxidized nitric oxide to a greater extent than the O-raffinose cross-linked hemoglobin solutions in all three assays. In summary, O-raffinose cross-linking substantially reduced the systemic vasoconstriction and the decrease in CO induced by unmodified hemoglobin and eliminated the deleterious effects of unmodified hemoglobin on renal hemodynamics and function. We hypothesize that O-raffinose cross-linking reduces the degree of oxidation of nitric oxide and that this contributes to the reduced vasoactivity of this modified hemoglobin.

Isolated MTAL cells produce an inhibitor of ouabain-sensitive oxygen consumption.

Dispersed medullary thick ascending limb (MTAL) cells of the rabbit kidney produce an inhibitor of transport-related oxygen consumption when incubated at 37 degrees C. Appearance of this inhibitor is enhanced by incubation with arachidonic acid. Its appearance can be prevented by either 10(-6) M nordihydroguaiaretic acid or 10(-4) M indomethacin, but not by 10(-6) M indomethacin. These results suggest that the transport inhibitor is an eicosanoid produced by a pathway other than that catalyzed by cyclooxygenase. It inhibits ouabain-sensitive respiration in intact cells treated with amphotericin, suggesting direct inhibition of the Na(+)-K(+)-ATPase.

Reversible changes in stress fiber expression and cell shape in regenerating rat and rabbit aortic endothelium.

The influence of intimal de-endothelialization on stress fiber expression in regenerating rat and rabbit aortic endothelium was studied using immunofluorescence microscopy. Rat thoracic and abdominal aortae were balloon de-endothelialized, and endothelial cell shape and stress fiber expression was studied in both uninjured and de-endothelialized animals. In control animals, the majority of thoracic endothelial cells did not contain stress fibers while the majority of abdominal endothelial cells did. One week after injury, all the endothelial cells distal to the regenerating edge contained very prominent stress fibers. In areas directly adjacent to the still de-endothelialized surface, the endothelial cells had an intense, diffuse cytoplasmic staining without stress fibers. Regenerating endothelium also had a substantially higher length-to-width ratio, but smaller cell areas. Six weeks after injury, the endothelium had completely regenerated, and stress fibers were lost from the majority of the thoracic endothelial cells. Changes in abdominal aorta stress fiber expression were not as marked. In the rabbit, all the control thoracic endothelial cells had stress fibers; however, cells at the leading edge of a narrow region of de-endothelialization had few stress fibers. The results suggest that stress fibers do not play a primary role in cellular migration in situ. The transient increase in stress fiber expression in the rat may result from a temporary demand for greater adhesive capabilities until the subendothelial extracellular matrix is remodeled.

Refractoriness of platelets to prostaglandins after infusion in rabbits.

Continuous intravenous infusion of prostacyclin (prostaglandin I2, PGI2) in rabbits induced refractoriness to PGI2-induced inhibition of platelet function. Although inhibited during earlier stages, platelet response to adenosine diphosphate and thrombin became normal within 24 hours of PGI2 infusion. Cross-mixing experiments with platelets and plasma from infused and control animals suggested that the altered response to PGI2 was caused by a defect intrinsic in the platelets. PGI2-stimulated cyclic adenosine monophosphate (cAMP) production was reduced in platelets from infused rabbits as compared with those from controls. Platelets refractory to PGI2 were refractory to PGE1 and PGD2, as well. Because PGE1 but not PGD2 shares the same platelet receptor as PGI2, the phenomenon could not be ascribed to receptor-specific downregulation, which was also shown by refractoriness of platelets from infused rabbits to the nonprostanoid inhibitor of platelet function adenosine. Either increased concentrations of ineffective inhibitors or their combination with phosphodiesterase inhibitors overcame refractoriness of resistant platelets, which also responded to inhibition by dibutyryl cAMP, indicating residual activity of adenylate cyclase. That at least the catalytic subunit of the enzyme was still working in refractory platelets was shown by inhibition of aggregation induced by forskolin, a non-receptor-mediated activator of adenylate cyclase. Impairment of the adenylate cyclase regulatory subunit, possibly accompanied by multireceptor downregulation, may explain the paradoxical refractoriness of platelets to prolonged infusion of PGI2. Such an effect may limit the benefit of PGI2 in treatment of thromboembolic disease.

Proliferation of smooth muscle cells in the thoracic aorta after injury to the abdominal aorta: evidence for a humoral mediator in experimental arteriosclerosis.

We tested the hypothesis that circulating humoral material(s) can induce vascular smooth muscle cells to synthesize DNA and to proliferate. Either the entire aorta or its abdominal segment was balloon de-endothelialized in four groups of rabbits. In the first group (control), the entire aorta was injured, and no further procedures were carried out. In a second group (reinjury), the abdominal aortic segment was reinjured 4 days after the initial de-endothelialization procedure. A third group (sham) had a second sham operation 4 days after initial injury. In a fourth group (abdominal only), the abdominal aortic segment was injured on two occasions 4 days apart. There was a rise in the specific activity of 3H-thymidine incorporation into smooth muscle cell DNA (DNA-SA) of the thoracic segments, which began 12 hours after reinjury, peaked within 24 hours at 335 +/- 63 dpm/micrograms DNA (+/- SEM), and returned to control level within 72 hours. The DNA-SA of the thoracic aorta of control rabbits and sham-operated animals 4.5 days after the initial injury was 86 +/- 19 and 48 +/- 8 dpm/micrograms DNA, respectively. There was no rise in DNA-SA in the thoracic aorta of animals in which the abdominal aorta was injured twice. Intimal cell nuclei per 0.1 mm internal elastic lamina were counted 3 days after the second injury and showed similar differences between doubly injured and control animals. Platelet accumulation, as measured by chromium 51 platelet attachment to the aortic surface, was increased in the abdominal segment 12 hours after reinjury.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular smooth muscle cell growth kinetics in vivo in aged rats.

Age is a risk factor in the development of atherosclerosis. In this study we investigated the hypothesis that proliferation of vascular smooth muscle cells (SMCs), an integral part of atherosclerotic plaque formation, changes with age. SMC growth kinetics of old rats (21-24 months) were compared to those of young adult rats (3-4 months). Rat aortas were denuded of their 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 age-related increase in response to injury rather than merely the accumulation of time-related intimal change.

In vivo aortic muscle cell growth kinetics. Differences between thoracic and abdominal segments after intimal injury in the rabbit.

Focal proliferation of smooth muscle cell (SMC) is an integral part of atherosclerotic plaque formation: characterization of regional variation in SMC growth kinetics is therefore important to the understanding of atherogenesis. SMC growth kinetics of rabbit abdominal and thoracic segments were compared. Rabbit aortas were denuded of endothelium and the animals killed after 3H-thymidine and Evans blue injections at 0 to 48 days after denudation. Incorporation of 3H-thymidine into both aortic segments peaked at 48 hours; no detectable incorporation occurred in the first 24 hours. Abdominal segment DNA specific activity (SA, dpm/micrograms DNA) and total kinetic activity (TKA, dpm/0.1 mm internal elastic lamina) at 48 hours were significantly greater than values for the thoracic aorta. Abdominal SA and TKA curves decreased exponentially after the 48-hour peak and parallel thoracic levels after day 7. SA and TKA values for each segment reflected the subsequent SMC intimal growth rates as measured morphometrically. Therefore, both segments share similar growth kinetic characteristics; however, the abdominal response to initimal injury is greater than the thoracic and leads to greater myointimal proliferation. The difference in response to injury in the two segments suggests regional variation in SMC's which are phenotypically similar.

Vascular smooth muscle cell kinetics: a new assay for studying patterns of cellular proliferation in vivo.

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.

Effects of compounds chemically related to salicylate on isolated antral mucosa of rabbits.

This study examines some of the gastric mucosal effects of compounds structurally related to salicylate (which consists of a hydroxyl and a carboxyl group attached to a benzene ring) in order to determine the importance of the various ligands of the salicylate molecule. The presence of carboxyl group increases mucosal permeability to acid. This increase in cation flow initially is associated with a decrease in anion permeability. These ion selective effects subsequently give way to a nonspecific increase in permeability. The presence of a carboxyl group also is associated with a more than 80% decrease in short circuit current. A similar decrease in short circuit current occurs when the mucosa is exposed to a pyridine molecule with an attached carboxyl group (pyridine-3-carboxylic acid), but in a relatively low concentration, this agent also decreases the back diffusion of acid. The above noted changes in mucosal permeability and short circuit current do not appear to be attributable to the benzene or phenolic ligands of the salicylate molecule. It is concluded that the presence of a sufficient concentration of an exposed carboxyl group on the mucosal side of the tissue causes an increase in the permeability of the mucosa to acid. The presence of a carboxyl group also appears to alter active ion transport, but this effect cannot be attributed to enhanced diffusion of acid into the tissue. The data implicate the carboxyl group of salicylate as being key to its damaging effects on the stomach.