Wilfred Lieberthal

We have examined the mechanisms of cell death induced by cisplatin in primary cultures of mouse proximal tubular cells. High concentrations of cisplatin (800 microM) led to necrotic cell death over a few hours. Much lower concentrations of cisplatin (8 microM) led to apoptosis, which caused loss of the cell monolayer over several days. Necrosis was characterized by a cytosolic swelling and early loss of plasma membrane integrity. In contrast, early features of cells undergoing apoptosis included cell shrinkage and loss of attachment to the monolayers. Nuclear chromatin became condensed and fragmented in apoptosing cells. These features were absent in necrotic cells. DNA electrophoresis of cells exposed to 800 microM cisplatin yielded a "smear" pattern, due to random DNA degradation. In contrast, the DNA of apoptosing cells demonstrated a "ladder" pattern resulting from internucleosomal DNA cleavage. Antioxidants delayed cisplatin-induced apoptosis but not necrosis. Thus the mechanism of cell death induced by cisplatin is concentration dependent. Reactive oxygen species play a role in mediating apoptosis but not necrosis induced by cisplatin.

The mechanisms of cell death induced by ATP depletion were studied in primary cultures of mouse proximal tubular (MPT) cells. Graded ATP depletion, ranging in severity from approximately 2 to 70% of control levels, was induced by incubating cells with either antimycin or 2-deoxyglucose, with varying concentrations of dextrose. We found that cells subjected to ATP depletion below approximately 15% of control died uniformly of necrosis. In contrast, cells subjected to ATP depletion between approximately 25 and 70% of control all died by apoptosis. The rapidity of cell death was proportional to the severity of reduction of cell ATP content and was independent of the mechanism of cell death. Renal growth factors, epidermal growth factor (EGF) and high-dose insulin, did not ameliorate apoptotic cell death induced by ATP depletion. We conclude that ATP depletion can cause either necrosis or apoptosis in MPT cells. Furthermore, we have identified a narrow range of ATP depletion (approximately 15 to 25% of control) representing a threshold that determines whether cells die by necrosis or apoptosis.

Cells can die by two distinct pathways: apoptosis or necrosis. Necrosis is associated with rapid metabolic collapse that leads to cell swelling, early loss of plasma membrane integrity, and ultimate cell rupture. Cytosolic contents leak from the necrotic cell causing injury and inflammation to surrounding tissue. In contrast, apoptosis is an energy-requiring, gene-directed process, which, when activated, results in cell "suicide." The morphological and biochemical characteristics of cells dying by apoptosis differ markedly from those of cells dying by necrosis. During apoptosis, cells decrease in size and round up. The nuclear chromatin undergoes condensation and fragmentation. The apoptotic cell then breaks apart into many plasma membrane-bound vesicles called "apoptotic bodies," which contain fragments of condensed chromatin and morphologically intact organelles such as mitochondria. Apoptotic cells and bodies are rapidly phagocytosed, thereby protecting surrounding tissues from injury. The rapid and efficient clearance of apoptotic cells makes apoptosis extremely difficult to detect in tissue sections. Recent studies show that multiple cytotoxic stimuli well known to cause necrosis can lead to apoptosis instead when cells are exposed to the same noxious agents at lower concentrations. This insight has led to an interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. These diseases include acute and chronic renal failure from exposure of the kidney to ischemia or to cytotoxic agents. In this review we discuss some relevant aspects of the differences between necrotic and apoptotic cell death. We also present evidence to support the hypothesis that apoptosis is an important pathogenic mechanism in those forms of acute and chronic renal failure in which the renal tubular epithelial cell is the primary target of ischemic or toxic injury.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a pivotal role in mediating cell size and mass, proliferation, and survival. mTOR has also emerged as an important modulator of several forms of renal disease. mTOR is activated after acute kidney injury and contributes to renal regeneration and repair. Inhibition of mTOR with rapamycin delays recovery of renal function after acute kidney injury. Activation of mTOR within the kidney also occurs in animal models of diabetic nephropathy and other causes of progressive kidney disease. Rapamycin ameliorates several key mechanisms believed to mediate changes associated with the progressive loss of GFR in chronic kidney disease. These include glomerular hypertrophy, intrarenal inflammation, and interstitial fibrosis. mTOR also plays an important role in mediating cyst formation and enlargement in autosomal dominant polycystic kidney disease. Inhibition of mTOR by rapamycin or one of its analogues represents a potentially novel treatment for autosomal dominant polycystic kidney disease. Finally, inhibitors of mTOR improve survival in patients with metastatic renal cell carcinoma.

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).