Rapamycin is a potent immunosuppressive agent that has been used for over a decade to treat recipients of renal allografts (1). The immunosuppressive effect of rapamycin has been attributed to inhibition of cytokine-induced proliferation and clonal expansion of T lymphocytes (2–4). The discovery of rapamycin led to the identification of the mammalian target of rapaymycin (mTOR), a serine threonine kinase shown to play a critical role in cell growth (increased cell size) and cell-cycle progression (5).
More recently, it has become evident that mTOR is a ubiquitous kinase that promotes the proliferation of most, if not all, cell types (6–9). We previously demonstrated that rapamycin profoundly inhibits the proliferation of renal tubular cells and severely impairs renal recovery from ARF during a 4-day period following renal injury (10). These findings have subsequently been confirmed by other investigators in a transplant kidney model of ARF (4). These findings are consistent with substantial evidence that proliferation of tubular cells is necessary for renal recovery following ARF (11–13).
In addition, a number of recent clinical studies have implicated rapamycin as a contributory factor for delayed graft function (DGF) in humans following renal transplantation (14–17). These findings are of considerable clinical significance, since rapamycin is one of the agents of choice in recipients of renal allografts with DGF, permitting later introduction of calcineurin inhibitors once the ARF has resolved (18, 19).
The objective of this study was to determine whether renal failure persists indefinitely in rapamycin-treated rats after acute renal injury, or alternatively, whether renal function eventually recovers despite continued rapamycin treatment. We confirmed our previous finding that rapamycin profoundly inhibits tubular regeneration as well as renal recovery after ischemic injury (10). In addition, we now demonstrate for the first time that renal recovery from ARF, although delayed, does eventually occur, despite continued administration of rapamycin. We also provide novel data demonstrating that cultured renal tubular cells acquire resistance to the anti-proliferative effects of rapamycin after prolonged exposure to this agent.
We speculate that the ability of the kidney to “escape” from the deleterious effects of rapamycin after ARF is due, at least in part, to an adaptive response of tubular cells characterized by the development of resistance to rapamycin after a few days of exposure.
MATERIALS AND METHODS
In Vivo Experiments: Experimental Model of ARF
We studied male Sprague Dawley rats (Harlan, IN) fed rat chow (Purina 5001) (Purina Mills, Chicago, IL) and allowed free access to water. The animal protocol was reviewed and approved by the Boston University Institute for Animal Care and Use Committee. ARF was induced under anesthesia by removing the left kidney and occluding the right renal artery with a vascular clamp for 40 min (as described previously) (10, 20, 21). After induction of ARF, rats were allowed to recover from the surgery and anesthesia. Rats were then anesthetized a second time at one of 4 time points: 2, 4, 6, or 7 days after induction of ARF. At each time point, we measured glomerular filtration rate (GFR), assessed morphologic renal injury, and determined the number of proliferating tubular cells in the outer medulla of kidneys. Eight rats were examined at each of the four time points (total n=32 rats).
Rapamycin or its vehicle (both kindly provided by Ayerst Laboratories Inc., St. Davids, PA) were administered by intraperitoneal (IP) injection. Rapamycin was given at a dose of 0.05 mg/300g body weight. The first dose was given three days prior to induction of ARF to allow a steady state of rapamycin to develop. Thereafter, rapamycin was given daily until the end of the experiment (i.e. 2, 4, 6, or 7 days after induction of ARF).
GFR (inulin clearance)
GFR was measured by inulin clearance using a method that is well established in our laboratory (10, 20, 21). After determination of inulin clearance, the kidney was removed for morphologic examination.
Coronal sections of kidney tissue were immersion-fixed in 10% formaldehyde (in PBS), paraffin-imbedded, and stained with periodic acid-Schiff. Four sections of kidney tissue from each rat were randomly photographed by a blinded investigator. Five photographs (40× magnification) of the outer medulla of each kidney section were taken by one of the investigators (RF) using a stereomicroscope (Nikon Opiphot, Melville, NY) and digital camera (Diagnostic Instruments, Sterling Heights, MI). Morphologic damage to the kidney was assessed by evaluating the severity of tubular cell injury and the extent of tubular obstruction by intra-tubular debris. We defined tubular cell injury as either tubular cell necrosis or the absence of cells from the tubular epithelium. The loss of tubular cells was used as an indirect indication of cell death or sub-lethal injury cells (22). One of the authors examined coded slides of kidney sections from each of the experimental groups in blinded fashion, and quantified the proportion of tubular cells injured and of tubule obstructed tubules per microscopic field. The data were decoded and analyzed statistically by another author. Cell injury and tubular obstruction were both expressed as a percentage of the total number of tubular cells per microscopic field.
Bromodeoxyuridine (BrdU) uptake
Rats received a single dose of 5-bromo-2′-deoxyuridine (BrdU) (50 mg/kg) 30 min prior to measurement of inulin clearance in order to label cells in the DNA synthetic (S) phase of the cell cycle (23). At the end of the study, paraffin embedded sections of the kidney were stained with antibody to BrdU and then counterstained with hematoxylin. Photographs (20X magnification) of the outer medulla were taken and the number of BrdU-positive cells in each photograph counted in a blinded fashion (23).
In Vitro Experiments
Primary cultures of mouse proximal tubular (MPT) cells
pMPT cells were cultured from collagenase-digested fragments of proximal tubules obtained from the cortices of C57BL/6 mice using previously described methods (24). We have previously characterized these cells as predominantly (>98%) of proximal tubular origin (25).
Counting of MPT cells
We examined the interaction of the duration of incubation and the presence of rapamycin on the total number of cells per plate using an assay which we have described previously. This assay determined the number of viable cells using a hemocytometer. Viable cells were defined as cells that were adherent and excluded trypan blue (10).
MPT cell proliferation was assessed by thymidine uptake using previously described methods (10, 26).
Western blotting to assess activity of p70S6 kinase (p70S6K)
Lysates of MPT cells were harvested. Aliquots of lysates containing comparable amounts of protein (25 μg) were separated by 10% polyacrylamide SDS gel under reducing conditions, and the proteins were then transferred electrophoretically to PVDF membranes as previously described (10, 27, 28). Blots were probed for active p70S6K using a phospho-specific antibody that recognizes the active form of the kinase. Membranes were then stripped, and a second antibody was used that recognizes total (inactive and activated) p70S6K (10, 27, 28).
All data are expressed as mean ± SEM. All comparisons between two groups were made using the Student's t test. When more than two groups were compared, the Bonferroni correction was used. A P value <0.05 was considered significant.
Effect of Rapamycin on GFR
GFR in the vehicle (control) and rapamycin groups was reduced to a comparable extent on day 2 following induction of ARF (0.40±0.02 and 0.34±0.07 ml/min, respectively) (Fig. 1). These data are consistent with our earlier study (10) and imply that rapamycin does not significantly affect the initial degree of renal injury in response to ischemic insult. Also in accord with our earlier study (10), the deleterious effects of rapamycin were confined to the recovery phase of ARF. In vehicle-treated rats, GFR increased between days 2 and 4 to 0.83±0.02 ml/min (P<0.01 vs. day 2) and again between days 4 and 6 to 1.50±0.12 ml/min (P<0.01 vs. day 4), with no further increase seen between days 6 and 7 (Fig. 1). In contrast, for rapamycin-treated rats, GFR remained unchanged between days 2 and 4 (0.38±0.02 ml/min on day 4), but increased between days 4 and 6 to 0.70±0.07 ml/min) (P<0.01 vs. day 4) and again between days 6 and 7 to 1.20±0.08 ml/min (P<0.01 vs. day 6) (Fig. 1). The GFR in rapamycin-treated rats was significantly lower than that of vehicle-treated rats on both days 4 and 6 (P<0.01). Only on day 7 did GFR in rapamycin- and vehicle-treated rats become comparable. Thus, rapamycin substantially delayed, but did not prevent, the full recovery of GFR following ARF induced by ischemia-reperfusion injury.
We did not examine the effects of rapamycin on GFR in sham-operated animals in this study. We have previously reported that rapamycin had no effect on GFR in rats with sham-operated kidneys (i.e., kidneys subjected to surgery but not ischemic injury) (10).
We next evaluated the severity of tubular injury in the outer medulla of kidneys. Two days after induction of ARF, morphologic injury was severe but comparable in vehicle- and rapamycin-treated rats (Table 1). In vehicle-treated rats, injury was less severe on day 4 vs. day 2 and was relatively normal by day 6 (Table 1). In marked contrast, there was no improvement in the severity of tubular cell injury in rapamycin-treated rats between days 2 and 4 (Table 1). Renal morphology eventually improved in rapamycin-treated rats between days 4 and 6, but was still not completely resolved (Table 1). By day 7 after induction of ARF, morphology in both groups was relatively normal and comparable. Thus, as in the case of GFR, rapamycin delayed, but did not prevent, resolution of tubular injury.
Effect of Rapamycin on Tubular Cell Proliferation In Vivo
We next examined the effect of rapamycin on tubular cell proliferation after ARF by assessing BrdU uptake in the outer medulla of the kidney. In vehicle-treated rats, cell proliferation was greatest on day 2, and then decreased progressively through days 4 and 6 (Fig. 2, Table 2). In contrast, in rapamycin-treated rats, there were relatively few proliferating cells on day 2, and then increased through days and 6 (Fig. 2, Table 2). By day 6 after ARF, the number of BrdU-positive cells in rapamycin-treated rats had increased to a value comparable to that in vehicle-treated rats on day 2 (Fig. 2, Table 2). Thus, just as for recovery of GFR and resolution of tubular injury, proliferation of tubular cells was delayed by several days in rapamycin-treated rats.
Effect of Rapamycin on Cell Proliferation
To explore possible mechanisms for the “escape” from the antiproliferative effects of rapamycin on tubular cells in vivo, we compared the effects of short- and long term preexposure to rapamycin on proliferation of cultured MPT cells. Confluent MPT cells were maintained in DMEM for 7 days, and the medium was replenished daily. To one set of cells, rapamycin was added daily at concentrations ranging from 0.1 to 1000 ng/ml for all 7 days. In another group of cells, no rapamycin was added to the medium until the last day. Thus, MPT cells were preexposed to rapamycin for either 1 or 7 days. Thymidine uptake was determined for each group of cells 24 hr following preexposure to rapamycin for 1 or 7 days (Fig. 4). The IC50 of rapamycin in MPT cells exposed to rapamycin for 1 and 7 days was ∼10 ng/ml and ∼100 ng/ml, respectively (Fig. 3). Thus, cells exposed to rapamycin for 7 days were >10-fold more resistant to rapamycin than cells exposed for 1 day. These data indicate that prolonged exposure of MPT cells to rapamycin induces marked resistance of MPT cells to the inhibitory effects of rapamycin on cell proliferation (Fig. 3).
We counted the number of cells/well in MPT monolayers incubated for 1 or 7 days in the presence or absence of rapamycin. The duration of exposure and the presence of rapamycin had no effect on the total number of viable cells/well (data not shown).
Effect of Rapamycin on p70S6K Activity
MPT cells were cultured in the continuous presence of varying concentrations of rapamycin (0.1 to 100 ng/ml) for either 1 or 7 days. Then, cell lysates were subjected to western blotting and probed with an antibody that recognizes the phosphorylated (active) form of p70S6K. The blots were then stripped and reprobed with an antibody that recognizes total (both active and inactive) p70S6K. The results of western blotting data closely paralleled those for proliferation (Fig. 4). Inhibition of p70S6K activity was substantially greater in cells exposed to rapamycin for 1 day compared to 7 days (Fig. 4). Thus, the activity of p70S6K in cells exposed to rapamycin for 1 day was significantly greater than in cells exposed to rapamycin for 7 days at all concentrations between 0.1 and 10 ng/ml (Fig. 4). These data indicate that prolonged exposure attenuates the inhibitory activity of rapamycin for p70S6k and may account for the resistance to the anti-proliferative effects of rapamycin acquired by prolonged exposure to this drug.
In this study, we confirm previous findings from our group that inhibition of mTOR with the highly specific inhibitor rapamycin markedly impairs recovery of renal function after ischemic ARF (10). We also provide evidence that the delayed recovery induced by rapamycin is due, at least in part, to inhibition of proliferation and regeneration of tubular cells. We also demonstrate for the first time that regeneration of tubular cells and recovery of GFR after experimental ischemic ARF, although delayed by rapamycin, does eventually occur, even in the face of continued administration of this drug (Figs. 1 and 2).
We hypothesize that the ability of rapamycin-treated tubular cells to regain the capacity for proliferation may reflect the development of true resistance to the metabolic effects of rapamycin rather than pharmacokinetic or other alterations. We tested this hypothesis in cultured MPT cells, a model we have used extensively in the past to examine the mechanisms responsible for acute tubular cell injury (25, 29). We compared the effects of short-term (1 day) versus long-term (7 days) exposure of MPT cells to rapamycin. In accord with our hypothesis, after 7 days of preexposure to rapamycin, MPT cells become relatively resistant to the antiproliferative effects of rapamycin as compared to MPT cells exposed to rapamycin for only 1 day. The IC50 for rapamycin in MPT cells exposed to rapamycin for 1 day was less than 10 ng/ml, whereas in cells exposed to rapamycin for 7 days it was significantly higher at ∼100 ng/ml. (Fig. 3). Thus, continuous exposure of MPT cells to rapamycin for 7 days results in a greater than 10-fold loss of sensitivity of MPT cells to the inhibitory effects of rapamycin on cell proliferation (Fig. 3). We recognize that rapamycin resistance was demonstrated in confluent monolayers in which the rate of DNA synthesis was probably relatively low. Additional studies need to be done to examine the role of the proliferative state of cells and other variables on the development of rapamycin resistance.
We next examined the effects of short- and long-term exposure of cells to rapamycin on the activity of p70S6K. We used p70S6K as a “readout” of mTOR activity, since p70S6K is an important downstream target of mTOR (5, 30). We confirmed previous observations that p70S6K activity is markedly inhibited in tubular cells exposed to rapamycin (10). In addition, we found that inhibition of p70S6K activity by rapamycin is substantially attenuated in cells continuously exposed to rapamycin for 7 days (Fig. 4). These data support the hypothesis that mTOR becomes relatively resistant to inhibition by rapamycin after many days of exposure.
Resistance of mTOR to rapamycin has been reported previously in cells with defined loss-of-function mutations of genes that encode proteins necessary for the inhibition of mTOR activity by rapamycin (such as FKBP12 (31), mTOR (32) and p70S6K (33, 34). Cell lines resistant to rapamycin have also been identified by selecting out clones of resistant tumor cells (6, 35). It is important to emphasize that the resistance to rapamycin we have described in this report cannot be ascribed to genetic mutations or to the selection of resistant clones of cells. We propose that our observation of the development of resistance to rapamycin after prolonged exposure represents an “adaptive” response of individual tubular cells to rapamycin that has not been described before.
These novel observations raise an interesting question. The immunosuppressive effects of rapamycin apparently persist as long as the drug is administered. How can this be explained if renal tubular cells are able to develop resistance to its effects? There are several possible explanations for this paradox. First, T cells, unlike renal tubular cells, may not be susceptible to the development of rapamycin resistance. Another possibility is that many parenchymal cells (such as renal tubular cells) are terminally differentiated and have an extremely long half-life, while the turnover of individual lymphocytes is relatively rapid. For this reason, the individual lymphocyte may not be exposed to rapamycin long enough to become resistant. In any case, the difference between the responses of the immune system and renal tubular cells to long-term exposure to rapamycin may explain the relative lack of renal toxicity associated with rapamycin. These interesting but speculative hypotheses warrant further investigation.
In summary, we have shown that rapamycin delays renal recovery from ARF. The impairment of renal recovery appears to be appears to be due, at least in part, to inhibition by rapamycin of tubular cell regeneration. We also provide novel information that the kidney is able eventually to regain renal function, despite continued administration of rapamycin. We also provide data in indicating that tubular cells develop resistance to rapamycin during prolonged continuous exposure. We speculate that the ultimate recovery of GFR after ARF, despite continued rapamycin treatment, represents an “adaptive” response of tubular cells characterized by the development of resistance to rapamycin. These effects of rapamycin on renal recovery following acute ischemic renal injury have important implications for allograft recipients with DGF.
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