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1Department of Nephrology and Center for Cardiovascular Research, Charité Universitätsmedizin Berlin, Charité Campus Mitte, Humboldt University, Berlin, Germany, and 2Department of Cell Biology and Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
Submitted 9 August 2007 ; accepted in final form 3 December 2007
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ABSTRACT |
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mesangioproliferative nephropathy; chronic kidney disease
Inhibition of the mammalian target of rapamycin (mTOR) has recently emerged as a novel mode of slowing down the progressive course in some experimental chronic kidney disorders (4, 18). mTOR is a major downstream component in the phospoinositine 3-kinase pathway (PI3K), classified as a central controller of cell growth (24). Inhibitors of mTOR, such as rapamycin or its derivative everolimus, are potent antiproliferative drugs. In clinical settings, they are used as efficacious maintenance immunosuppressants in the prevention of graft rejection. The key action of mTOR inhibitors is to block clonal proliferation and expansion of stimulated lymphocytes by blocking G1-to-S transition in the cell cycle. In addition to lymphocytes, mTOR inhibitors act antiproliferatively for several other cell types such as vascular smooth muscle cells, mesangial cells, and endothelial cells (4, 18, 24). Whether mTOR inhibition might be of benefit in chronic mesangioproliferative nephropathy is unknown and has even been doubted. In acute models of mesangioproliferative glomerulonephritis induced by anti-thy1 antibody injection, the administration of the mTOR inhibitor everolimus unexpectedly turned out to be detrimental, as documented by decreased kidney function, increased proteinuria, and persistent glomerular fibrotic lesions (7, 8).
This study was designed to characterize the role of mTOR signaling in a progressive model of mesangioproliferative nephropathy, using anti-thy1-induced chronic-progressive glomerulosclerosis of the rat. In this model, a single injection of a high dose of anti-thy1 antibody into uninephrectomized rats leads to a short-term complement- and nitric oxide-mediated acute glomerulonephritis, which then, over months, progresses autonomously toward persistent glomerulosclerosis, marked tubulointerstitial fibrosis, and renal insufficiency in a not primarily immune-mediated manner (17, 21, 27). This model has been termed anti-thy1-induced chronic glomerulosclerosis or anti-thy1-induced renal fibrosis (17, 27). The course of experimental progressive anti-thy1-induced chronic glomerulosclerosis mimics to a relevant degree advancing mesangioproliferative nephropathy in humans and thus can act as an experimental model for studying human disease. Treatment with low-dose rapamycin was started 10 days after antibody injection, i.e., after the initial marked glomerular proliferation of mesangial cells had passed. Proteinuria was determined monthly as a disease follow-up. In week 20 after disease induction, the actions of rapamycin on key pathways of renal disease progression, such as proteinuria, fibrogenesis, cell proliferation, and round cell infiltration, were measured. Blood pressure and renal function served as additional measures of disease severity and therapeutic efficacy.
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METHODS |
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Animals. Male Wistar rats (150–180 g) were obtained from Charles River (Sulzfeld, Germany). The animals were fed a normal protein diet (22.5% protein, A1311, Altromin, Lage, Germany) for at least 3 days before the experiment to allow equilibration. Animals were housed in a constant-temperature room with a 12:12-h dark-light cycle. Body weight was determined weekly. Food and water intakes were monitored daily. Animal care and treatment were in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by local authorities.
Model of anti-thy1-induced chronic-progressive glomerulosclerosis.
Chronic-progressive glomerulosclerosis (cGS) was produced by surgically removing one kidney and intravenously injecting the monoclonal antibody mAb 1-22-3 (5 mg/kg body wt in PBS, pH = 7.4) 3 days later (17, 21, 27). The mAb 1-22-3 antibody binds to a thy1-like antigen on the surface of mesangial cells of the kidney and causes a fast complement-dependent and NO-dependent mesangial cell lysis within the next 24 h (17, 27). The progression in cGS is linked to the uninephrectomy that is performed before anti-thy1 antibody injection, since the glomerular disease resolves over 
4 wk in animals with two kidneys (17, 21, 27). Control animals were injected with equal volumes of PBS only.
Treatment with rapamycin. Rapamycin (sirolimus) was given with food at a daily dose of 2.5 mg·kg–1·body wt–1. Rapamycin is a bacterial macrolide widely used as an immunosuppressive drug to prevent rejection in organ transplantation (18). The rapamycin-containing food was produced in our laboratory by using the flour of the standard rat chow (22.5% protein, A1311, Altromin). The drug was mixed into the dry food flour in appropriate amounts, water was added to form pellets, and the air-dried pellets were subsequently given to the rapamycin-treated animals.
Experimental groups and design. One week after anti-thy1 antibody injection, 24-h urine was collected. On the basis of the actual 24-h proteinuria achieved, the diseased animals were randomly assigned to the experimental groups. The following groups were formed: 1) animals with PBS injection as controls (Con; n = 5); 2) animals with anti-thy1-induced cGS, with no treatment (cGS; n = 18); 3) animals with anti-thy1-induced cGS treated with rapamycin (cGS+Rapa; n = 19).
Treatment with rapamycin was started on day 10 after anti-thy1 antibody injection. Day 10 was chosen because, in a pilot study, we were able to demonstrate that the acute proliferative phase is over at this point. On day 10, in anti-thy1-injected animals, the glomerular number of proliferation nuclear factor antigen (PCNA)-positive cells was even 31% lower than in normal controls. By contrast and as a positive control, the peak of proliferation was seen on day 5 after antibody injection, when the PCNA-positive cell count was 534% higher in controls.
In week 20, i.e., after 18 wk of rapamycin administration, the effects of treatment on proteinuria, blood pressure, plasma markers of renal function, as well as on glomerular and tubulointerstitial fibrosis and cell infiltration were determined. Blood creatinine and blood urea concentrations and a creatinine clearance-based glomerular filtration rate estimation served as markers of renal function. Glomeruli were isolated by a graded sieving technique. Since the renal cortex consists mainly of tubulointerstitial tissue (>95%), it was used as representative for the tubulointerstitium. Analysis of glomerular and tubulointerstitial fibrosis involved histological scoring of matrix accumulation, molecular analyses of transforming growth factor (TGF)-β1 as a mediator of fibrosis, collagen I as an indicator of matrix production, and the protease inhibitor tissue inhibitor of metalloproteinases (TIMP)-1 as a marker of matrix protein degradation. Tubulointerstitial and glomerular cell infiltrations and cell proliferation were analyzed by immunohistochemistry, using an ED1 antibody for macrophages, CD3 antibodies for T lymphocytes, and PCNA as a proliferation marker.
Proteinuria and systolic blood pressure. During the experiment, animals were housed individually in metabolic cages for a 24-h urine collection in weeks 1, 4, 8, 12, 16, and finally on the day before euthanasia in week 20. Urinary protein was measured using a pyrogallol red microtiter plate technique as previously described (17, 27). Proteinuria is expressed as milligrams protein per 24 hours. Systolic blood pressure was assessed before euthanasia in trained conscious animals by a tail-cuff method as previously described (17, 27).
Blood rapamycin levels. Blood mTOR concentrations were analyzed in weeks 12 and 20 after disease induction. Rapamycin blood levels were determined in whole-blood samples using a rapid, sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as previously described (25). Eluates from HPLC columns were introduced into a Sciex API 2000 triple-quadrupole mass spectrometer (PerkinElmer Applied Biosystems, Foster City, CA) and calculated (integration of peak areas; calculation of peak area ratios, calibration curve and drug concentration) with PE Sciex TurboQuan (Ver.1.0) software.
Euthanasia. The animals were anesthetized with 0.1 mg ketanest/0.01 mg xylazine per 100 g body wt (ketamine 10%, WDT, Garbsen, Germany; rompun 2%, Bayer Vital, Leverkusen, Germany). Following a midline abdominal incision, 5–10 ml of heparinized blood was drawn from the abdominal aorta, and kidneys were subsequently perfused with 30 ml of ice-cold PBS. Materials and tissues were subsequently processed as described in the following sections.
Renal function. Plasma and urine creatinine and plasma urea were measured spectrometrically in enzyme-based assays (17, 27). Creatinine clearance was used to estimate glomerular filtration rate and was calculated on the basis of plasma and urinary creatinine concentrations and the corresponding urine volume and is expressed as milliliters per minute per 100 grams body weight.
Lipid levels. Plasma cholesterol and triglyceride levels were measured by standard enzymatic methods following a 5-h fasting period. Blood lipid concentrations were analyzed by enzymatic photometric assays (CHOD-PAP method for cholesterol and GPO-PAP method for triglyceride) using an Hitachi 917 analyzer (Roche Diagnostics, Basel, Switzerland) (16).
Light and immunohistochemical microscopy. For histological examination, cortical tissue was fixed in Carnoy's solution. All histological studies were performed in a blinded fashion. Paraffin sections (3 µm) were stained with periodic acid-Schiff reaction (PAS) to determine glomerular and tubulointerstitial fibrosis by computer-based morphometric analysis (17, 28). Renal sections were examined on a Zeiss Axio Imager A.1 light microscope connected to a PL-A662 video camera and the Axiovision 4.1 image-analysis system (all Karl Zeiss Vision, Munich, Germany), with a 10 x 10 orthographic grid superimposed on digital images. The relative degree of glomerular matrix expansion was evaluated by rating the mesangial matrix occupying the area of 15 glomeruli/animal, using a scoring system from 0 to 100% (17, 27). The relative degree of tubulointerstitial fibrotic lesions, i.e., matrix deposition, tubular atrophy, and dilation, was calculated in 15 randomly selected tubulointerstitial areas per animal observed at x200 magnification and graded from 0 to 100% of the tubulointerstitial area (17, 27).
Immunohistology for detecting type I collagen was performed by using goat anti-type I collagen primary antibody (Southern Biotech, distributed by Biozol, Eching, Germany). As a secondary antibody, horseradish peroxidase-conjugated rabbit anti-goat antibody was used and visualized with AEC reagent (all DakoCytomation, Glostrup, Denmark). Glomerular collagen I-positive staining was analyzed by examining at least 15 glomerular areas/animal. Tubulointerstitial collagen I-positive staining was analyzed by randomly selecting at least 15 cortical areas/animal using x200 magnification. Collagen I-positive staining was expressed as percentage per area by applying the histomorphometric computer-based Axiovision 4.1 image-analysis system.
For analysis of cell infiltration, renal tissue was immunohistologically stained for ED1 (macrophages)- and CD3 (lymphocytes)-positive cells, and proliferation was monitored by analysis of PCNA (17, 27). Paraffin-embedded tissues were incubated with a primary mouse anti-CD3, mouse anti-ED1 (both Serotec, Oxford, UK), and mouse anti-PCNA (DakoCytomation) antibody. For detecting CD3-positive cells, further staining was performed using a secondary goat horseradish peroxidase-conjugated anti-mouse IgM antibody and APAAP technology for visualization, as previously described (17, 27). ED1- and PCNA-positive cells were detected with an alkaline phosphatase (AP)-conjugated secondary goat anti-mouse antibody, using fast red as a substrate (all DakoCytomation). Cell infiltration was evaluated by counting infiltrating cells in at least 15 glomerular sections (glomerular cell infiltration) and at least 15 randomly selected cortical areas (tubulointerstitial cell infiltration) from each rat (17, 27). Tissue sections were observed at x200 magnification.
Tubulointerstitial and glomerular TGF-β1 and TIMP-1 protein expression. Glomeruli from individual rats were isolated by a graded sieving technique (160-, 125-, and 71-µm mesh metal sieves), as described previously (17, 22, 27). For cultures of renal cortical tissue, a piece of cortical tissue was weighed and minced extensively with a razor blade. Glomeruli or cortical tissues were suspended in DMEM supplemented with 0.1 U/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Glomeruli were cultured at a density of 2,000 glomeruli/ml for 48 h, and minced cortical tissue at a density of 10 mg/ml, respectively. After 48-h incubation at 37°C/5% CO2, supernatants were harvested and stored at –70°C until analysis of TGF-β1 or TIMP-1 content. TGF-β1 and TIMP-1 content of the culture supernatant was measured after acid activation, using commercially available ELISA kits (TGF-β1 and TIMP-1 Duoset, R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions (17, 22, 27). Three samples from each rat were analyzed.
Statistical analysis. The results are expressed as means ± SE. Statistical analysis between groups was performed by a Kruskal-Wallis test and subsequent Mann-Whitney U-testing. A P value <0.05 was considered significant.
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RESULTS |
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One week after disease induction, proteinuria (Fig. 1A) was significantly elevated in the nephritic animals, which pointed out the efficacy of the randomization procedure (cGS 148 ± 12 mg/day; cGS+Rapa 147 ± 11 mg/day). Administration of rapamycin started on day 10 after disease induction showed a significant reduction of protein excretion (–38%, cGS+Rapa 183 ± 26 mg/day, P < 0.01) in week 4. In the nephritic control group, a continuous increase in proteinuria was observed, reaching values of 550 ± 46 mg/day at the end of the experiment in week 20. In comparison, final proteinuria was stabilized in the rapamycin-treated group and significantly lower than in the untreated animals (–62%, cGS+Rapa 213 ± 25 mg/day, P < 0.001). Proteinuria of normal control group was constantly low (week 20: 26 ± 3 mg/day).
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Effects of rapamycin on renal matrix accumulation. The histological photographs in Fig. 2 provide a characteristic overview on the impact of rapamycin administration on renal matrix accumulation in anti-thy1-induced cGS. The most pronounced actions of rapamycin were noted in the tubulointerstitial compartment.
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To further explore the molecular and cellular mechanisms underlying the beneficial effects of rapamycin, renal cell proliferation and round cell infiltration (macrophages and lymphocytes) were analyzed in anti-thy1-induced cGS.
Effects of rapamycin on renal cell proliferation. As shown in Fig. 7, anti-thy1-induced cGS shows marked tubulointerstitial (271 ± 71 vs. Con 7 ± 4 PCNA-positive cells/tubulointerstitial section) and glomerular proliferation (18 ± 5 vs. Con 2 ± 1 PCNA-positive cells/glomerular section), respectively. In week 20, PCNA-positive renal cell staining in animals treated with rapamycin was reduced by –90% in the tubulointerstitial (27 ± 6 PCNA-positive cells/section; P < 0.05) and by –76% in the glomerular compartment (5 ± 1 PCNA-positive cells/section; P < 0.05).
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DISCUSSION |
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The present study was designed to explore the renoprotective potential of low-dose rapamycin in a chronic model of progressive mesangioproliferative nephropathy, i.e., anti-thy1-induced cGS in the rat. The major findings of the present study are 1) rapamycin remarkably limits the progressive course of chronic anti-thy1 antibody-induced renal disease towards glomerulosclerosis, tubulointerstitial fibrosis, and renal insufficiency; 2) this outcome contrasts to the one previously described in acute, short-term anti-thy1-induced glomerulonephritis in the rat (7, 8); and 3) renoprotection by low-dose rapamycin involved beneficial effects on a number of key pathways of renal disease progression, i.e., proteinuria, renal matrix protein accumulation, cell proliferation, and leukocyte infiltration (15, 17, 23, 28). Both the therapeutic and mechanistic implications of these clinically relevant findings are discussed in the following paragraphs.
The beneficial effects of mTOR inhibition have recently been reported in several rat models of chronic kidney disease, i.e., hypertensive nephrectomy, diabetic nephropathy, hypertrophy following unilateral nephrectomy, tubulointerstitial fibrosis due to urethral obstruction or nephrotic syndrome, and polycystic kidney disease (2, 5, 10, 19, 26, 29). These beneficial results are now expanded toward a progressive model of human mesangioproliferative nephropathy as a leading cause of end-stage kidney disease worldwide (13, 14). Since the experimental studies mentioned above represent the vast majority of human chronic kidney disease, their findings jointly imply that mTOR signaling acts as a rather common key pathway in the progression of renal disease. Furthermore, the studies jointly suggest that inhibition of mTOR might be a novel, generally effective therapeutic approach to chronic kidney disease. The latter option may especially apply to early preventive treatment, since this represents generally the timing of mTOR inhibition in the experimental rat studies mentioned above.
The outcome of mTor inhibition in anti-thy1-induced cGS contrasts with the one previously reported in anti-thy1-induced acute glomerulonephritis (7, 8). In the latter, the mTOR inhibitor everolimus before or during the early marked mesangial cell proliferation turned out to be detrimental, manifesting aggravation of proteinuria, impaired self-healing, increased uremic mortality, and persistent glomerular fibrotic changes. These effects were observed both with high- and low-dose everolimus (7). The studies in anti-thy1 acute and chronic renal disease unanimously indicate that unaffected mTOR signaling is critical for the very early and marked mesangial cell proliferation and subsequent normal glomerular repair of acute anti-thy1 glomerulonephritis (7, 8). In the further course of the disease, inhibition of mTOR even acts beneficially and prevents chronic disease progression. As shown in the present work, this turning point already occurs 10 days after antibody injection, when the mesangial cell proliferation has come to a halt. Furthermore, the consensus on the tandem model of anti-thy1-induced acute and chronic renal disease reveals that findings in acute anti-thy1 glomerulonephritis cannot in all cases be extrapolated toward the chronic anti-thy1 glomerulosclerosis model and vice versa.
The contrasting actions of mTOR inhibition on experimental acute and chronic anti-thy1-induced renal disease mirror in principle the one seen in patients with primary chronic glomerulopathies or renal transplantation. In renal transplant recipients, preventive use of mTOR-based and calcineurin inhibitor-free immunosuppressive strategies has been shown to improve kidney function and transplant histology (12). However, in later stages of chronic allograft nephropathy, the switch to an mTOR inhibitor was associated with adverse effects, such as marked aggravation of proteinuria (9) and eventually deterioration of renal function. Predictors of adverse outcome included preexisting proteinuria >800 mg/day (9). In human primary glomerular disease, mTOR inhibition has been tried in more advanced disease states. In parallel to chronic allograft nephropathy, introduction of mTOR-based treatment was associated with acceleration of proteinuria and decline in renal function in some patients, but was at least to some degree beneficial in others (1, 6, 11). With regard to the relatively early start of mTOR inhibition in this experimental study, the early preventive use of mTOR inhibitors in chronic primary human glomerulopathies has not been determined yet. As in acute and chronic anti-thy1-induced renal disease, the emerging human data indicate that timing, the degree of preexisting disease, and maybe dose appear to be critical for the outcome in rapamycin treatment.
The key action of rapamycin is inhibition of cell proliferation. The present study proves that this pathway was actually operating. Using PCNA staining, low-dose rapamycin treatment went along with significantly lower numbers of both tubulointerstitial and glomerular proliferating cells. Furthermore, this investigation documents that rapamycin interferes with several other key pathways of renal disease progression. This involved reductions in 1) proteinuria, as shown by periodically measured 24-h urinary protein excretion; 2) renal matrix accumulation and fibrosis, as described by tubulointerstitial and glomerular matrix protein and collagen I deposition and the key fibrosis mediator TGF-β; and 3) renal round cell infiltration, as documented by tubulointerstitial and glomerular numbers of infiltrating macrophages and lymphocytes. Thus this study in chronic anti-thy1 glomerulosclerosis indicates that rapamycin directly or indirectly interferes with multiple key pathways in the progression of chronic renal disease, including mesangioproliferative nephropathy.
Systolic blood pressure was significantly lower in the rapamycin-treated animals than in the untreated chronic anti-thy1 animals in week 20 after disease induction. This may have contributed to the renoprotection observed with rapamycin treatment. With regard to their primary mode of pharmacological action (10, 30), mTOR inhibitors pose no direct effect on blood pressure. Therefore, it is likely that the lower blood pressure with rapamycin in this study was mediated indirectly through less renal damage and fibrosis.
mTOR treatment in humans, mainly in patients with solid organ transplantation, has been found to increase markedly blood lipid levels and thereby to adversely affect the cardiovascular risk profile (20). In the present investigation, blood triglyceride and cholesterol concentrations were found to be elevated by the chronic anti-thy1 disease itself. Rapamycin treatment went along with even lower triglyceride levels, while cholesterol levels were unaffected. How this translates into the human situation remains to be defined.
In conclusion, in a model of chronic anti-thy1-induced glomerulosclerosis, low-dose rapamycin significantly slows its progressive course toward chronic renal fibrosis and insufficiency. mTOR inhibition involves joint actions on renal cell proliferation, fibrogenesis, tissue cell infiltration, and proteinuria as key pathways in mediating the progression of chronic renal disease.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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