NDT Advance Access originally published online on June 5, 2007
Nephrology Dialysis Transplantation 2007 22(8):2175-2182; doi:10.1093/ndt/gfm191
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Renoprotective effects of sirolimus in non-immune initiated focal segmental glomerulosclerosis
Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney at Westmead Hospital, Westmead NSW 2145, Sydney, Australia 2145
Correspondence and offprint requests to: Dr G. Rangan, Department of Renal Medicine & Transplantation, Westmead Hospital, Westmead, Sydney, Australia 2145. Email: g.rangan{at}wmi.usyd.edu.au
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Abstract |
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Background. In this study we tested the hypothesis that sirolimus (a target of rapamycin inhibitor that attenuates intrinsic renal and immune cell proliferation) reduces glomerular hypertrophy and tubular epithelial cell (TEC) proliferation, and attenuates the progression of renal scarring and dysfunction, in a non-immune initiated model of focal segmental glomerulosclerosis (FSGS).
Methods. Adult male Wistar rats with adriamycin nephropathy (AN) were stratified into two groups, according to proteinuria on day 12, and received either vehicle (dimethylsulphoxide) or sirolimus (0.1 mg/kg) by daily subcutaneous injection, from day 14 until day 49 (n = 8 each). Control animals were also examined (n = 3 each).
Results. Sirolimus did not affect the progression of proteinuria, renal dysfunction, hypercholesterolaemia, body weight or alter intraluminal cast formation in AN. Sirolimus prevented the increase in kidney enlargement in AN, and attenuated glomerular capillary tuft expansion, glomerulosclerosis and periglomerular myofibroblast accumulation. In the tubulointerstitium, sirolimus attenuated tubular dilatation, TEC proliferation and interstitial fibrosis. This was accompanied by a reduction in renal cortical TGF-ß1, but peritubular myofibroblast accumulation and renal inflammation (glomerular and interstitial ED-1 and CD3-positive cell accumulation), were unaffected.
Conclusion. The anti-renotrophic properties of sirolimus were correlated with a reduction in renal scarring in AN. These data suggest that sirolimus has renoprotective effects when administered during the early stages of an FSGS pattern of chronic renal injury.
Keywords: glomerular disease; nephrotic syndrome; rapamycin
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Introduction |
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Focal segmental glomerulosclerosis (FSGS) is a specific pattern of chronic renal injury resulting from the progressive depletion of podocytes through immune and non-immune mechanisms [1]. It is a heterogeneous disorder and a range of causative factors have been postulated including viral infection, toxins, genetic mutations, metabolic diseases such as obesity, and hyperfiltration-mediated glomerular injury in chronic kidney disease [1]. A clear understanding of the consequences of podocyte loss and the cellular pathogenesis of the FSGS lesion has emerged in recent years, leading to the potential for new therapies to treat this pattern of glomerular injury [2,3].
Kidney growth [characterized by glomerular hypertrophy and tubular epithelial cell (TEC) proliferation], due to the effects of chronic nephrotic-range proteinuria [4], local growth factor production [5] and/or nephron loss [6], is also a feature of early FSGS [7, 8] and has been postulated to contribute to the progression to end-stage kidney failure [6]. For example, in animal models of FSGS, aggravation of glomerular hypertrophy leads to further podocyte detachment and perpetuates glomerular injury [6]. Sirolimus (rapamycin) is a novel immunosuppressant with potent anti-trophic effects on intrinsic renal as well as immune cells, through its ability to specifically inhibit the rapamycin-sensitive (or raptor containing) mammalian target of rapamycin (mTOR) protein kinase complex [9–10]. These properties, as well as its theoretical advantages over calcineurin inhibitors [11], make sirolimus a promising candidate drug along with other targets of rapamycin inhibitors (TORi), to treat FSGS. In support of this possibility, a 6-month open-label prospective clinical trial of steroid-resistant and calcineurin-dependant FSGS, recently reported by Tumlin and colleagues [12], found that sirolimus reduced proteinuria and stabilized renal function in 12 out of 21 treated patients. In contrast, in other small pilot studies [13,14] sirolimus was associated with detrimental effects in FSGS, for reasons that are presently not clear.
To further understand the potential role of TORi in the treatment of FSGS, the main aim of this study was to test the hypothesis that sirolimus reduces kidney enlargement, glomerular hypertrophy and TEC proliferation in rats with early adriamycin nephropathy (AN) (a non-immune initiated model of FSGS induced with the podocyte and glomerular capillary wall toxin, doxorubicin hydrochloride) [15]. The secondary aims were to determine whether chronic sirolimus treatment attenuates renal dysfunction, fibrosis and inflammation or alters intraluminal protein cast formation, in this model.
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Induction of Adriamycin nephropathy
Adult male Wistar rats [
12 weeks in age; initial body weight (BW) 384 ± 6 g, mean ± SE] (n = 28)] were provided by the breeding colony at the Westmead Hospital Animal Care Department. All rats were housed in groups of two to three per cage in a temperature-and light-controlled environment, with free access to food and water. AN was induced by a single intravenous injection of doxorubicin hydrochloride (4.0 mg/kg) under anaesthesia (a single intraperitoneal injection of ketamine: xylazine 90 : 10 mg/kg) on day 0 [16]. A separate group of animals received a single injection of saline instead of doxorubicin and were designated control animals. All protocols were approved by the Animal Ethics Committee, Westmead Hospital, The University of Sydney (Protocol number 135.02-07). One rat died on day 0 following injection with doxorubicin, but otherwise there was no other mortality throughout the study.
Experimental design
On day 12, non-fasting rats were placed in metabolic cages for 14 h to determine baseline proteinuria. This was to ensure that the groups with AN were matched according to disease severity prior to treatment with sirolimus or the vehicle. Rats that did not develop proteinuria or were unable to be matched into two groups according to proteinuria were excluded from the study and all further analysis (n = 5). To eliminate the potential confounding effects of sirolimus on modulating acute podocyte injury (and causing concomitant alterations in proteinuria), as well as to mimic the clinical scenario more closely, the drug was administered to rats with established proteinuria. Therefore, on day 14, rats with AN were stratified into two groups (n = 8) according to BW and proteinuria, and received either daily subcutaneous injections of sirolimus (0.1 mg/kg, LC Laboratories, MA, USA) or an equal volume of the vehicle (dimethylsulphoxide, DMSO) until day 49. Groups of control rats were also divided into two groups (n = 3 each) according to BW, and also received either sirolimus or DMSO, under identical conditions. Sirolimus and the vehicle were prepared under sterile conditions and administered once daily (1200 h) by subcutaneous injection (100 µl volume).
Rats were monitored daily, and the BW and food intake were measured weekly. On day 48, animals were placed in metabolic cages for 14 h to determine urine protein and creatinine excretion. On day 49, animals received a single intraperitoneal injection of bromodeoxyuridine (BrdU, 50 mg/kg). Three hours later, animals were anaesthetized with ketamine and xylazine. A mid-line laparotomy was performed, blood was collected and nephrectomies were performed.
Renal function
Renal function and assessment of nephrotic syndrome was determined by urinary protein and creatinine excretion, and the serum creatinine, albumin and cholesterol. Serum or urine from each animal was analysed by an automated random access analyser (Roche/Hitachi Modular System, Sydney, Australia) by staff at the Institute of Clinical Pathology and Medical Research at Westmead Hospital. Creatinine was measured by the buffered kinetic Jaffe reaction; total protein was measured by the Biuret method; the total cholesterol determined by an enzymatic colourimetric assay measuring the production of 4-cholestenone; and the albumin was determined using an enzymatic colourimetric assay using bromocresol green as an anionic dye.
Renal histology and immunohistochemistry
Coronal sections of the kidney were immersion-fixed in methyl Carnoy's solution or 10% neutral-buffered formalin and embedded in paraffin. Light microscopy was performed on 4-µm sections of tissue stained with periodic acid–Schiff's (PAS) reagent and counterstained with haematoxylin or Gomory trichrome. For immunohistochemistry, sections were incubated with the primary antibody (16 h, 4°C) as previously described [10,12]. The following primary antibodies were used: (i) a murine anti-BrdU monoclonal IgG1 antibody (1 : 100, Clone BU-33, Amersham Biosciences, UK) (ii) a murine monoclonal IgG2 antibody against 
-smooth muscle actin (SMA), a cytoskeletal marker of myofibroblastic differentiation (1 : 1000, Sigma-Aldrich, St Louis, MO, USA); (iii) a murine monoclonal IgG1 antibody against ED-1, a cytoplasmic antigen present in monocytes, macrophages and dendritic cells (1 : 500, Serotec, UK); (iv) a murine anti-rat CD3 monoclonal IgM antibody (1 : 100, Clone 1F4, Serotec, UK) that detects the CD3 antigen on T lymphocytes.
Quantification of renal histology and immunohistochemistry
The number of tubules filled with casts was counted using a graticule (10 x 10, 0.5 mm2) fitted in the eyepiece of a microscope (BX51, Olympus Australia, Welshpool, WA, Australia) at a magnification of 100x. The cortex and medulla were assessed separately and the data were expressed as the number of casts per square millimetre in each section.
To assess the glomerular capillary tuft area, digital images were taken of glomeruli (magnification 400x) using a digital camera (Olympus DP11, 2MP, Olympus Australia, Welshpool, WA, Australia) and downloaded to a computer. The glomerular capillary tuft was carefully traced and the area in arbitrary units was determined using an image analysis program (Image J, NIH software). At least 20 glomeruli per section were assessed and the mean determined for each experimental group. To assess tubular dilatation, at least twenty cortical fields of sections from each animal were examined at 200 x magnification and graded according to a scale of 0–4 according to the proportion of the field occupied by dilated tubules, with 0 indicating no tubular dilatation, and 1, 2, 3, and 4, designated as <25%, 25–50%, 51–75% or 76–100% of the field, respectively. Finally, the number of BrdU-positive glomerular or TECs was counted in 20 glomerular cross-sections or tubulointerstitial fields.
Glomerulosclerosis was defined as collapse and/or obliteration of glomerular capillary tuft accompanied by hyaline material and/or an increase in matrix [17]. The severity of sclerosis for each glomerulus was graded from 0 to 4 as follows: 0, no lesion; 1+ sclerosis <25% of the glomerulus; 2+, 3+ and 4+ indicating sclerosis of 25–50%, >50–75%, and >75% of the glomerulus, respectively. A whole kidney average glomerusclerotic index was determined for all glomeruli on one section. Glomerular collagen content was assessed by computer-assisted image analysis of sections stained with Gomory trichrome. Digitalized images of glomeruli (at magnification 400x) were viewed on a computer, and the glomerular capillary tuft area was traced as the region of interest, and staining that was positive for collagen in this area was quantitated using image analysis software (Media Cybernetics, Silver Spring, MD, USA). At least 20 glomeruli per section were assessed. Interstitial collagen was assessed by semiquantitative scoring, as described for tubular dilatation.
For quantitation of periglomerular -SMA the number of positive cells in direct apposition to the glomerular compartment, was counted in at least 20 glomerular cross-sections per animal (magnification x 400). For interstitial -SMA, the percentage area occupied by positive staining was calculated using image analysis software, as described for glomerular collagen. The number of ED-1 and CD3-positive cells were counted in at least 20 glomeruli or 20 tubulointerstitial fields using a graticule, as described earlier.
Renal cortical transforming growth factor (TGF)-ß1
Protein extracts from each animal were prepared by homogenization of renal cortical tissue in a cold lysis buffer including protease and phosphatase inhibitors (pH 7.4; 50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Igepal, 0.01% SDS, 0.5 mM DTT, 1 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin A, 1 mM sodium orthovanadate, 1 mM sodium fluoride). TGF-ß1 was assessed in protein extracts, by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kit (Catalogue number MB 100, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. Samples were incubated in a 96-well plate pre-coated with anti-rat TGF-ß1 antibody. After washing, an alkaline phosphatase conjugate probe was then incubated in the wells, followed by a tetramethyl benzidine (TMB) substrate solution for colour development. The reaction was stopped with hydrochloric acid, and colour intensity was measured using a Multiskan EX plate reader (Thermo Electron, Waltham, MA, USA) at 450 nm with a correction factor at 570 nm. The concentration of TGF-ß1 was expressed as picograms per milligrams of total protein.
Statistical analysis
The data were analysed with JMP statistical software package (version 4.04, SAS institute, Carey, NC, USA). Data are expressed as mean ± SE. Comparisons between the control + vehicle and the AN + vehicle groups were performed with the Kruskal–Wallis test followed by a post-hoc analysis with the Tukey–Kramer HSD test. If the latter indicated a difference, the P-value was determined using the Wilcoxon test. A similar method of analysis was undertaken to determine the effect of sirolimus in AN. A P value of less than 0.05 indicated statistical significance.
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Sirolimus does not alter proteinuria, renal function or markers of nephrotic syndrome
By design, proteinuria on day 12 (prior to treatment with either the vehicle or sirolimus) was similar in the two groups of rats with AN (24-h protein excretion: AN + vehicle: 294 ± 28 vs AN ± sirolimus: 290 ± 38 mg/day, P = 0.93) (urine protein to creatinine ratio: AN + vehicle: 4539.6 ± 295.0 vs AN + sirolimus: 4506.1 ± 422.0 mg/mmol; P = 0.95). On day 49, the progression of renal dysfunction and proteinuria, and markers of nephrotic syndrome (hypoalbuminaemia and hypercholesterolaemia) were not altered by sirolimus in AN (Table 1). The mean percentage increase in the urine protein to creatinine ratio from day 12 to day 49 in AN was similar in both groups (AN + vehicle: 92 ± 23 vs AN + sirolimus: 104 ± 22%, P = 0.72).
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Sirolimus suppresses kidney enlargement in adriamycin nephropathy
Treatment with sirolimus did not significantly affect body growth (Figure 1A) or food intake (AN + vehicle: 19 ± 0 vs AN + sirolimus: 17 ± 2 g/day/rat, P = 0.32). However, the increase in kidney size in AN was almost completely prevented by treatment with sirolimus (P = 0.0016 and 0.0046 when compared with AN + vehicle for kidney weight and kidney to body weight ratio, respectively) (Figure 1B and C).
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Sirolimus does not potentiate intratubular cast formation in adriamycin nephropathy
Based on our previous observations on the effects of sirolimus in rats with protein-overload nephropathy (PON) [18], we first assessed whether sirolimus altered intratubular cast formation in AN. The formation of proteinaceous casts in distal tubules was a very common pathological finding in both the cortex and medulla of rats with AN. Casts had a homogenous appearance and were not present in proximal tubules. In some vehicle-treated rats with AN, casts containing distal tubules were significantly dilated and lined by flattened epithelial cells, suggesting intranephronal obstruction. Treatment with sirolimus did not potentiate or reduce protein-cast formation in AN in either the cortex or medulla (cortex: AN + vehicle 6.2 ± 1.7 vs AN + sirolimus 6.4 ± 1.3 casts/mm2, P = 0.93; Medulla: AN + vehicle 13.8 ± 4.3 vs AN + sirolimus: 9.1 ± 1.9 casts/mm2, P = 0.34).
Sirolimus attenuates glomerular capillary tuft enlargement, glomerulosclerosis and periglomerular myofibroblast accumulation in adriamycin nephropathy
The enlargement of the glomerular capillary tuft area in the AN + vehicle group (P < 0.05 compared with the control + vehicle group) was almost prevented by treatment with sirolimus (P = 0.0008 when compared with the AN + vehicle; Figure 2A and upper panel in Figure 3). The glomerulosclerotic index (Figure 2B) and periglomerular myofibroblast (-SMA-positive cell) accumulation (Figure 2C and lower panel in Figure 3) were reduced by 39 and 63%, respectively, in AN + sirolimus group when compared with the AN + vehicle (both P < 0.05). Glomerular cell proliferation, as assessed by the number of positive for BrdU-positive glomerular cells, was rare and similar in all groups, as was the glomerular collagen content (data not shown).
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Sirolimus attenuates tubular dilatation, tubular cell proliferation and interstitial fibrosis in adriamycin nephropathy
In the tubulointerstitium, there were focal areas of tubular dilatation in the AN + vehicle group, particularly in the outer medulla, and this was significantly reduced by sirolimus (P < 0.05 when compared with the AN + vehicle group (Figure 4A and upper panel Figure 5). The increase in TEC proliferation in AN + vehicle (P < 0.05, when compared with control + vehicle), as assessed by the number of BrdU-positive TECs, was reduced by 64% with sirolimus treatment in AN (P < 0.05 when compared with AN + vehicle, Figure 4B and middle panel Figure 5). Interstitial fibrosis in vehicle-treated rats with AN was characterized by focal areas of interstitial collagen deposition, and this was attenuated by 35% with sirolimus treatment (P < 0.01, Figure 4C). The interstitial fibrosis was also accompanied by the accumulation of peritubular myofibroblasts (
-SMA-positive cell) in the cortical interstitium, but this was not significantly altered by sirolimus (P = 0.40 compared with AN + vehicle) (Figure 4D and lower panel Figure 5).
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Sirolimus reduces renal cortical TGF-ß1 in adriamycin nephropathy
Renal cortical TGF- ß1 was increased by 6.7-fold in vehicle-treated AN rats compared with the control + vehicle group (P < 0.01), and reduced by 35% with sirolimus treatment (P < 0.01, compared with AN + vehicle, Figure 6).
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Sirolimus does not alter glomerular or interstitial inflammation in adriamycin nephropathy
The infiltration of the glomerulus and interstitium with monocytes (ED-1-positive cell) and lymphocytes (CD3-positive cell) in AN, was not significantly altered by sirolimus treatment (Figure 7).
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Discussion |
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The effects of sirolimus in chronic proteinuric renal diseases have received little attention. To our knowledge, this is the first report of the functional and pathological effects of sirolimus on the progression of experimental FSGS. The major finding of the present study was that treatment with sirolimus almost completely abrogated kidney and glomerular capillary tuft enlargement, and reduced tubule dilatation and TEC proliferation in rats with established AN. Sirolimus also exhibited anti-fibrotic effects in AN, as shown by reductions in glomerulosclerosis, periglomerular myofibroblast accumulation, interstitial fibrosis and renal cortical TGF-ß1 protein. The reduction in interstitial fibrosis in sirolimus-treated rats with AN was not associated with a decrease in peritubular myofibroblast infiltration. These findings are in agreement with in vitro studies of rat fibroblasts, where everolimus reduced mitogenesis, collagen and TGF-ß1 production but promoted myofibroblastic differentiation, as determined by SMA expression [19]. The clinical implications of these contradictory effects of mTOR inhibition on fibroblast turnover are not yet known.
Despite the attenuation in kidney hypertrophy and reduction in renal fibrosis, treatment with sirolimus did not alter the progression of renal dysfunction in AN. The most likely explanation for this is that sirolimus was not anti-proteinuric in AN. Because of this, progression promoters, such as hypercholesterolaemia, protein-mediated tubular injury and interstitial inflammation, as well as factors that determine impaired glomerular ultrafiltration [4,20], were probably not attenuated. The combination of sirolimus with conventional anti-proteinuric/renoprotective agents, such as angiotensin converting enzyme inhibitors and/or HMG CoA reductase inhibitors, might, however, overcome this neutral effect.
It is important to highlight that our study examined the effects of sirolimus during the early stages of AN, when tubulointerstitial scarring and glomerulosclerosis are minimal. This is relevant because the observations of Fervenza et al. [13] and Tumlin et al. [12] in humans suggest that sirolimus-unresponsive patients with FSGS tend to have a longer duration of disease and established interstitial fibrosis. Moreover, under these circumstances, sirolimus may be associated with a deterioration in renal function [12–14]. The mechanisms of these observations are presently unknown and warrant further investigation in animal models of FSGS, such as AN. One hypothesis to investigate is whether inhibition of renal cell proliferation impairs glomerular and tubulointerstitial repair during the later stages of disease [10]. Consistent with this possibility, in a model of mesangioproliferative glomerulonephritis induced in rats by the anti-thy1.1 antibody [21], inhibition of endothelial cell proliferation with everolimus was followed by glomerular capillary microaneurysm formation, glomerulosclerosis, crescent formation and worsening of the renal function. In FSGS, several experimental studies suggest that promotion of new glomerular capillary growth is important in the regression of established glomerulosclerotic lesions [22]. Therefore, it will be important to determine whether delaying treatment with sirolimus will remain beneficial in AN.
Although the serum levels of sirolimus were not measured in this study, in previous experiments [18], we have verified that these doses result in therapeutic concentrations (relevant to humans) in whole blood, and also do not cause kidney injury in normal rats [18]. Although these doses are much higher than that used in humans, such amounts are required to reduce cell proliferation in the kidney and mediate immunosuppression in small animals [23].
In a separate study, we have also investigated the effects of sirolimus on TEC proliferation in PON [18]. The latter is a non-immune-mediated model of proteinuria, induced by the repeated intraperitoneal injections of heterologous protein. In contrast to AN, reducing TEC proliferation with sirolimus in PON, unexpectedly caused acute renal failure due to a marked increase in intraluminal cast formation [18]. In the present study, however, intraluminal cast formation in AN was not affected by sirolimus. One likely explanation for this difference is that the increase in proteinuria and TEC proliferation are approximately 10–100-fold greater and have a more rapid onset in PON, than AN. Therefore, sirolimus-induced suppression of TEC proliferation may have greater adverse effects on tubule protein handling and intraluminal cast formation in PON. This effect may simply be due to a reduction in the tubule surface area available for intraluminal protein absorption, because we have not found evidence to suggest that alters the receptor-mediated endocytosis of albumin in cultured TECs [24].
In conclusion, the present study has demonstrated that the anti-renotrophic properties of sirolimus in AN was correlated with a reduction in renal scarring, suggesting that TORi have renoprotective effects when administered during the early stages of an FSGS pattern of chronic renal injury. These findings are similar to recent observations in the Heymann nephritis model of membranous nephropathy [25], but the unique observation of the current study was that the prevention in renal hypertrophy was demonstrated in a non-immune initiated model and independent of proteinuria [4,25]. Additional studies in AN are needed to address the effects of sirolimus on the podocyte-endothelial cell axis and angiopoietic growth factor production; whether combination therapy with angiotensin-converting enzyme inhibitors can further attenuate renal injury; and the efficacy of delayed treatment with sirolimus when glomerulosclerotic lesions and interstitial fibrosis are present at the time of drug initiation. These additional experimental studies will help further define the role of TORi in the management of human FSGS.
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This study was supported by research grants from the National Health and Medical Research Council of Australia (Grant no. 230500), the Medical Research Fund of Western Australia and the Fremantle Hospital Medical Research Foundation. Light microscope and digital imaging equipment and software for image analysis were provided by grants from Janssen–Cilag and Pfizer, respectively, to G.K.R. Portions of this work were presented in abstract form at the Annual Scientific Meetings of the Australian and New Zealand Society of Nephrology (Wellington, NZ, September 2005) and American Society of Nephrology (Philadelphia, November 2005). G.K.R. has received grant support from Wyeth-Ayerst (commercial manufacturer of sirolimus) to evaluate the efficacy of sirolimus in glomerulonephritis.
Conflict of interest statement. Mr Coombes reports no involvements that might raise the question of bias in the work reported or in the conclusions, implications or opinions stated.
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Accepted in revised form: 12. 3.07
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