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Keywords:

  • Kidney transplantation;
  • mTOR inhibitor;
  • proteinuria

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References

The development of proteinuria has been observed in kidney-transplanted patients on m-TOR inhibitor (m-TORi) treatment. Recent studies suggest that m-TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney-transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit-diaphragm. In a group of patients on ‘de novo’ m-TORi-treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m-TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m-TORi-treatment, a protocol biopsy performed before introduction of m-TORi was also available. The expression of nephrin in the pre-m-TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m-TORi(s). Proteinuria increased after the m-TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin. In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m-TORi(s) may affect nephrin expression in kidney-transplanted patients, consistently with the observation in vitro on cultured podocytes.


Abbreviations: 
ACE

angiotensin-converting enzyme inhibitor

ARB

angiotensinreceptor blacker

ATN

acute tubular necrosis

CNI

calcineurin inhibitor

m-TORi

mammalian target of rapamycininhibitor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References

The glomerulus is the filtering unit of the kidney, allowing the passage of water and small solutes but retaining proteins within the circulation. Several disease processes may cause impairment of the glomerular filtration barrier, resulting in glomerular leakage of proteins. In the past decade, several advances in the understanding of the mechanisms responsible for the glomerular perme-selectivity have been made. In particular, the unraveling of podocyte slit-diaphragm proteins has been viewed as a milestone in the understanding of glomerular function mechanisms (1). The podocyte slit diaphragm is a specialized cell–cell junction that spans the gap between adjacent interdigitating podocyte foot processes and forms the final component of the filtration barrier to macromolecules (2). The ‘slit-diaphragm complex’ consists of molecular components of the slit diaphragm, including nephrin and neph1, and slit diaphragm-associated proteins including podocin, CD2AP, ZO1 and a-actinin 4, which link the slit diaphragm to the actin cytoskeleton (2).

Besides genetic diseases with mutation of slit-diaphragm proteins, expression of these proteins has been found altered in several acquired glomerular diseases characterized by proteinuria such as membranous nephropathy, focal segmental glomerular sclerosis (FSGS) and diabetic nephropathy (3–6).

MTOR inhibitors (m-TORi) are a class of drugs with a high immunosuppressive activity, currently used in the field of transplantation (7). In addition, based on their antiproliferative activity they are now also used in neoplastic conditions (8).

Proteinuria was revealed as a complication of m-TORi therapy in renal transplantation as well as in other organ transplantation, in some cases resulting in nephrotic-range urinary protein excretion (9–12). Most of the initial observations derive from studies of conversion to m-TORi from calcineurin inhibitor (CNI)-based therapy, whose withdrawal may eventually unmask a preexisting altered glomerular perme-selectivity to proteins by increased renal blood flow and glomerular pressure (13,14). However, an increase of proteinuria was also described in patients converted to sirolimus from an azathioprine-based protocol (15) or in patients with de novo sirolimus-based CNI-free immunosuppressive regimen (16).

These data suggest a direct role of m-TORi(s) in the development of proteinuria. However, the mechanisms of its development are so far poorly elucidated. Recent studies provide evidence of a direct effect of sirolimus on podocyte behavior and integrity (17–19). Indeed, Letavernier et al. observed changes in cell phenotype and cytoskeleton reorganization as well as decreased vascular endothelial growth factor (VEGF) synthesis, Akt phosphorylation and WT1 gene and protein expression in primary cultures of human podocytes exposed to sirolimus (18). Moreover, prolonged sirolimus treatment reduces the expression of m-TOR and rictor, and formation of m-TORC2, resulting in a reduced phosphorylation of protein kinase B. In addition, the expression level of nephrin and transient receptor potential cation channel 6 as well as the cytoskeletal adaptor protein Nck significantly decreased in cultured human podocytes after incubation with sirolimus (17). However, ‘in vivo’ the administration of rapamycin was shown to have dual opposing effects on proteinuric experimental nephropathies (20). In a puromycin-induced proteinuria model in rats, rapamycin treatment further reduced the expression of podocin (nephrin was already in an extremely low expression in controls). Conversely in a chronic hyperfiltration and inflammatory model by mass reduction, rapamycin restored nephrin expression within glomeruli (20).

In this study, we analyzed the expression of nephrin in transplanted kidney biopsies of patients treated with m-TORi(s).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References

Patients

This retrospective study included renal biopsies of 23 renal transplants from deceased donor. In detail, we considered a group of 7 patients under treatment with m-TORi(s) since the beginning of the transplant (‘de novo’) (group A) with available a pretransplant donor biopsy and a posttransplant biopsy performed in occasion of increase of serum creatinine or appearance of proteinuria. Control transplant kidney biopsies of patients not on m-TORi(s) (group B) and matched for donor age, length of immunosuppressive therapy before biopsy and renal function, were used (n = 7). Then, we analyzed a third group of 9 patients (group C) started on a CNI-based immunosuppressive protocol with subsequent introduction of an m-TORi with pre-m-TORi and post-m-TORi addition/conversion biopsy performed in occasion of increase of serum creatinine or appearance of proteinuria, as well as donor kidney biopsy (the latter performed in 7 out of 9 patients). The main reasons of m-TORi conversion/addition were calcineurin inhibitor toxicity, the presence of past-history of neoplasia or, in one case, as part of a clinical trial protocol. In addition, 10 specimens were obtained from normal kidney portions of patients who underwent surgery for cancer and used as nephrin expression control baseline. These patients were selected for the absence of proteinuria and lack of glomerular lesions. All the subjects were informed about the nature, purposes, procedures and possible risks of the renal biopsy before their informed consent was obtained. The procedures were in accordance with the Helsinki declaration. The protein content of 24-h urinary samples was measured by pyrogallol red method. The creatinine concentration in plasma was analyzed by kinetic Jaffé method with a Beckman Synchron CX3. Analytical chemistry technique used to determine m-TOR-i levels was HPLC-MS/MS (ng/mL; liquid chromatography-mass spectromery; Waters Laboratories. Milford, MA, USA) for sirolimus and MEIA (ng/mL; Abbott Laboratories. Abbott Park, IL, USA) for everolimus.

Reagents

Polyclonal anti-human nephrin guinea pig antibodies to the extracellular fibronectin domain (GP-N1) and to the intracellular domain (GP-N2) were purchased from Progen Biotechnik (Heidelberg, Germany). Alexa Fluor 488 anti-guinea pig IgG were supplied by Molecular Probes (Leiden, The Netherlands). DMEM, BSA fraction V (tested for not more than 1 ng endotoxin/mg) and Sirolimus were purchased from Sigma Chemical Company (St Louis, MO, USA). FITC-conjugated secondary antibodies were purchased from Invitrogen (San Diego, CA, USA). FCS was from Euroclone Ltd (Wetherby, West Yorkshire, UK).

Immunofluorescence (IF) studies on kidney biopsies

Kidney biopsies were rapidly frozen in liquid nitrogen, and 2-μm-thick cryostat sections were fixed in 3.5% paraformaldehyde for 15 min and washed in PBS. The sections were blocked and labeled overnight at 4°C with a 1:100 dilution of antinephrin antibodies, washed in PBS, and incubated with Alexa Fluor 488 anti-guinea pig IgG for 30 min at room temperature. Control sections were stained with the secondary antibody only. Hoechst 33258 dye (Sigma) was added for nuclear staining. Confocal microscopy analysis was performed using a Zeiss LSM5 Pa model confocal microscope (Carl Zeiss International, Germany). The number of glomeruli available on each biopsy section for analysis of nephrin expression ranged between three and seven. Globally sclerosed glomeruli were not considered for IF analysis. Three nonsequential sections were examined for each specimen. The intensity of glomerular IF was detected in a 180-μm-diameter field (approximate size of glomeruli). Nephrin expression was analyzed semiquantitatively by measuring fluorescence intensity by digital image analysis after subtraction of the background fluorescence of tissue, using the LSM image analysis program (Carl Zeiss International) as previously described (6). Banff 07 classification of renal pathology was used (21). Vimentin staining with anti-human vimentin monoclonal antibody (Sigma) and relative expression quantitation within biopsy glomeruli were also performed.

‘In vitro’ studies on cultured podocytes

Primary cultures of human podocytes were established and lines of differentiated podocytes were obtained by infection with a hybrid Adeno5/SV40 virus. Podocytes were characterized and cultured in DMEM containing 10% FCS as described previously (22). Immunofluorescence on cultured podocytes was performed as described (6). Briefly, podocytes were plated in eight-well Permanox slide at a density of 30 000 cells per well. The following day, cell layers were rinsed with PBS. Sirolimus was solubilized in DMSO and stored at −20°C in the dark as previously described (23). Cells were incubated in the presence of sirolimus at 10 ng/mL for 1, 18, 24 hrs. Then, cells, fixed in 3.5% paraformaldehyde containing 2% sucrose for 15 min at 4°C, were incubated either with the antinephrin polyclonal antibody (10 μg/mL) or the irrelevant IgG1 isotypic control for 1h, followed by FITC-conjugated anti-guinea pig IgG for 40 min. All samples were counterstained by 0,5 μg/mL Hoechst for 30 s, mounted with antifade mounting medium (Vector Laboratories, Burlingame, CA, USA). Membrane localization of nephrin was analyzed by confocal microscopy.

For FACS analysis, after appropriate stimulation cells were detached with EDTA, fixed in 4% paraformaldehyde for 15 min at 4°C and stained with the antinephrin polyclonal antibody (10 μg/mL) or the irrelevant IgG1 isotypic control for 1h at 4°C, followed by FITC-conjugated anti-guinea pig IgG for 40 min. All the incubation periods and washings were performed using a medium containing 0,25% BSA. At the end of the staining, cells were newly washed and subjected to FACS analysis (Becton Dickinson, Mountain View, CA, USA).

For evaluating gene nephrin expression on podocytes in culture, quantitative reverse-transcription PCR was performed as described previously (23). RT-PCR was performed using total RNA from cells plated in 25 cm2 dishes at a concentration of 300 000 cells per dish, grown for 1 day, and then exposed to 10 ng/mL sirolimus for 1h and 24 h. Total RNA was extracted using TRI reagent according to the manufacturer's directions. The final RNA pellet was dissolved in 10 μL of diethyl pyrocarbonate water and stored at −70°C; 1 μg of total RNA was reverse-transcribed using a First Strand Synthesis Kit (Boehringer Mannheim, Indianapolis, IN, USA). Relative quantization by real-time PCR was performed using SYBR-green detection of PCR products in real-time using the iCycler from Biorad (Hercules, MA, USA). Sequence-specific oligonucleotide primers (purchased from MWG-Biotech AG, Ebersberg, Germany) were previously described (24): human nephrin: forward, 5′-AGGACCGAGTCAGGAACGAAT-3′; reverse, 5′-CTGTGAAACCTCGGGAATAAGACA-3′; Beta-2 microglobulin was used as experimental control. IQ SYBR Green Supermix was purchased from Biorad. Thermal cycling conditions were as follows: Activation of iTaq DNA polymerase at 95°C for 3 min, followed by 50 cycles of amplification at 95°C for 30 s, 61°C (for nephrin) or 60°C (for Beta-2 microglobulin) for 30 s and 72°C for 30 s. The relative expression of different mRNAs was determined by relative quantification: delta Ct = Ct target – Ct beta 2-microglobulin, where beta 2-microglobulin represents the reference housekeeping gene. The target quantity is given by x target = 2−deltaCt. Experiments were performed in triplicate.

For cytotoxicity assay, podocytes were cultured on 24-well plates (Falcon Labware, Oxnard, CA, USA) at a concentration of 5 × 104 cells/well and incubated with appropriate agonists and 250 μg/mL XTT (Sigma) in a medium lacking phenol red. The absorption values at 450 nm were measured in an automated spectrophotometer at different time points. All experiments were performed in triplicate.

Statistical methods

Statistical analysis was performed with SPSS (SPSS Inc. Chicago IL, vers. 12.01). Normal Continuous variables, not normally distributed, are presented as median (min-max). The difference between groups was analyzed with Mann–Whitney test. Categorical variables are presented as fraction and Pearson's χ2 test was employed to compare groups. Significance level for all tests was set at p < 0.05. Wilcoxon test was used where indicated.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References

In this study, we analyzed renal biopsies from kidney-transplanted patients and evaluated the expression of nephrin within glomeruli.

A first series of patients treated with m-TORi (group A) was analyzed comparing pretransplant donor biopsies with biopsies obtained posttransplant under these immunosuppressors (Table 1). All the patients in group A were on a m-TORi-based ‘de novo’ therapy and one out of five was also on CNI at low doses. In pretransplant biopsies, nephrin exhibited a glomerular epithelial staining pattern with a punctate/linear distribution along the peripheral capillary loops, similar to specimens from normal kidneys (Figure 1A and B). Control sections incubated with the nonimmune isotypic control antibody or with the appropriate labeled secondary reagent without the primary antibody were always negative (data not shown). In glomeruli of the same kidneys biopsied in the posttransplant period during treatment with m-TORi, an extensive reduction of staining of nephrin was observed (Figure 1C). Accordingly semiquantitative analysis of nephrin expression documented a significative decrease in nephrin expression (Figure 2). This finding was present in all patients, treated either with sirolimus (4 out of 7) with an average sirolimus blood level at biopsy of 7.7 ± 0.5 ng/mL, or everolimus (3 out of 7) with an average everolimus blood level at biopsy of 5±1 ng/mL. Histologically, there were no lesions compatible with a diagnosis of FSGS or other glomerulopathy in this series (Table 1).

Table 1.  Patients characteristics
GroupABC
  1. Data are reported as median (min-max). Abbreviations: mTORi = mammalian target of rapamycin inhibitor; CNI = calcineurin inhibitor; srl = sirolimus; eve = everolimus; MMF = mycophenolate mophetil; tac = tacrolimus; CsA = cyclosporine A; ATN = acute tubular necrosis; sCr = serum creatinine; st = steroid.

TypologyDe novo’ m-TORiControl m-TORi-untreatedSwitch to m-TORi
Number of patients779
Recipient age (years)60 (53–73)55 (43–58)57 (31–66)
Donor age (years)67 (56–76)67 (61–74)63 (45–77)
Immunosuppressive treatment at time of renal biopsy (no. of patients)-Eve, MMF, st (3)-Tac, MMF, st (4)PreswitchPostswitch
-Srl, MMF, st (1)-Tac, st (1)-Tac, MMF, st (4)-Eve, MMF, st (1)
-Srl, MMF (1)-CsA, MMF, st (1)-Tac, st (2)-Srl, MMF, st (2)
-Srl, st (1)-CsA, Aza, st (1)-CsA, MMF, st (3)-Srl, st (2)
-Srl, tac (1)  -Srl, tac, st (2)
   -Srl, tac (1)
   -Eve, tac, st (1)
Length of treatment before biopsy (days)m-TORi: 125 (8–1826)CNI: 122 (30–1644)mTORi : 154 (22–1769)
sCr (mg/dL) at biopsy1.7 (0.9–7)2.6 (1.68–4.8)PreswitchPostswitch
3 (1.1–5)3 (1.4–5)
Proteinuria (g/24 h) at biopsy0.48 (0.1–1.7)0.4 (0.1–1.8)PreswitchPostswitch
0.23 (0.11–2)1.8 (011–4.3)
Histologic findings at biopsy-ATN, interstitial fibrosis (1)-CNI toxicity (1)PreswitchPostswitch
- CNI toxicity (4)- CNI toxicity (2)
-interstitial fibrosis (2)-ATN (1)- interstitial fibrosis (3)- ATN (1)
- acute T cell-mediated rejection (grade IA)(1)-interstitial fibrosis, and artheriosclerosis (3)-cholesterinic embolia (1)- interstitial fibrosis (2)
- acute T cell-mediated rejection (grade IB) and ATN (1)-intersitial fibrosis (1)- ATN (1)- ischemic glomerular lesions (1)
- acute T cell-mediated rejection (grade IB) (1)-artheriosclerosis (1) - acute T cell mediated rejection (grade IB) (1)
- acute T cell-mediated rejection (grade IIA) (1)  - acute T cell-mediated rejection (grade IIA) (1)
   -chronic allograft arteriopathy (1)
image

Figure 1. IF staining for nephrin in glomeruli of: normal kidney specimen (A); Group A, pretransplant donor kidney biopsy (B) and posttransplant kidney biopsy of the same patient under ‘de novo’ mTORi treatment (C); Group B, pretransplant donor kidney biopsy (D) and posttransplant kidney biopsy of the same control patient untreated with mTORi (E); Group C, pretransplant donor kidney biopsy (F), posttransplant kidney biopsy before (G) and after introduction of m-TORi therapy (H) in a representative patient. Original magnification×400. Hoechst 33258 blue dye was added for nuclear staining. Control sections incubated with the nonimmune isotypic control antibody were always negative (I).

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image

Figure 2. Semiquantitative analysis of nephrin expression as detected by IF staining in glomeruli of patients under ‘de novo’ m-TORi treatment (Group A), and of control kidney-transplanted subjects untreated with m-TORi (Group B), measured in the pretransplant donor kidney biopsies and in the posttransplant kidney biopsies. In addition, nephrin in glomeruli of Group C patients measured in the pretransplant donor kidney biopsy, and in the biopsies pre- and post-m-TORi introduction is shown.

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Control transplant biopsies of patients untreated with m-TORi (group B) and matched for donor age, length of treatment before posttransplant biopsy, failed to present a significative loss of nephrin expression in comparison to donor kidney biopsy and showed a similar pattern and amount of nephrin expression, that was in turn superimposable to that of normal kidney specimens (Figure 1D, E and A, respectively). In contrast to nephrin expression at the posttransplant biopsy, the expression of vimentin, a constitutive cytoskeleton component, was not different in relative fluorescence intensity between group A (44.2 ± 4.5) and B (48.5 ± 2.1). The histological diagnosis of the posttransplant biopsies are shown in Table 1.

In these two groups, the extent of proteinuria was not significantly different among groups and was not related to the expression of nephrin (data not shown) and only one patient per group was on angiotensin-converting enzyme inhibitor (ACEi) or angiotensin receptor blocker (ARB).

Then, we analyzed a third group of 9 patients (group C) started on a CNI-based immunosuppressive protocol with subsequent introduction of an m-TORi. In 4 out of 9 patients the CNI was continued at low doses after addition of the m-TORi, whereas in the others a full conversion was performed. A biopsy performed before the m-TORi introduction was currently included in our m-TORi clinical protocol and thus it was possible to evaluate a pre-m-TORi and post-m-TORi addition/conversion biopsy as well as donor kidney biopsy (the latter performed in 7 out of 9 patients). In pre-mTORi biopsies, the glomerular pattern of nephrin expression was similar to pretransplant donor biopsies and to normal kidney tissue specimens (Figure 1G, F and A, respectively). The expression of nephrin was significantly reduced after the beginning of the m-TORi (Figures 1H and 2C), with a granular pattern along the peripheral capillary loops. In none of these biopsies, the features of FSGS or other glomerular nephropathies were observed. The histological diagnosis of the posttransplant biopsies on m-TORi(s) are described in Table 1. Out of 9, 7 patients were on sirolimus with an average sirolimus level ± standard deviation at biopsy of 7.8 ± 1.9 ng/mL, and 2 on everolimus with an average everolimus level ± standard deviation at biopsy of 4.5 ± 0.7 ng/mL.

Since in some patients within the study groups acute rejection or ATN were present in the bioptic diagnosis (Table 1), we analyzed whether these conditions may themselves influence the reduction of nephrin in glomeruli in a separate set of biopsies from transplanted patients not on m-TORi but with a diagnosis of acute T cell-mediated rejection (6 cases) or ATN (3 cases, 2 of which also with CNI nephrotoxicity). Nephrin expression in these biopsies (relative fluorescence intensity: 53.2 ± 8.4 acute rejection biopsies and 58.5 ± 6.8 for ATN biopsies) was not significantly different from normal kidney controls or group B posttransplant biopsies.

As shown in Figure 3, there was a significative difference in the amount of proteinuria before and after mTORi initiation, despite the fact that 4 out of 9 patients were on ACEi or ARB. Notably, 2 patients displayed normal protein excretion (0.13 and 0.1 g/24 h) at the post-m-TORi biopsy time despite a reduction of nephrin expression on m-TORi treatment.

image

Figure 3. Significant change (p = 0.012, Wilcoxon test) in proteinuria extent at time of the kidney biopsies performed before and after switch to m-TORi in Group C patients.

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Finally, we performed ‘in vitro’ experiments with a human podocyte cell line to study changes of nephrin expression under culture with sirolimus. Unstimulated podocytes expressed nephrin in a fine punctuate diffuse pattern (Figure 4A). Nephrin was analyzed by confocal microscopy on the surface of nonpermeabilized cells. To determine whether nephrin expression was affected by sirolimus, we incubated podocytes with sirolimus at 10 ng/mL for 1, 18 and 24 h. At 1 h, sirolimus slightly modified the surface expression of nephrin (Figure 4C). Conversely, at 18 and 24 hrs sirolimus induced a pronounced reduction of nephrin expression (Figure 4B and C). Quantitative PCR analysis of mRNA showed a reduction in nephrin mRNA extracted from podocyte cultures on sirolimus challenge (Figure 4D). The effect was present after 1 h incubation and became more relevant after 24 h (Figure 4D). This set of experiments suggests reduced synthesis as the cause of nephrin loss by sirolimus. Notably, no significant cytotoxic effect was exerted by sirolimus at doses reflecting the current levels adopted in the clinical setting (Figure 4E).

image

Figure 4. A and B: IF staining for nephrin in unstimulated cultured podocytes (A) or podocytes incubated with 10 ng/mL sirolimus for 24 h (B). C: FACS analysis of nephrin expression (solid plot) by podocytes unstimulated (CTRL) or incubated with 10 ng/mL sirolimus for different periods of time (1 h, 18 h and 24 h). Control staining with irrelevant antibody is shown in line plot. D: comparison of expression of nephrin mRNA extracted from cultured podocytes unstimulated (CTRL) or incubated with 10 ng/mL sirolimus for different periods of time (1 h and 24 h). E: cytotoxicity assay on cultured podocytes incubated with vehicle alone or sirolimus at different concentrations for 48 hrs.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References

Several lines of evidence rising from ‘in vitro’ studies and ‘in vivo’ experimental animal models indicate a direct effect of m-TORi(s) on the glomerular pathophysiology (17–20). In this study, we report the reduction of expression of glomerular nephrin in biopsy specimen of kidney-transplanted patients treated with m-TORi(s). This phenomenon is also observed ‘in vitro’ where expression of nephrin is impaired in cultured human podocytes after incubation with sirolimus. In ‘in vivo’ experimental models of nephropathies, the administration of rapamycin was shown to have dual opposing effects (20). In a puromycin-induced proteinuria model in rats, rapamycin treatment further reduced the expression of podocin (nephrin was already in an extremely low expression in controls). Conversely in a chronic hyperfiltration and inflammatory model by mass reduction, rapamycin restored nephrin expression within glomeruli. These results may suggest that different factors may contribute to vectorially define the effect of m-TORi(s) on glomerular podocytes. In the human transplantation setting that we analyzed, several conditions may be copresent at the time of initiation of the m-TORi therapy particularly in kidneys from marginal donors, such as hyperfiltration and preexisting proteinuria. However, these factors ‘alone’ did not supposedly influenced nephrin expression as suggested by the analysis of the control transplant and pre-mTORi biopsies, in contrast to the findings observed under m-TORi treatment. Anyway, we cannot rule out their contribution to facilitate the effect of m-TORi in reducing nephrin expression. Indeed several observations suggest that proteinuria induced by sirolimus occurs mainly in the setting of a preexisting graft damage (25–28).

In a recent work, Letavernier et al. demonstrated that sirolimus at high dosage is able to induce de novo FSGS in renal transplant patients (29). In that study immunohistochemistry analysis showed podocyte alterations such as absent or diminished expression of the podocyte-specific epitopes synaptopodin and p57, reflecting dedifferentiation, and acquired expression of cytokeratin and PAX2 reflecting a immature fetal phenotype (29). We observed a reduction of nephrin expression in the absence of FSGS histologic lesions, suggesting that there are multiple levels of damage that may result from m-TOR inhibitor activity on glomerular podocytes and this may probably depend also on the intensity of exposure to sirolimus. In our study, both sirolimus and everolimus levels at biopsy were in ranges non considered high for current protocols. With the limits of what in vitro experiments may reflect in vivo events, these doses were not associated to direct cell cytotoxicity on cultured podocytes.

Interestingly, a part of m-TORi treated patients had no significant proteinuria at the time of biopsy, although they had reduced nephrin expression. This observation may potentially have different explications: (1) partial nephrin reduction alone may not suffice for loss of perme-selectivity by the glomerular capillary wall; (2) an individual threshold of nephrin expression may exist to allow development of proteinuria. These hypothesis are supported by the fact that nephrin reduction has been found already present in the early stages of certain glomerulopathies, such as the diabetic one, in the presence of microalbuminuria only (6). The function of this transmembrane protein is not only exerted by its extracellular portion in the slit diaphragm but also by its intracellular domain that interact with the cytoskeleton and with several adaptor-signaling proteins (2), and therefore the impact of the physical loss of this molecule on podocyte activities may depend not only on its extent but also on the functional impairment of the signal transduction network within the cell.

Last, our study involved transplants from marginal donor kidney and many patients at time of biopsy had suboptimal graft function and preexisting proteinuria. Therefore, it remains to be determined if our observation may be extended to optimal donor kidney transplantation. However, most part of our current deceased donor transplants are from marginal donors and, in addition, it is a frequent practice to switch to m-TORi when chronic allograft nephropathy is suspected. Thus, examining glomerular effect of m-TORi may be relevant particularly in the kidney transplant patient category that we studied. Further studies should be aimed to determine if nephrin loss in the absence of frank proteinuria may be predictive of proteinuria development in the long term.

In conclusion, our results suggest that m-TORi(s) may affect nephrin expression in kidney-transplanted patients, consistently with the observation in vitro on cultured podocytes. However, these findings were not correlated to any clinical effect, suggesting that nephrin downregulation by m-TORi(s) either requires other cofactors to lead to proteinuria or, as ‘minimal hypothesis’, is an epiphenomenon unlinked to proteinuria, but anyway demonstrating drug-induced podocyte alterations.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References

Funding Sources: This work was supported by Italian Government Miur PRIN project 2008 to L.B. and FIRB project to G.C., ‘Ricerca Finalizzata – Regione Piemonte’ to L.B., G.C., B.B. and G.P.S, and Local University Grants (‘ex60%’public Funds from the University of Torino, Italy) to L.B., V.C., G.C., G.P.S., B.B. and G.M.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. References