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J Am Soc Nephrol. Dec 2010; 21(12): 2157–2168.
PMCID: PMC3014029

The Anti-Fibrotic Effect of Mycophenolic Acid–Induced Neutral Endopeptidase

Abstract

Mycophenolic acid (MPA) appears to have anti-fibrotic effects, but the molecular mechanisms underlying this are unknown. We prospectively studied 35 stable kidney transplant recipients maintained on cyclosporine and azathioprine. We converted 20 patients from azathioprine to enteric-coated mycophenolate sodium (EC-MPS) and continued the remaining 15 patients on azathioprine. Exploratory mRNA expression profiling, performed on five randomly selected EC-MPS patients, revealed significant upregulation of neutral endopeptidase (NEP), which is an enzyme that degrades angiotensin II. We confirmed these microarray data by measuring levels of NEP expression in all subjects; in addition, we found that NEP gene expression correlated inversely with proteinuria. In an additional 33 patients, glomerular and tubular NEP protein levels from renal graft biopsies were significantly higher among the 13 patients receiving cyclosporine + EC-MPS than among the 12 patients receiving cyclosporine + azathioprine or 8 patients receiving cyclosporine alone. Glomerular NEP expression inversely correlated with glomerulosclerosis and proteinuria, and tubular NEP expression inversely correlated with interstitial fibrosis. Incubation of human proximal tubular cells with MPA increased NEP gene expression in a dose- and time-dependent manner. Moreover, MPA reduced angiotensin II–induced expression of the profibrotic factor plasminogen activator inhibitor-1, and a specific NEP inhibitor completely reversed this effect. Taken together, our data suggest that MPA directly induces expression of neutral endopeptidase, which may reduce proteinuria and slow the progression of renal damage in kidney transplant recipients.

Over the last decade, the continuous progress in the development of immunosuppressive agents has enhanced both the efficacy and safety of anti-rejection regimens after renal transplantation, leading to a dramatic reduction in the incidence of acute rejection.1 This significant improvement in short-term graft outcome was not followed by a similar advance in preventing graft loss caused by chronic graft injury.2,3 The addition of mycophenolic acid (MPA) into contemporary immunosuppressive regimens represented an important step forward in the development of new therapeutic protocols in kidney transplantation. MPA exerts its immunosuppressive effect by inhibiting the inosine 5′ monophosphate dehydrogenase, a key enzyme involved in the first step of de novo synthetic pathway of guanine nucleotides.46 Inosine 5′ monophosphate dehydrogenase inhibition has a potent cytostatic effect on both T and B lymphocytes, because these cells are critically dependent for their proliferation on de novo purine synthesis, differently from other cell types that can use salvage pathways.4 In addition, MPA suppresses glycosylation of adhesion molecules, limiting recruitment of lymphocytes and monocytes, and depletes tetrahydrobiopterin, reducing inducible nitric oxide synthase activity, nitric oxide production, and peroxynitrite-induced tissue damage.7

Two MPA formulations, mycophenolate mofetil and enteric-coated mycophenolate sodium (EC-MPS), have been extensively introduced in association with standard or low-dose calcineurin inhibitors (CNIs) and corticosteroids. They have largely replaced the purine antagonist azathioprine (AZA) in post-transplant immunosuppressive therapy.4,8,9 The use of MPA, instead of AZA, has significantly reduced the incidence of biopsy-proven acute rejection in the early post-transplant period10 and has significantly improved long-term patient and graft survival.11,12 In addition, the introduction of MPA along with CNI minimization/withdrawal has been successfully applied to patients with chronic allograft dysfunction and CNI toxicity, resulting in a long-term stabilization of their renal function.1316

Several in vivo studies reported that MPA significantly reduces mesangial matrix deposition, interstitial fibrosis, myofibroblast infiltration, and glomerular sclerosis in patients with chronic graft rejection, CNI nephrotoxicity, and glomerulonephritides.1720 Even in different models of non–immune-mediated renal diseases, including unilateral ureteric obstruction and the 5/6-remnant kidney, MPA was shown to reduce glomerular sclerosis and interstitial fibrosis.21,22 These results suggest that MPA may exert an anti-fibrotic action, although the molecular mechanisms underlying this effect are still largely unknown. Thus, the aim of this study was to investigate such mechanisms, using an exploratory high-throughput genomic approach.

RESULTS

Genomic Profile of Peripheral Blood Mononuclear Cells after Conversion from AZA to EC-MPS

The first step of the study was to perform an exploratory/hypothesis-generating analysis, using the microarray technology, in five randomly selected patients included in the training group. Statistical analysis showed that 16 genes were significantly modulated after 3 months of treatment with MPA. Among these genes, 6 were up-regulated and 10 were down-regulated 3 months after conversion (T1) compared with baseline (T0) (Table 1). Principal component analysis, using the 16 genes, clearly showed the degree of genomic change after conversion from AZA to EC-MPS treatment (Figure 1A).

Table 1.
Top-ranked genes selected by microarray analysis
Figure 1.
Conversion from azathioprine to EC-MPS modifies PBMC transcriptomic profile and induces NEP gene expression. Principal components analysis (PCA) (A) and NEP gene expression by real-time PCR in PBMCs (B). (A) PCA plot using the 16 selected gene probe sets ...

Neutral endopeptidase (NEP) was among the most significantly up-regulated genes. This result was confirmed measuring NEP expression by real-time PCR in the patients converted to EC-MPS (excluding those selected for the microarray study; Figure 1B). No difference between T0 and T1 in NEP expression was found in those patients continuing AZA treatment (Figure 1B).

Correlation between NEP Expression, Proteinuria, and Serum Creatinine Levels in the Training Group

Interestingly, NEP gene expression levels, measured in PBMCs from all patients (n = 35) in the training group, were significantly and inversely correlated with daily proteinuria at both T0 (R2 = 0.21, P = 0.005) and T1 (R2 = 0.12, P = 0.042; (Figure 2, A and B, respectively). No significant correlations were observed between NEP expression and serum creatinine levels at both time points (Figure 2, C and D).

Figure 2.
PBMC NEP gene expression correlates with proteinuria but not with graft function. Plots represent the correlations between NEP gene expression, proteinuria (A and B), and serum creatinine levels (C and D) in 35 patients included in the training group ...

Graft Expression for NEP in the Independent Test Group

Because NEP is known to be expressed at the renal level, we investigated whether intragraft NEP protein levels were influenced by long-term MPA treatment. Glomerular NEP protein expression, localized at the podocyte level, was significantly higher in the cyclosporine (CsA) + EC-MPS group compared with CsA + AZA (P = 0.02) and CsA-only patients (P = 0.03). No statistically significant difference was observed between CsA + EC-MPS patients and deceased donors (P = 0.4), as well as in the comparison between CsA + AZA and CsA-only groups (P = 0.9; Figure 3). Tubular NEP protein expression, mainly localized at the brush border level, was significantly higher in the CsA + EC-MPS group compared with the other two groups of patients. Deceased donor renal biopsies presented higher tubular NEP protein levels compared with the CsA + EC-MPS group (P = 0.01; Figure 4).

Figure 3.
Glomerular NEP protein expression is increased in EC-MPS–treated patients. NEP protein expression at the glomerular level in renal graft biopsy of deceased donors (DD)- (A), CsA only– (B), CsA + AZA– (C), and CsA + EC-MPS– ...
Figure 4.
Tubular NEP protein expression is increased in EC-MPS–treated patients. Tubulointerstitial NEP protein expression (A–D) and Masson trichrome staining (E–H) of graft biopsies of deceased donors (DD)- (A and E), CsA only– ...

Correlation between NEP Expression and Clinical and Histologic Features in the Independent Test Group

Glomerular NEP protein levels measured in the independent test group (33 patients) were significantly and inversely correlated with daily proteinuria evaluated at the time of graft biopsy (R2 = 0.29, P = 0.001; Figure 5A) and with the degree of glomerular sclerosis (R2 = 0.21, P = 0.007; Figure 5B). The area of specific tubular staining was inversely correlated with the extent of interstitial fibrosis (R2 = 0.27, P = 0.0045) quantified in graft biopsies of the independent test group (Figure 5C).

Figure 5.
Glomerular and tubular NEP expression correlate with the degree of glomerulosclerosis and interstitial fibrosis, respectively. Plots represent the correlation between glomerular NEP protein expression and proteinuria (A) and glomerular sclerosis levels ...

Concordance between PBMCs and Renal NEP Expression

We next evaluated NEP gene expression in PBMCs harvested at the time of graft biopsies along with NEP protein expression within the graft in an independent validation group (10 patients). Interestingly, NEP gene expression measured in PBMCs was significantly correlated with both glomerular (R2 = 0.21, P = 0.004) and tubular (R2 = 0.86, P = 0.001) NEP protein level.

Effect of MPA on NEP mRNA Expression in Stimulated Cultured Human Proximal Tubular Cells (HK2)

To clarify whether NEP up-regulation observed in PBMCs and glomerular and renal tubular cells in vivo was caused by a direct effect of MPA, we evaluated the influence of this drug on NEP gene expression in HK2, an immortalized line of human proximal tubular cells. MPA induced a dose- (Figure 6A) and time-dependent increase of NEP gene expression (Figure 6B), whereas CsA incubation did not influence its mRNA abundance (data not shown).

Figure 6.
MPA induces NEP expression in cultured HK2 cells and NEP activity is necessary for MPA-induced inhibition of angiotensin II-stimulated PAI-1 gene expression. Dose response (A) and time course (B) of MPA effect on NEP gene expression in HK2 cells. (A) ...

Effect of MPA and NEP Inhibition on Angiotensin II–Induced Plasminogen Activator Inhibitor-1 Gene Expression in HK2

Because NEP is an angiotensin II–degrading enzyme,23 in an attempt to define the molecular link between NEP overexpression and reduced interstitial fibrosis observed in MPA-treated patients, we investigated in vitro the effects of MPA on angiotensin II–induced expression of plasminogen activator inhibitor-1 (PAI-1), a powerful profibrotic mediator. As previously shown,24 angiotensin II induced a marked increase of PAI-1 mRNA abundance (P = 0.003). MPA preincubation significantly reduced angiotensin II–induced PAI-1 gene expression (P = 0.007). Phosphoramidon, a potent NEP inhibitor, at a concentration of 5 (P = 0.005) and 10 μM (P = 0.006), completely reversed this MPA effect (Figure 6C).

DISCUSSION

Despite the introduction of several new immunosuppressive drugs and treatment strategies, chronic allograft damage remains the main obstacle to successful long-term graft outcome.25 In vitro and in vivo studies indicate that MPA may have a positive effect in preventing progressive glomerular and tubulointerstitial changes, leading to chronic graft damage.10,20,2630 The introduction of MPA along with CNI withdrawal or dose reduction improves renal transplant dysfunction, especially when CNI nephrotoxicity is the primary cause of graft functional impairment.31,32 Although the reduction in CNI exposure allowed by MPA may play a role, the mechanisms underlying these clinical and histologic effects of MPA are still largely unclear. Experimental evidence suggests a direct effect of MPA on the pathogenic mechanisms of progressive renal damage. Indeed, different groups showed that MPA reduces the extent of interstitial fibrosis and glomerular sclerosis also in the 5/6-nephrectomy model, a non–immune-mediated model of progressive renal damage.22,3335

To investigate the possible biologic mechanisms underlying the clinical action of this immunosuppressive drug, we decided to apply an exploratory genomic analysis performed as an hypothesis-generating approach. The transcriptomic screening has been extensively used in transplantation to identify biomarkers for an early diagnosis of acute36 and chronic rejection,37 but presently, little is known about the change in the genomic profile induced by pharmacologic intervention in transplant recipients. Microarray analysis showed that 16 genes were modulated by the conversion from AZA to EC-MPS in the immunosuppressive regimen of our renal transplant population. The number of patients included in the genomic analysis may be considered a limitation of our study. However, investigating the changes induced by EC-MPS introduction in the same patient, using a pre- and a postconversion biologic sample, significantly increased the statistical power of our analysis. In addition, to overcome this issue, we applied a novel statistical approach previously validated by our group.38 Finally, the microarray results were confirmed by real-time PCR on a larger patient population.

Among the top selected genes, we focused our attention on NEP. Interestingly, our results showed that chronic MPA treatment could cause a significant increase of NEP protein expression also within the graft. NEP is a zinc-containing metallo-endopeptidase involved in the degradation of several regulatory peptides and plays an important role in turning off peptide signaling events on the cell surface.23,39,40 It is expressed on the surface of polymorphonuclear leukocytes and in several organs, including the kidney, in which it is localized, with a high density, on the brush border of proximal tubular cells and on the cell surface of glomerular podocytes.41,42 Debiec et al.42,43 showed that NEP may represent the target antigen in a rare subset of patients with alloimmune antenatal membranous nephropathy.

Additionally, NEP is primarily involved in the enzymatic degradation of angiotensin II.23 Thus, an up-regulation of NEP expression may induce increased angiotensin II turnover. The inhibition of angiotensin II generation is well known to reduce proteinuria and slow down the progression of renal damage in experimental models of progressive renal disease and in human nephropathies.44 Several studies on transplant and glomerular diseases described a significant improvement in proteinuria after MPA introduction, being equally effective to angiotensin converting enzyme (ACE) inhibitors.45 Therefore, it is conceivable that glomerular NEP up-regulation induced by EC-MPS treatment may play a role in this beneficial effect of MPA. This hypothesis is supported by a previous observation showing that inhibition of NEP by candoxatrilat may induce a significant increase in circulating angiotensin II together with a marked raise in urine albumin excretion in stable renal transplant patients.46 A similar proteinuric effect was observed with NEP inhibition also in patients with chronic renal failure.47

Angiotensin II has been suggested to also play a key role in the progression of tubulointerstitial damage in chronic kidney disease.48 NEP may also play a role in the modulation of local level of angiotensin II within the tubulointerstitial area because it is closely colocalized with ACE on the brush border of proximal tubular cells.49 The hypothesis that MPA may influence renal damage progression through the modulation of angiotensin II degradation is also suggested by the observation that MPA reduces the progression of renal failure in an animal model of angiotensin II–dependent renal injury induced by the simultaneous overexpression of renin and angiotensinogen genes.50 Finally, the in vitro data on the ability of NEP inhibition to reverse the effect of MPA on angiotensin II–induced PAI-1 gene expression further confirm our hypothesis. Interestingly, we confirmed in vivo the effect of MPA on PAI-1 expression. Indeed, we evaluated by immunohistochemistry PAI-1 protein expression in the graft biopsies of CsA + AZA– and CsA + EC-MPS–treated patients and observed a decreased in kidney PAI-1–specific staining in the latter group, although the difference did not reach the statistical significance (P = 0.12; data not shown).

It is conceivable that the increased NEP expression at the glomerular and tubular levels may be the results of a reduced CNI nephrotoxicity. However, our observation, that MPA can directly induce NEP expression in cultured tubular cells, clearly suggests a direct renal effect of the drug. A potential bias of this study may be represented by the fact that analyses were carried out in different sets of patients. Nevertheless, we believe that our study design, confirming the observation in independent groups of patients (training, test, and validation group), addresses this potential bias and further validates the consistency of our observation. Although our data suggest that NEP overexpression might correlate with better clinical outcomes, this effect may not be always beneficial. In fact, clinical evidence suggests that simultaneous NEP and ACE inhibition, increasing natriuretic and vasodilatory peptides, is extremely effective in reducing BP. In addition, this double treatment has been also shown to slow down the progression of vascular damage.51

Taken together, our findings contribute to better understand the molecular mechanisms of MPA in solid organ transplantation and also underlie the promising use of this drug in the treatment of a variety of proteinuric glomerulonephritides.

CONCISE METHODS

Patients and Treatment

A total of 68 stable renal transplant recipients, after signing an informed consent form, were included in this study and divided into a training-group (n = 35) and an independent test group (n = 33). A third independent group (n = 12) was used only for biologic validation.

Patients with biopsy-proven acute rejection in the last 6 months before the study or active infections, gastrointestinal disorders, or malignancies at the time of enrollment (T0) were excluded from the study.

Training Group.

This population was used for the microarray and gene expression studies on PBMCs. Thirty-five patients included in this group were, at T0, on standard maintenance immunosuppression with CsA (mean ± SD of daily dose: 160.5 ± 36.44 mg, Neoral; Novartis, Basel, Switzerland), prednisone (5 mg daily), and AZA (50 mg daily). Twenty patients with hyperuricemia were converted, at T0, from AZA to EC-MPS (720 mg twice daily, Myfortic; Novartis) because of their need to start allopurinol therapy to avoid clinical complications (e.g., gout). This strategy was planed to prevent the development of allopurinol adverse events (e.g., leukopenia) because of the inhibition of the purine catabolic enzyme xanthine oxidase. However, to avoid confounding factors, allopurinol treatment did not start until the end of our study (3 months). The remaining 15 patients continued AZA treatment. The main baseline clinical and demographic characteristics of the two groups are summarized in Table 2. Follow-up time was 3 months (T1). We did not observe any significant difference in the main clinical and laboratory parameters between T0 and T1 in both AZA and EC-MPS treatments (Table 2). For the microarray analysis, we randomly selected five patients converted from AZA to EC-MPS treatment. The main clinical and laboratory findings were not statistically different between these five patients and the whole group of patients converted to EC-MPS (data not shown).

Table 2.
Clinical and laboratory findings of the training group at the two different time points of the independent test group and the independent validation group

Independent Test Group.

This group was used for the gene expression study on the graft. Among the patients included in this group, 8 were treated with CsA and prednisone (CsA only), 12 with CsA, prednisone, and AZA (CsA + AZA), and 13 with CsA, prednisone, and EC-MPS (CsA + EC-MPS). All patients underwent renal biopsy for chronic allograft dysfunction. The histologic diagnosis was chronic active humoral rejection in 6 patients, chronic active T cell–mediated rejection in 7, chronic CNI toxicity in 10, and interstitial fibrosis/tubular atrophy not otherwise specified in 10 cases, according to Banff 2007 criteria.52 Six deceased donor biopsies were used as control for the gene expression study on the graft. At the time of biopsy, all patients included in the independent test group did not differ for the main clinical and demographic features (Table 2). However, patients included in this group had significantly higher serum creatinine and proteinuria levels compared with those included in the training group (P < 0.01). Semiquantitative scoring for mononuclear cell interstitial inflammation (ti-score [Ti]) in total parenchyma of renal biopsy from the patients included in the independent test group was performed according to Banff 2007 criteria.52 This analysis showed that eight patients had Ti-0 (n = 5 in CsA only, n = 1 in CsA + AZA, n = 2 in CsA + EC-MPS), nine patients had Ti-1 (n = 2 in CsA only, n = 3 in CsA + AZA, n = 4 in CsA + EC-MPS), seven patients had Ti-2 (n = 1 in CsA only, n = 2 in CsA + AZA, n = 4 in CsA + EC-MPS), and four patients had Ti-3 (n = 2 in CsA + AZA and n = 2 in CsA + EC-MPS).

Independent Validation Group.

This group of 10 patients, matching with the two initial study groups for demographic and clinical features (Table 2), was used to confirm the relationship between peripheral and tissue gene expression.

The study was carried out according to Declaration of Helsinki principles and was approved by our institutional ethic review board.

PBMC Isolation and RNA Extraction

For all patients included in the training group, 20 ml whole blood was collected at T0 and after 3 months from conversion (T1). In the independent validation group, the blood sample was obtained at the time of renal biopsy. PBMCs were isolated by density separation over a Ficoll–Hypaque gradient (Flow-Laboratories, Irvine, UK) and washed three times with PBS pH 7.4/1 mM EDTA (Sigma, Milan, Italy). Cells were counted, and their viability was determined by Trypan blue exclusion (>90% PBMCs were viable). Total RNA was extracted by the RNeasy mini kit (Qiagen, Valencia, CA) from a minimum of 5 × 106 cryopreserved PBMCs. Total RNA was quantified by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and its integrity was assessed by electrophoresis, using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).

Gene Expression Profiling

For the exploratory microarray analysis, we studied five patients converted from AZA to EC-MPS randomly selected from the training group. RNA, isolated from PBMCs at T0 and T1, was processed and hybridized to the GeneChip Human Genome U133A oligonucleotide microarray (Affymetrix, Santa Clara, CA), which contained 22,283 gene probe sets, representing 12,357 human genes, plus approximately 3800 expressed sequence tag clones (Affymetrix; see manufacturer's manual for detailed protocol). We used the default settings of Affymetrix Microarray Suite software version 5 (MAS 5.0) to calculate scaled gene expression values. Results of the microarray experiments are available in Gene Expression Omnibus (accession number GSE14630).

Real-Time Quantitative RT-PCR

Twenty microliters of a reaction mixture containing 500 ng of total RNA, 10× RT-PCR buffer, 25× dNTPs mix, 10× RT random primers, RNase inhibitor, and 50 U of Multiscribe reverse transcriptase (PE Applied Biosystems, Foster City, CA) was incubated at 25°C for 10 minutes and heated at 37°C for 120 minutes and then at 85°C for 5 seconds.

Real-time quantitative RT-PCR was performed with two separate sets of oligonucleotide primers specific for human NEP (upstream: 5′-GAT GAC AAT GGC AGA AAC TT-3′; downstream: 5′-CCT GAA ATT GCC TGG ACT GT-3′; Invitrogen, Milan, Italy) and glyceraldehyde-3 phosphate dehydrogenase (GAPDH), respectively (upstream 5′-GAA GGT GAA GGT CGG AGT CA-3′; downstream: 5′-CAT GGG TGG AAT CAT ATT GGA A-3′; Invitrogen). NEP and GAPDH cDNA amplification was performed, in two separate sets of reactions, using iQ SYBR Green Supermix buffer (6 mM MgCl2, dNTPs, iTaq DNA polymerase, SYBR Green I, fluorescein, and stabilizers; BIO-RAD Laboratories) and 100 nM of NEP primers or 100 nM of GAPDH primers, in a total volume of 25 μl. Amplification was carried out with an MiniOpticon real-time PCR detection system (BIO-RAD Laboratories) programmed with an initial step of 3 minutes at 95°C, followed by 40 thermal cycles of 10 seconds at 95°C and 30 seconds at 60°C for GAPDH or at 58°C for NEP. Melting curves were generated through 60 additional cycles (65°C for 5 seconds, with an increment of 0.5°C/cycle). Gene expression results were obtained as average CT (threshold cycle) values of duplicate samples. NEP expression levels in each sample were normalized with expression of GAPDH by CFX Manager software version 1.5 (BIO-RAD Laboratories) using the 2ΔΔCt method.

Morphometric Analysis

Morphometric analysis was carried out to analyze the extent of glomerular sclerosis and interstitial fibrosis. For each biopsy, all glomeruli (mean, 10 glomeruli/biopsies; range, 5 to 20), including sclerotic and nonsclerotic, were analyzed. The percentage of glomerular sclerosis was calculated as the ratio between the number of glomeruli with global sclerosis plus glomeruli with segmental sclerosis and the number of all glomeruli for each biopsy.

Extent of interstitial fibrosis was assessed on Masson's trichrome–stained slides using Adobe Photoshop 6.0 (Adobe System, San Jose, CA) in a MS Windows 98 environment (Microsoft, Redmond, WA).53 The cortical area of the entire biopsy was analyzed in a stepwise fashion as a series of consecutive fields, with a 20× objective, avoiding the capsule, the subcapsular areas, and the arterial adventitia. The percentage of green-stained area ([fibrous tissue]/total area) was measured. Values from all consecutive images for each biopsy were averaged.

Immunohistochemistry

Immunohistochemical evaluation of NEP protein expression in the glomeruli and proximal tubules of renal biopsy from deceased donors and transplant patients was carried out on paraffin-embedded renal biopsy sections according to standard procedures. Briefly, thin (2 μm) sections of paraffin-embedded tissue were hydrated through xylenes and graded alcohol series. After antigen retrieval, the sections were blocked with protein block serum-free (Dako, Glostrup, Denmark) at room temperature for 10 minutes. The slides were incubated with a mouse monoclonal anti-human NEP antibody (1:50, 1-hour incubation at room temperature; Abcam, Cambridge, UK). The binding of the secondary biotinylated antibody was detected by the Dako Real EnVision, Peroxidase/DAB kit (Dako), according to the manufacturer's instructions. Visualization of peroxidase was performed by incubation in DAB chromogen solution, giving a brown precipitate. The sections were counterstained with Mayer hematoxylin (blue) and mounted with glycerol (Dako Cytomation). Negative controls were obtained incubating serial sections with the blocking solution and then omitting the primary antibody. For each biopsy, all glomeruli were analyzed, and an average of 10 glomeruli/biopsy were present (range, 5 to 20). Digital images of each glomerulus were captured with a 40× objective using a Leica (Leitz, Wetzlar, Germany) microscope fitted with a Coolpix990 digital camera (Nikon, Calenzano, Italy). To quantify NEP immunostaining at the tubular level, for each biopsy, the entire cortical region was analyzed in a stepwise fashion as a series of consecutive fields with a 20× objective, avoiding the capsule, the subcapsular areas, the arterial adventitia, and the glomeruli. Image processing and computer operations were performed on Adobe Photoshop 6.0. The percentage of positive stained area/total area was calculated. Values from all consecutive images for each biopsy were averaged. NEP glomerular and tubular expression was evaluated in each group of treated patients.

Cell Isolation and Culture

HK2 cells, an immortalized human proximal tubular epithelial cell line,54 were obtained from American Type Culture Collection (Rockville, MD). Cells were grown to confluence in DMEM/F12 (Sigma) medium supplemented with 10% FBS (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine (all from Life Technologies, Milan, Italy). For each passage, confluent cells were washed with PBS, detached with 0.05% trypsin/0.02% EDTA (Sigma) in PBS, and plated in DMEM/F12.

RNA Isolation and Real-Time PCR

HK2 cells were plated in 6-well dishes at 15 × 104 /well and grown to confluence in DMEM/F12 (Sigma) supplemented with 10% FBS. The cells were starved 48 hours in serum-free medium and, for the first set of experiments, exposed for the indicated time periods to MPA (Sigma) at concentrations of 1, 5, 10, and 25 μg/ml. At the end of the treatment, the cell monolayer was rapidly washed twice with PBS and lysed with 350 μl of RLT buffer (Qiagen), and total RNA was extracted by QIACUBE (Qiagen; see manufacturer's manual for detailed protocol). NEP RNA expression was investigated by real-time PCR and, to this purpose, 500 ng of RNA was used in a reverse transcription reaction. In the second set of experiments, confluent cultures of HK2 cells were washed, serum starved for 48 hours, and preincubated for 24 hours with MPA (10 μg/ml), and then angiotensin II (20 nM; Sigma) was added for 6 hours in the presence or absence of phosphoramidon (2, 5, and 10 μM; Sigma) for 6, 12, and 24 hours at 37°C. At the end of the incubation period, cells were washed with PBS and lysed with RLT buffer, and total RNA was extracted. Human PAI-1 gene expression was investigated by real-time PCR using one set of specific oligonucleotide primers (upstream: 5′-CAG ACC AAG AGC CTC TCC AC-3′; downstream: 5′-ATG CGG GCT GAG ACT ATG AC-3′; Invitrogen SRL) with an annealing temperature of 60°C. Real-time PCR was performed as described above.

Statistical Analysis and Bioinformatics

ANOVA, t test, and Fisher's exact test were used to assess differences in clinical and demographic features among patients included in both the training and test groups. Results were expressed as mean ± SD. Spearman correlation tests were used for continuous variable assessment. A value of P < 0.05 was considered to be statistically significant.

For microarray analysis, the gene expression profile of each subject was composed of the log-transformed levels of 22,283 probe sets. Statistical analysis of the expression profiles of the five subjects enrolled in the study was carried out using the standard t test and Wilcoxon test. Because of the small number of subjects analyzed and, more importantly, the limited number of genes truly associated to the phenotype, these classical methods may not reliably identify any statistically significant differentially expressed gene. To overcome the problems related to multiple comparisons, we used a different statistical approach. The fold change (FC) of each probe was evaluated as the difference of the log-transformed values between the two experimental conditions analyzed. The probes having an FC value simultaneously greater than (up-regulated) or less than (down-regulated) 0.7 in all of the five analyzed subjects were considered as candidates to be differentially expressed (DE) probes. We obtained a list of 6 up-regulated and 10 down-regulated probes (P < 0.001). The statistical significance of the obtained lists was assessed with nonparametric permutation tests. The test consists in assigning randomly the FC values to probes in gene expression profile of each analyzed subject and counting the number of probes up- or down-regulated simultaneously in all of the subjects. We carried out 1000 random permutations of the FC values. The P value associated with the list of DE genes was evaluated as the number of times in which we obtain larger lists of DE genes by chance divided by the total number of permutations.

Principal component analysis was performed using Spotfire DecisionSite 9.0 (www.spotfire.com).

DISCLOSURES

None.

Acknowledgments

This work was funded by Ministero della Salute (Ricerca Finalizzata 2006, granted to G.G.) and an unrestricted research grant from Novartis. We thank Vincenzo Gesualdo (Renal, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari) for the excellent technical assistance, Giovanni Pannarale, MD, for the helpful discussion on the histologic findings and Mariella Mastronardo for her editorial assistance and language revision of the manuscript.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

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