Estimation of Serum and Urinary Profibrotic Cytokines in Renal Allograft Recipients

Salwa Ibrahim Department of Medicine, Cairo University

Gamal Saadi Department of Medicine, Cairo University

Mervat Al-Ansary Department of Clinical Pathology, Cairo University

Citation: S. Ibrahim, G. Saadi, M. Al-Ansary: Estimation of Serum and Urinary Profibrotic Cytokines in Renal Allograft Recipients. The Internet Journal of Nephrology. 2007 Volume 4 Number 1

Keywords: TGF-1, PDGF, cyclosporine A, chronic allograft dysfunction

Abstract

Background: Chronic allograft nephropathy (CAN) is an important cause for graft failure after the first year of renal transplantation. Recent data confirmed the involvement of the key fibrogenetic cytokines such as transforming growth factor-beta 1(TGF-ß1) and platelet derived growth factor (PDGF) in the pathogenesis of CAN. We evaluated the potential contribution of TGF-ß1 and PDGF in the development of renal allograft dysfunction as well as the impact of cyclosporine A (CsA) therapy on serum and urinary levels of these cytokines. Methods: Serum and urinary TGF-ß1 and PDGF were determined by enzyme-linked immunosorbent assay (ELISA) in 10 renal allograft recipients for more than one year with normal renal function (group I), 10 renal allograft recipients for more than one year with impaired renal function (group II), 10 patients with chronic renal failure (CRF) under conservative therapy (group III) and the measurements were compared with the levels of 10 healthy controls (group IV). Results:Serum and urinary TGF-ß1 and PDGF levels in the transplanted groups with normal or impaired renal function (group I&II) as well as in CRF patients (group III) were significantly increased compared to healthy controls (P<0.01). The impact of cyclosporine A and immune stimulation in the transplanted cases was manifested by higher levels of serum and urinary TGF-ß1 and PDGF in the transplanted group with normal kidney function (group I) when compared to healthy controls (P<0.01). Serum and urinary TGF-ß1 and PDGF levels were significantly elevated among transplanted cases with impaired renal function (group II) compared to transplanted cases with normal renal function (group I) (P<0.01). Serum and urinary TGF-ß1 and PDGF levels showed significant positive correlations with serum creatinine levels in the patients groups (P<0.001). Conclusion: Our data confirm the crucial contribution of the profibrotic cytokines TGF-ß1 and PDGF in the development of chronic graft dysfunction that could be further augmented by cyclosporine A therapy. Future studies are needed to examine the effect of manipulation of immunosuppressive regimen on the extent of profibrotic gene expression as well as the long term graft survival.

Introduction

Chronic allograft nephropathy (CAN) is the principal cause of late renal allograft loss after renal transplantation (1). Although chronic rejection has traditionally been seen as a repeated low grade immune response directed against allogenic tissue, recent evidence indicates that non-immunological factors, principally ischemia/reperfusion injury, donor age, lipid abnormalities and cyclosporine nephrotoxicity, might contribute to its pathogenesis (2). Independently of any triggering events, the cause of tissue destruction in chronic allograft nephropathy is fibrogenesis with subsequent loss of allograft function via a glomerular hyperfiltration syndrome (3). This hyperfiltration may result in endothelial activation followed by an up-regulation of adhesion molecules, such as intercellular adhesion molecule-1(ICAM-1), a release of cytokines including interleukin-1(IL-1), tumor necrosis factor-α (TNF-α) and growth factors as insulin-like growth factor-1 (IGF-1), platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β₁), which in turn induce cell migration and proliferation of leukocytes and smooth muscle cells (SMCs) with subsequent extracellular matrix accumulation (3,4).

TGF-β is a cytokine that exists structurally as a homodimer (5). TGF-β can either stimulate or inhibit cell growth and proliferation (5). It has been seen as a regulatory molecule, acting to restore cellular growth balance after deviations from normal (5). It prevents the adhesion of neutrophils to endothelium (6) and their subsequent transmigration (7). It also inhibits the proliferation of monocytes and lymphocytes and can induce lymphocyte apoptosis (8). TGF-β seem to trigger the reduction of inflammation, in preparation for healing, and this tie in with its role in extracellular matrix turnover and fibrosis (5). TGF-β₁ directly stimulates the synthesis of extracellular matrix components and blocks matrix degradation by stimulating protease inhibitors such as plasminogen activator inhibitor-1 (PAI-1) (9).

It is increasingly recognized that modern immunosuppressive drugs can have adverse effects on renal allograft (10). In experimental models, cyclosporine A (CsA) administration produced similar histological appearance to those found in transplant recipients with chronic allograft nephropathy that was associated with increased TGF-β₁ expression (11). It was hypothesized that CsA might function in vivo as an immunosuppressant not only by inhibiting the expression of proinflammatory cytokines such as interleukin-2, but also by stimulating the expression of TGF-β₁, a potent immunosuppressive cytokine (11).

Platelet-derived growth factor (PDGF) is one of the most ubiquitous of the peptide regulatory growth factors. Recently, PDGF has been observed to be induced in chronic rejection both in experimental and clinical studies (12). In addition, one study has shown that PDGF was already induced in acute rat renal allograft rejection indicating the link between acute and chronic rejection (13). Of interest, CsA treatment, either at high or low dose, failed to inhibit the expression of PDGF ligands during this pathological process (14).

The aim of the present study was to evaluate serum and urinary TGF-β₁ and PDGF levels in renal allograft recipients and to explore their potential contribution to the impaired graft function occurring one year after renal transplantation. In addition, the impact of CsA treatment on their levels was further assessed.

Patients and Methods

The patients selected for the present study were recruited from the outpatient transplant clinic at the Cairo University Hospital during the period February to October 2001. The study protocol was approved by the University of Cairo Research Committee. All subjects gave informed consent and they were divided into four groups:

Group I : 10 patients with transplanted renal allograft for > 1 year and serum creatinine less than 1.4 mg/dl. They were 6 males and 4 females with mean age of 37±12 years. The mean time after renal transplantation was 18.10±6.06 months (13 to 30 months).

Group II : 10 patients with transplanted renal allograft for > 1 year and serum creatinine more than 1.4 mg/dl. They were 5 males and 5 females with mean age of 34 ±10 years. The mean time after renal transplantation was 20.10±8.92 months (14 to 41 months).

Group III: 10 chronic renal failure (CRF) patients under conservative treatment. They were 6 males and 4 females with mean age of 36±9 years.

Group IV: 10 normal individuals serving as controls. They were 5 males and 5 females with mean age of 34±11 years.

Biochemical Evaluation

All groups were assessed for the following:

Measurement of blood urea, serum creatinine and creatinine clearance.
Measurement of trough whole blood cyclosporine level for the renal transplant groups (Sandimmun-Kit, Novartis, Switzerland).
Measurement of serum and urinary TGF-β1 levels using a solid-phase specific sandwich enzyme-linked immunosorbent assay (ELISA; Quantykine; R&D Systems, Minneapolis, MN, USA) (15).
Measurement of serum and urinary PDGF levels using the quantitative sandwich enzyme immunoassay technique (EIA) (16).

Statistics

Data were shown as mean±SD. Data were analyzed using SPSS software version 11. For statistical analysis, Student's t-test and analysis of variance (ANOVA) was used to detect the differences in variables among study groups. Correlation analysis was performed using Pearson's correlation. P value <0.05 was statistically significant.

Results

Serum TGF-β₁ levels were significantly increased in transplanted groups with normal or impaired renal function as well as in CRF patients compared to healthy controls (317.77±94.61, 712.53±45.52, 662.72±47.29, 209.63±68.96 ng/ml respectively, P<0.05)(Table 1). TGF-β₁ levels were increased in transplanted groups with normal or impaired renal function as well as in CRF patients compared to healthy controls (415.46±101.64, 653.51±101.05, 610.80±126.23, 148.94±29.16 ng/ml respectively, P <0.05). Serum PDGF levels were significantly increased in transplanted groups with normal or impaired renal function as well as in CRF patients compared to healthy controls (1664.55±126.46, 4201.33±1469.78, 3800.04±1598.22, 688.34±127.06 pg/ml respectively, P<0.05).

Urinary PDGF levels were significantly increased in transplant recipients with impaired renal function and in CRF group compared to the healthy controls (437.99+117.58, 460.21+111.66, 214.08+36.46 pg/ml respectively, P<0.05). Serum and urinary TGF-β₁ and PDGF levels were significantly increased in renal allograft recipients with impaired renal function and in CRF patients (group II&III) compared to their levels in transplanted group with normal renal function (group I) (P<0.01). Serum and urinary TGF-β₁ and PDGF levels showed significant positive correlations with serum creatinine levels in the study groups (P<0.001) as well in the transplantation groups (P<0.05)( Fig. 1A-D).

Table 1: The baseline characteristics of the study groups(mean±SD), *P<0.05

Figure 1a: Correlation between serum creatinine and serum TGF-β1 levels (ng/ml) in the study groups

Figure 1b: Correlation between serum creatinine and serum PDGF (pg/ml) levels in the study groups

Figure 1c: Correlation between serum creatinine and serum TGF-β1 levels in the transplantation groups

Figure 1c: Correlation between serum creatinine and serum TGF-β₁ levels in the transplantation groups

Figure 1d: Correlation between serum creatinine and serum PDGF levels in the transplantation groups

Discussion

Our results showed increased serum and urinary TGF-β₁ and PDGF levels in the transplanted groups with normal or impaired renal function as well as in CRF patients compared to healthy controls (P<0.01). The impact of CsA and immune stimulation in the transplanted cases was manifested by higher levels of serum and urinary TGF-β₁ and PDGF in the transplanted group with normal kidney function (group I) when compared to healthy controls (P<0.01).

These findings could be explained by the experimental data reporting the development of structural injury early in the natural history of renal allograft which was mediated, in part, by the early upregulation of profibrotic growth factors such as TGF-β₁ and PDGF (17,18). CsA levels and acute rejection episodes were demonstrated to correlate closely with the TGF-β₁ mRNA expression in serial renal allografts biopsies (18). Sequential protocol biopsies from renal allograft recipients had shown significant TGF-β₁ over-expression that started as early as one week after renal transplantation and was very significant 6 months after transplantation in a group of stable renal allograft recipients (1). Thus, the higher levels of serum and urinary TGF-β₁ in transplanted cases with normal serum creatinine in the present study could be attributed to the effect of the calcineurin inhibitor as well as to the incompletely suppressed immune reaction in renal allografts. Recently, CsA was found to induce PDGF both in vivo and in vitro (19), explaining, in part, the higher level of PDGF in this CsA treated transplant group with normal renal function.

Serum and urinary TGF-β₁ and PDGF levels were significantly increased in transplanted cases with impaired renal function (group II) compared to healthy controls (P<0.01). This was consistently reported in experimental studies showing that CAN is associated with upregulation of TGF-β₁ expression with subsequent extracellular matrix accumulation ending in allograft fibrosis (3). One study has also found elevated levels of intragraft TGF-β₁ in cases of biopsy proven chronic rejection that correlated with increased rate of decline in renal function (20). PDGF over-expression was demonstrated in cases with chronic allograft nephropathy (21). PDGF-A chain peptide and mRNA were over-expressed by both intimal and medial smooth muscle cells in arteries with chronic rejection (21).

Serum and urinary TGF-β₁ and PDGF levels were significantly elevated among transplanted cases with impaired renal function (group II) compared to transplanted cases with normal renal function (group I) (P<0.01). This finding could not be explained by the CsA level effect alone, since it was comparable in both groups. This signifies an ongoing chronic rejection process in group II as compared to partially suppressed immune reaction in group I. Although initially following transplantation, the immunosuppressive actions of TGF-β₁ are potentially beneficial and may contribute to the inhibition of acute allograft rejection, it is possible that prolonged over-expression of TGF-β ₁ within the vessel walls and /or in the interstitium of the graft may drive a fibrotic process that contribute to progressive allograft nephropathy.

Serum and urinary TGF-β₁ and PDGF levels were also increased in CRF group (group III) compared to healthy controls (P<0.01). The elevation of serum TGF-β₁ and PDGF could be explained by either retention of these cytokines secondary to renal impairment or an ongoing chronic inflammatory reaction, the latter possibility is further supported by increased urinary TGF-β₁and PDGF levels as well. Increased urinary TGF-β₁ excretion has been demonstrated in patients with chronic renal impairment secondary to diabetic nephropathy and membranous nephropathy (22, 23). Similarly, an increased urinary PDGF excretion in patients with insulin dependent diabetes mellitus and micro/macroalbuminuria was reported (24).

Serum and urinary TGF-β₁ and PDGF levels showed significant positive correlations with serum creatinine levels in the study groups (P<0.001). In subgroup analysis, serum and urinary TGF-β₁ and PDGF levels were significantly correlated with serum creatinine in groups not receiving CsA (group III&IV) and CsA treated groups(group I&II)(P<0.05). This finding confirms the role of these profibrotic cytokines in the development and progression of chronic allograft dysfunction.

In summary, our data indicate that TGF-β₁ and PDGF could be the principal mediators of progressive renal allograft dysfunction that might be induced by cyclosporine A. As the deleterious consequences of chronic immunosuppression on the development of chronic allograft are now recognized, newer immunosuppressive drugs that were reported to be associated with less profibrotic gene expression such as rapamycin and mycophenolate mofetil, may offer better long term allograft survival. Future clinical studies, however, are needed to verify their usefulness in improving the long term outcome of renal transplantation.

Correspondence to

Dr Salwa Ibrahim, MD MRCP Address 3 Refaa street, flat 14, Dokki, Giza, Egypt 12311 Email salwaibrahim@hotmail.com

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Journal

The Internet Journal of Nephrology ISSN: 1540-2665