Increased Serum Levels of Insulin-Like Growth Factor (IGF)-1 and IGF-Binding Protein-3 in Henoch-Schonlein Purpura

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1 Tohoku J. Exp. Med., 2008, 214, IGF-1 and IGFBP-3 in HSP 333 Increased Serum Levels of Insulin-Like Growth Factor (IGF)-1 and IGF-Binding Protein-3 in Henoch-Schonlein Purpura BILAL YILDIZ, 1 NURDAN KURAL, 1 BANU AYDIN 2 and OMER COLAK 3 1 Department of Pediatric Nephrology and Rheumatology, Eskisehir Osmangazi University, Faculty of Medicine, Eskisehir, Turkey 2 Department of Pediatrics, Eskisehir Osmangazi University, Faculty of Medicine, Eskisehir, Turkey 3 Department of Biochemistry, Eskisehir Osmangazi University, Faculty of Medicine, Eskisehir, Turkey Henoch-Schönlein purpura (HSP) is a common systemic vasculitis of childhood, and may affect the kidney. Endothelial cell dysfunction and fibrosis is an important part of HSP vasculitis and may account for renal involvement in HSP. Insulin-like growth factor (IGF)-1 enhances the cytokine-induced expression of adhesion molecules in endothelial cells (EC). Besides, IGF-1 may stimulate angiogenesis, fibrosis and tubular formation in EC and IGF-1 increases glomerular filtration rate. We, therefore, investigated the role of IGF-1 and IGF-binding protein-3 (IGFBP-3) in HSP. The study included 44 patients with HSP (30 boys and 14 girls), including 13 patients with proteinuria, 15 patients with hematuria and 16 patients with positive stool occult blood (SOB), and 26 healthy children. Serum levels of IGF-1 and IGFBP-3 levels were significantly higher in HSP than in the controls ( vs ng/ml, p = and vs μg/ml, p = 0.001, respectively). Serum IGF-1 levels were significantly higher in proteinuria than those without proteinuria and controls ( p = and p = 0.001, respectively). Also, IGFBP-3 levels were greater in proteinuria compared to those without proteinuria and controls ( p = and p = ). Serum immunoglobulin-a/ complement-c3 ratio was higher in HSP than in the controls (p = ) but this ratio did not change according to proteinuria, hematuria or positive SOB. In conclusion, IGF-1 and IGFBP-3 levels could be new markers for determination of renal involvement in HSP. Henoch-Schönlein purpura; insulin-like growth factor-1; insulin-like growth factor-binding protein-3; proteinuria; hematuria. Tohoku J. Exp. Med., 2008, 214 (4), Tohoku University Medical Press As an inflammatory disease, Henoch- Schöenlein purpura (HSP) is the most common systemic vasculitis in children characterized by narrowing of the small vessels lumen and IgA deposition (Tizard 1999). Although there seems to be a temporal relationship between HSP and recent respiratory infections, exposure to allergens, drugs, cold and insect bites, pathophysiolo- Received October 26, 2007; revision accepted for publication February 18, Correspondence: Bilal Yildiz, M.D., Pediatric Nephrology and Rheumatology, Eskisehir Osmangazi University, Faculty of Medicine, TR-26480, Eskisehir, Turkey. bilalyn@yahoo.com 333

2 334 B. Yildiz et al. gy of HSP is still unknown (Szer 1994; Kraft et al. 1998). The uncertainty about the pathophysiology of HSP led us to consider other factors that could be affective on HSP. Insulin-like growth factor (IGF-1) is thought to be one of them. IGF-1 is a polypeptide growth factor that plays a significant role in cellular growth and survival, regarding pathological states in particular. IGF-1 binds to the type I IGF-1 receptor existing in many cell types, including endothelial cells. The primary role of IGF-1 in endothelial cells is to stimulate angiogenesis and tube formation. On the other hand, endothelial cells, which produce cytokines including tumor necrosis factor (TNF)- α, play a central role in inflammation. Although it has been shown that IGF-1 enhances TNF-α and enhances TNF-α induced adhesion molecule expression in endothelial cells resulting in increased adhesion of monocytes, the role of IGF-1 in vasculitis, including HSP, is unclear (Che et al. 2002). The aim of the present study was to investigate the potential role of serum levels of IGF-1 and IGF binding protein-3 (IGFBP-3) in HSP. MATERIAL AND METHODS Patients and controls Forty-four children (30 boys, 14 girls; mean age years) diagnosed with HSP between January 2004 and May 2007 were taken as the study population. All the patients were evaluated within the first week of their presentation. All of the patients had purpura and joint involvement. 13 of the 44 patients (29.5%) had proteinuria while 15 of the 44 patients (34%) had hematuria. Further, 16 of the 44 patients (36.3%) had gastrointestinal involvement with positive guaiac-based stool occult blood (SOB). 8 of the 44 patients (18.2%) had renal involvement with proteinuria and hematuria. Interestingly enough, one of the patients had radial arterial thrombosis with Factor V Leiden (FVL) mutation and positive test for activated protein C resistance and two patients had Familial Mediterranean Fever (FMF). The inclusion criteria adopted were the American College of Rheumatology (ACR) criteria (palpable purpura, bowel angina, gastrointestinal bleeding, hematuria, age < 20 years at the disease onset, and no history of medication before the onset) (Trapani et al. 2005). Those patients that matched three or more of the criteria were deemed eligible for the study. The patients were identified as hematuria when over five red blood cells per high power microscopic field in a centrifuged urine specimen was determined. The patients with proteinuria was defined as a small amount of protein (+) on dipstick testing or proteinuria greater than 4 mg/m²/hr obtained from 24-hr collected urine. The control group consisted of 26 healthy individuals (16 boys, 10 girls; mean age years). The control group consisted of the healthy population referring to our clinic. Study protocol Serum IGF-1 and IGFBP-3 levels were measured both in patient and control groups by means of a commercial kit. All the blood samples were obtained within the first week of presentation of the patients. Blood samples were kept at 70 C until the time of hormone assay. The levels of IGF-1 were measured with IGF-1, IMMULITE 1000 Analyzer (Diagnostic Products Corporation, Los Angeles, CA, USA). Assays were carried out by the solid phase, enzyme-labeled chemiluminescent immunomonometric method. IGFBP-3 assays were carried out with a kit that used solid phase, enzymelabeled chemiluminescent immunomonometric method (IGBP-3, IMMULITE 1000 Analyzer, Diagnostic Products Corporation). Also, serum immunoglobulin levels (Ig A, G and M), anti-neutrophil cytoplasmic antibodies (p-anca and c-anca), fibrinogen, complement C3 and C4, albumin, and C-reactive protein (CRP) were measured. Anti-streptolysin O (ASO) test, erythrocyte sedimentation rate (ESR), complete blood count, urine analysis and guaiac-based stool occult blood examinations were performed. Glomerular filtration rates (GFR) with clearance of creatinine, proteinuria (mg/m 2 /hr) in the 24-hr urine. Body mass index (BMI) and IgA/C3 ratio was calculated. Creatinine clearances were calculated with the Schwartz formula in controls. Renal biopsies were performed in patients with persistent proteinuria, persistent hematuria, nephrotic or nephritic syndrome. Statistical analysis Results are presented as mean S.D. and median. Continuous variable were compared using Mann- Whitney s U, t-tests or Pearson correlation tests as appropriate. Chi-Square tests were used to compare proportions. The statistical analyses were conducted using

3 IGF-1 and IGFBP-3 in HSP 335 SPSS 13.0 software. Values of p < 0.05 were taken as significant. Ethics The study was approved by the Research Ethics Committee of Eskisehir Osmangazi Medical Faculty, Eskisehir Osmangazi University. Informed consent was obtained from the parents or guardians of the patients and the healthy individuals. RESULTS Serum IGF-1 levels were significantly higher in HSP patients than those in the control group ( / , p = 0.024). We also determined that serum IGFBP-3 levels were considerably higher in HSP patients when compared to those in the control group ( / , p = 0.001). Serum IGF-1 and IGFBP-3 levels were higher in HSP patients with proteinuria than in those without proteinuria ( p = and p = 0.005, respectively, Table 2). We also found that there was positive correlation between proteinuria and serum IGF-1 and IGFBP-3 levels (Figs. 1 and 2). Both IGF-1 and IGFBP-3 levels were higher in proteinuria than in controls (p < 0.001, Table 4). On the contrary, IGF-1 and IGFBP-3 levels did not differ between or without hematuria (p > 0.05). IGF-1 and IGFBP-3 levels were higher in HSP patients with hematuria than in controls (p = 0.05 and p = 0.03, respectively). difference was detected in IGF-1 levels of HSP patients with positive SOB compared to the ones with negative SOB and between these patients and the controls (p > 0.05). Whereas IGFBP-3 levels were lower in HSP patients with positive SOB than those with negative SOB (p < 0.022) but IGFBP-3 levels were higher in HSP patients both with positive and negative SOB than were in the control group (Table 3). We found that serum IgA, IgG, fibrinogen, complement C3, C4, CRP levels, leukocyte and thrombocyte counts and IgA/C3 ratio were significantly higher in HSP than were in controls (Table 4). Positive correlation was detected between Ig A and ESR and CRP (p < 0.05). Hemoglobin, complement C4, IgM and albumin levels in HSP were the same as those of the control group (p > 0.05). Serum levels of immunoglobulin, fibrinogen, complement C3 and C4 and CRP did not vary according to the presence of proteinuria or hematuria (p > 0.05). Hemoglobin levels, thrombocyte and leukocyte counts did not vary according to the presence of proteinuria and hematuria either (p > 0.05). Serum immunoglobulin, fibrinogen, complement C3 and C4 levels showed no variation in accordance with the presence of positive SOB (p > 0.05) but CRP levels and leukocyte counts were higher in patients with positive SOB than were in those without positive SOB (Table 3). IgA/C3 ratio did not change according to the presence of the proteinuria, hematuria or positive SOB (Table 3). Anti-nuclear antibody, anti-dna, rheumatoid factor, p-anca and c-anca levels were negative in our patients. 14 out of the 44 patients (31.8%) underwent the ASO test and all these 14 patients had positive ASO. Clearance of creatinine and BMI did not differ in HSP when compared to that of controls (p > 0.05). Eight of the 44 patients underwent kidney biopsies, which revealed that only one patient had Grade IV findings (> 50% crescent/segmental lesion) according to classification of the International Study of Kidney Disease in Children. During the study, this patient was found to have a chronic renal disease (2.3%). The other patients had minimal glomerular abnormalities as Grade I and none of these patients had biochemical and/or urinary abnormalities. DISCUSSION HSP is a kind of systemic small vessel vasculitis and usually benign and self-limited; however, internal organs such as gastrointestinal tract and kidney may be inflicted (Yang et al. 2005). Clinical findings of our patients are presented in Table 1. Joint involvement (100%) and gastrointestinal bleeding ratios (40.9%) were higher in our patients than those in the literature. As for the patients with FVL and FMF we obtained quite remarkable results. To the best of our best knowledge, the association of HSP and

4 336 B. Yildiz et al. TABLE 1. Clinical features of patients with HSP. (Total n = 44) n % Purpura Joint involvement Gastrointestinal involvement Recent history of infection Hematuria Proteinuria Hypertension 4 9 Familial Mediterranean Fever with HSP Radial arterial thrombosis with Factor V leiden mutation HSP, Henoch-Schönlein purpura; n, number of subjects. FVL mutation has not been reported in the literature so far. This association suggests that FVL mutation may have a part in the development of the vascular trombosis in patients with HSP. Also, we determined FMF in 4.5% of the 44 HSP patients, a ratio exceeding the determined ratio for the general Turkish population ( %). Thus, we suggest that the clinical findings of FMF including positive SOB should be carefully investigated in patients with HSP because of FMF and vasculitis have a remarkable similarity. We found that serum IGF-I and IGFBP-3 were significantly higher in HSP than in controls (Table 2). other study has reported these findings up to this time. The increased IGF-1 and IGFBP-3 levels in HSP probably reflect the reactive growth processes in the affected tissue. Possible reasons for increased IGF-I and IGFBP-3 levels in HSP could be stated as follows: (i) IGF-1 might have a contributory effect upon the vascular inflammatory process developing in HSP. IGF-1 is an enhancing factor for cytokineinduced endothelial cell inflammation in HSP vasculitis. In fact, IGF-1 enhances c-jun and nuclear factor nuclear factor (NF-κB) activation, which is regulated by TNF-α, and enhances TNFα-induced adhesion molecule expression including intercellular adhesion molecule (ICAM)-1 in endothelial cells. Also, it has been reported that high concentrations of IGF-1 increase ICAM-1 expression but that the effect of IGF-1 on ICAM-1 expression is much weaker than that of TNF-α TABLE 2. IGF-1 and IGFBP-3 levels in proteinuria, hematuria and positive SOB. (Mean S.D.) (n = 13) Proteinuria (n = 31) p (n = 15) Hematuria (n = 29) Positive SOB (n = 16) (n = 28) Controls (n = 26) p IGF-1 (ng/ml) P1 < P2 < 0.05 IGFBP-3 (μ g/ml) P1 : P2 : 0.03 P3 : HSP, Henoch-Schönlein purpura; P, significance levels for comparison between parameters (P1, HSP patient with proteinuria vs controls; P2, HSP patient with hematuria vs controls; P3, HSP patient with positive SOB vs controls); p < 0.05 was taken as significant. SOB, stool occult blood; n, number of subjects.

5 IGF-1 and IGFBP-3 in HSP 337 (Balaram et al. 1999). All of these effects increase adhesion of monocytes to the endothelial cells. Taken together, these novel findings suggest a role for IGF-1 during the HSP vasculitis, related to vascular inflammation. (ii) IGF-I and IGFBP-3 levels in our study could have increased as secondary reaction to the HSP nephritis, in which diffuse endocapillary proliferation and fibrin deposits can occur. These processes may result in glomerular crescents. Therefore, increased IGF-I and IGFBP-3 might protect kidney from such effects in our patients. Vijayan et al. (1999) showed that inulin clearances in patients with end-stage renal disease who received IGF-I were significantly higher than in those who received placebo. The increases of Fig. 1. Positive correlation between serum IGF-1 levels (ng/ml) and proteinuria (mg/day). Fig. 2. Positive correlation between serum IGFBP-3 levels (μg/ml) and proteinuria (mg/day).

6 338 B. Yildiz et al. GFR with IGF-I may be explained with the renal vascular effects of IGF-I, which causes activation of two endogenous vasodilators, nitric oxide and prostaglandins (Tonshoff et al. 1998). (iii) IGF-1 could have increased due to anti-fibrotic factors. IGF-1 has fibrotic effects: IGF-1 increases collagen and fibronectin protein, in rat mesangial cells in-vitro, enhancing collagen transcription, whereas in human mesangial cells IGF-I and transforming growth factor-β have a synergistic effect on matrix protein accumulation. It also has antifibrotic effects: IGF-I has been suggested to reduce fibrosis, while its ability to inhibit apoptosis or vasodilate blood vessels could also inhibit the fibrotic process (Oldroyd et al. 2006). We concluded that IGF-1 might serve as an antifibrotic agent during the onset of HSP. Serum IGF-I and IGFBP-3 levels were higher in proteinuria than in without proteinuria in the present study (Table 2). Also, there was a positive correlation between degree of proteinuria and IGF-I and IGFBP-3 (Figs. 1 and 2). TABLE 3. Laboratory features of patients with HSP (Mean). Proteinuria (n = 13) (n = 31) Hematuria (n = 15) (n = 29) Positive SOB (n = 16) (n = 28) Controls (n = 26) Hemoglobin P1: 0.05 Leukocyte count (mm 3 ) 10 3 Thrombocyte count (mm 3 ) P1: P2: P3: P1: P2: P3: Fibrinogen P1: P2: P3: CRP P3: 0.02 ESR P1: P2: P3: C P1: P3: IgG 1, , , , , , P1: IgA P1: P2: P3: IgA/C P1: P2: P3: Albumin P3: 0.05 HSP, Henoch-Schönlein purpura; P, significance levels for comparison between parameters (P1, HSP patient with positive SOB vs controls; P2, HSP patient with hematuria vs controls; P3, HSP patient with proteinuria vs controls); p < 0.05 was taken as significant. SOB, stool occult blood; CRP, C-reactive protein; C3, complement C3; Ig, immunoglobulin; n, number of subjects. p

7 IGF-1 and IGFBP-3 in HSP 339 The relationship between serum levels of IGF-1, IGFBP-3 and proteinuria has not been reported in HSP before. We think that proteinuria may result from glomerulo-scaring (Hara et al. 2007). Also, proteinuria could be related to vasoconstrictive agents, especially angiotensins (ANG). Although IGF-1 therapy did not lead to proteinuria, IGF-1 stimulates ANG II receptor expression at the transcriptional level in kidney and may stimulate other undetermined vasoconstrictive agents (Tönshoff et al. 1998; Li et al. 1999). ANG II has growth-promoting properties in the kidney (Wolf and Neilson 1993). IGF-1 and IGFBP-3 may be related to a renal disease in HSP. However, our study has some limitations. IGF-1 and IGFBP-3 were not studied in the kidney tissue in the present study. We, therefore, suggest that pathologic correlations should be investigated in large series. Although no diagnostic laboratory abnormalities have been proposed for HSP, hemoglobin, fibrinogen, CRP, complement C3, immunoglobin G and A levels were significantly higher in HSP than those in controls in the present study. We found that these parameters did not change in or without proteinuria and hematuria. Nevertheless, leukocyte count and CRP levels were higher in SOB than in those without SOB. Our study suggests that hemoglobin, fibrinogen, CRP, complement C3, IgG, IgM and IgA levels, thrombocyte and leukocyte counts are not useful markers to distinguish between presence and absence of renal and gastrointestinal involvement. According to pathological and laboratory findings of IgA deposits on small vessel wall and increased serum levels of IgA, it is speculated that HSP is an immune-mediated vasculitis (Ballinger 2003). Therefore, IgA/C3 ratio has been proposed as a marker to predict disease activity (Shin et al. 2005). Our study found that IgA/C3 ratio was higher in HSP than that in controls but this ratio was similar in HSP patients with or without proteinuria, hematuria and SOB (Tables 3 and 4). These findings suggest that the serum IgA/C3 ratio could be a useful marker for prediction of disease activity, although the serum IgA/C3 ratio does not seem be offer much help in determining whether the involved area is the kidney or the gastrointestinal tract. In conclusion, increased IGF-1 and IGFBP-3 were observed in the acute onset of HSP. Also, these increased levels could be related to degree of the proteinuria. Therefore, IGF-1 and IGFBP-3 levels may be indicative of a renal involvement. TABLE 4. Laboratory features of HSP patients and controls. All patients (n = 44) Controls (n = 26) Leukocyte count (mm 3 ) Thrombocyte count (mm 3 ) Fibrinogen (mg/dl) CRP (mg/dl) ESR (mm/hr) C3 (mg/dl) IgG (mg/dl) IgA (mg/dl) IgA/C3 ratio HSP, Henoch-Schönlein purpura; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; C3, complement C3; Ig, immunoglobulin; n, number of subjects; p, significance levels, p < 0.05 was taken as significant. p

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