Study Of Apolipoprotein H In Children With Nephrotic Syndrome

BackgroundPrimary nephrotic syndrome (PNS) is a common disease during childhood. Although most of the patients were sensitive to steroid treatment, some were prone to frequent-relapse or resistant to steroid treatment. Moreover, Some of them would likely to progress to chronic renal insufficency ultimately, consequently influenced children's health and on the other hand, impose burden on thier families and country. It is necessary to prevent the renal damage and inhibit the progression of chronic renal insufficiency in children with nephrotic syndrome.Hyperlipidemia is an important characteristic of nephrotic syndrome in children. Dyslipidemia is predominantly reflectd in changes of concentration and compositions of individual lipoprotein, lipids deposited in diseased kidneys have more contribution to the tubulointerstitial lesions that lead to the progression of chronic renal interstitial fibrosis. Apolipoprotein (Apo) plays an essential role in maintaining lipid homeostasis brought about by the regulation of lipoprotein trafficking, and contributes to renal damage in children with NS as well. But they have different role in producing renal lesion in virtue of their combined lipid compositions and the expression in renal tissues. ApoH plays an important role in lipid metabolism due to its high affinity for triglyceride (TG) rich lipoprotein particles, and activating of lipoprotein lipase (LPL) as well. But the mechanism of changes in serum ApoH, and the expression in renal tissues of NS and role of its expression, and ApoH could be expressed in the kidneys by itself have not been elucidated, The present study aimed at evaluating the role and mechanism of ApoH in the progression of renal lesions in children with NS.Part Ⅰ Changes of scrum apolipoprotein H in children with idiopathic nephroticsyndrome and its impactobjective: Hyperlipemia characterizes nephrotic syndrome (NS) and contributes to the progression of the underlying nephropathy, but the mechanisms remain hypothetical. ApoH is a main plasma protein that plays an essential role in maintaining lipid homeostatis brought about by regulation of lipoprotein trafficking. But its role in NS is elucidated. The present study aimed at studying the changes and significance ofserum ApoH in children with NS.Methods: 78 cases with PNS were studisd. The age of onset was from 1 year and 3 months to 14 years with an average of 6 years and 5 months. All of them were in active stage ( irrespective their at disease onset or at recurrence) . According to (heir sensitivity to steroid who had been treated with standard therapeutic protocols, 68 were steroid-responsive (46 were frequently relaps , 22 were nonfrequently relaps) , 10 were steroid-resistent. Of which including 31 of MCNS, 26 of MsPGN, 15 of FSGS, and 6 of MN. 40 healthy young children were enrolled as control. Serum and urine ApoH were determind by dot-polt and immunostaining with specific mouse anti-human ApoH monoclonal antibody.Results: (1) Concentration of serum ApoH ( mg/dl ) in active stage ( 26.67±2.32 ) with NS were higher than those of in remission stage( 22.36±1.85 ) and control (20.73±1.21) , the levels of serum ApoH in remission stage were higher than those of control group, all P < 0.01; Same results have been found between steroid-resistent group (22.55±2.09) and steroid-sensitive group ( 25.90±3.14 ), between frequently relaps group (27.63±1.66) and infrequently relaps group (22.28±2.28), P < 0.01; There was significant positive correlation between serum ApoH and proteinuria , between serum ApoH and Ch and between ApoH and LDL in NS (r was 0.851,0.782, 0.775, respectively, P < 0.01 ), the level of serum ApoH and albumin reached negative correlation ( r =-0.715, P < 0.01). But no significance was found of concentration in serum ApoH and lipid among different kinds of renal biopsies. (2) Concentration of urine ApoH (mg/dl) in MsPGN (1.45±0.41) and FSGS (1.53±0.12 ) group were higher than those in MCNS (1.16±0.12), MN (1.11 ±0.05), respectively, And in steroid -sensitive group (1.29±0.24) were lower than those of in steroid-resistent group (1.50±0.21), in frequently relaps group (1.41±0.25 ) were higher than those of in nonfrequently relaps group ( 1.23±0.22 ), all P < 0.01. No significance was found in urine ApoH concentration between different stages. (3) No significant correlation was found between concentration of ApoH in urine and serum (r = 0.07, P > 0.05) . Conclusions : (1) ApoH is dysregulated in NS with marked increment in serum, which is part of the complex lipid metabolism. Serum levels of apoH in NS is higher than in normal serum and being correlated with hypoalbuminemia. (2) There were differences in concentration of serum ApoH in different stages and in different responsiveness to steroids. (3) There were differences of the level of urine ApoH in different stages and response different to steroids, the level of urine ApoH was notcorrelated to the level of serum ApoH, might be related to the tubulointerstitial lesions.Part II Expression and role of apolipoprotein H in renal tissues of childhoodwith nephrotic syndromeObjective To study the expression and distribution of apolipoprotein H in renal tissues of childhood with nephrotic syndrome and to find out it's role in renal damage. Methods Immunohistochemistry staining, in suit hybridation and real-time quantitative polymerase chain reaction ( RT-PCR ) were used to study the expression of ApoM in renal tissues of 78 patients with NS. Meanwhile, tubulointerstitial lesions were investigated. According to their sensitivity to steroid who had been treated with standard therapeutic protocols, 68 were steroid-responsive ( 46 were frequently relaps , 22 were nonfrequently relaps ) , 10 were steroid-resistent. Of which 31 were MCNS, 26 were MsPGN, 15 were FSGS, and 6 were MN. 14 normal controls were evaluated as well.Results (1) There were positive expression of ApoH mRNA and protein in renal tissues of NS patients and normal controls, distribution of ApoH mRNA was corresponds to protein, mainly in cytoplasm of proximal tubules epithelium, no expression was shown in other nephron. Relative weakly intensity of expression of ApoH mRNA and protein were found in some tissues, in which higher degree of renal tubules lesions and interstitial damage was found. (2) The level of ApoH mRNA expression and its protein expression in renal tissue of minimal change nephrotic syndrome (MCNS), memberanous nephropathy (MN) decreased slightly ( 4.95±0.40, 4.73±0.60, and 12.06±2.04, 12.35±0.61, respectively ), but not significantly compared to normal controls (5.44±1.56 and 12.69±1.89, P > 0.05). The expression in mesangial proliferative glomerulonephritis (MsPGN) and focal segmental glomersclerosis ( FSGS ) decreased significantly ( 3.30±0.28,2.82±0.36, and 10.13±3.09, 10.12±1.02, respectively ), when compare to MCNS, MN and the controls, P < 0.01. (3) There were also decreased expression of ApoH mRNA in steroid resistant group comparing to sreroid sensitive group ( 4.27±0.30 vs 4.97±0.99, P < 0.05 ). And lower expression in frequent relapse group than in infrequent relapse group ( 4.15±0.72 vs 5.03±0.32, P < 0.05 ) . (4) The lower expression of ApoH were shown in patients of higher degree of tubules lessons group and higher degree of interstitial damage group ( P < 0.01, respectively). (5) Significantly positive correlation was found about the levels ofApoH expression between mRNA and protein, r = 0.83, P < 0.01. There was significant negative correlation between the levels of ApoH mRNA, protein and urine ApoH , RBP and proteinuria in NS ( r was -0.56, ~0.60, -0.46, respectively, P < 0.01), No correlation was found between the levels of ApoH mRNA, protein and serum ApoH, r = 0.17, P>0.05.Conclusions: (1) There were positive expression of ApoH mRNA and protein in the renal tissues of NS patients and normal controls, ApoH could be expressed in the kidneys by itself. The proximal tubulars epithelium is a major source of ApoH synthesis. (2) Relative weak intensity of expression of ApoH mRNAand protein were found in some of the renal tissues, of which higher degree of renal tubulars lesions and interstitial damages was occurred, ApoH might play some role in preventing interstitial fibrosis in children with nephrotic syndrome.(3) The levels of ApoH mRNA and protein reflected the pathologic patten, effect of responses to steroids and the progression of nephrotic syndrome in children.Part HI Pilot study on the mechanisms of ApoH in inhibiting renal lesionsin children with nephrotic syndromeObjective To observe the relationships among the expressions of ApoH and cytokines TGF-pV IL-ip and LDL in renal tissues with various degree of damage, Pilot study the mechanism of ApoH in preventing interstitial fibrosis in children with nephrotic syndrome.Methods 60 specimens of renal boipsies with NS were collected, including paraffin sections, frozen sections and the unfixed renal tissue was used to extractive RNA. 22 cases with MCNS, 17 with MsPGN, 15 with FSGS and 6 with MN. 14 normal renal tissue as control. Using immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR) to detect ApoH, TGF-p,, IL-lp, LDL expression and distribution, and ApoH, TGF-pi, IL-ip mRNA expression. Double immuno-fluorescence to observe the influence of ApoH expression on TGF-Pi, IL-lp. Results (1) No TGF-Pi and IL-ip positive stain was found in normal renal tissue. Contrasted to normal renal tissue, TGF-Pi and IL-ip was detected mainly in tubulo-interstitium and in a few glomeruli with NS renal tissue. The higher expression of TGF-Pi , IL-lp of protein and mRNA were shown in patients of higher degree of tubules lesions group and higher degree of interstitial damage group, P < 0.01. The...