C5b - 9 NO TNF 3. ( anti - thymocyte serum nephritis, ATSN) ( mesangial proliferative glomerulonephritis, MPGN)

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1 54 Chinese Journal of Pathophysiology 2004,20 (1) :54-59 [ ] (2004) C5b - 9 NO TNF 3 1, 1, 2, 2 ( 1, ; 2, ) [ ] : (ATSN) C5b - 9 : (NO) ( TNF ) : (ATS) ATSN,ATSN C5b - 9 ;C5b - 9 (MC) ATSN NO (inos) mrna NO (NO 2 - / NO 3 - ) TNF : ATSN MC, ( ) C5b - 9 MC ;,C5b - 9 MC, ATSN MC inos mrna, NO - 3 TNF ATSN ( 7 d ), : ATSN MC C5b - 9 NO TNF [] ; ; ; ; [] R ; R69213 [] A ( anti - thymocyte serum nephritis, ATSN) ( mesangial proliferative glomerulonephritis, MPGN) (tumor necrosis factor, TNF ) MC Rat - [6 ],C5b - 9 [ ] [ ] [ ] 333 (No ) ;(No. BK97154) TNF, 1 (mesangial cells, MC), FITC - IgG FITC - C3 ABC, [8 ], MC (lytic) (proliferating cell nuclear anti2 C5b - 9 gen, PCNA) - (smooth muscle actin,- SMA) C5b - 9,( sublytic) C5b - 9, Dako ( Bam HI) [1-3 ], ATSN KRNA (digoxinin, DIG) RNA (SP 6 / T 7 ) MC (nitric ox2 B1M1 ide, NO) [4,5 ], OCT Amresco ELISA TNF Endogen NO2 - / NO3 - MC NO TNF,TNF inos cdna Nor2 MC NO [7 ],C5b - 9 vatis SD NO ATSN BK1 [8 ] ATSN C5b (anti - thymocyte serum, ATSN,ATSN ATS) [9 ] (ATS, C5b - 9 NO 1 320) 212 ATSN (1) SD 24 ( g), 2 ATSN ( n = 18) :

2 55 ATS,015 ml/ 100 g BW(2) : A ( n = 6) : - SMA,ATS (:ATS 3, 6,, 6) (2) : 24 h,ats,nmol/ h 48 h 96 h 7 d 14 d :, 218 TNF ATS 24 h 96 h 4 %, ELISA TNF ; OCT ; TNF24 h, TNF,ng/ 24 h ATS 2h 24 h 48 h 96 h 7 d 14 d (: 24 h) 213, 24 h 24 h, mg/ 24 h 214 (1) HE,,,20, (2) FITC - IgG C3 30 min,pbs IgG C3 HCl 012 % Triton X min,k Yamamoto [10 ] (3) 215 (1) PCNA ABC : 2 SSC, 50 % 1 40 PCNA,37 30 min,pbs 3 ; ABC,37 30 min,pbs 3 ; ABC 30 min,dab 5 min,, PCNA, h buffer 1,buffer 3 PCNA, (2)- SMA SMA, ABC PCNA - SMA MD 20 Leitz (C) (:3 ),20 3 ( A ) (:20 DAB q, A ) 216 C5b - 9 (1) : C5b - 9 (1 25),ABC,PCNA (3) :ATS 2h 24 h 48 h, C5b - 9, PCNA 217 NO - 3 h 219 NO (inducible NO synthase, inos) mrna (1) inos crna [11 ] (2) :SD 6 ( g) 5 ATS,015 ml/ 100 g BW,1 ATS 3 h 011 mol L - 1 PBS (ph714), 4 % 20 min :,,012 mol L - 1 (1 g L - 1 ) min, 012 % PBS 10 min,5 mmol MgCl 2 PBS 2 4 %15 min,, 4 : 15 min, 42 3 h DIG - antisense RNA(cRNA) h 2 SSC 011 SSC buffer 1 15 min, buffer 2 30 min DIG NBT/ BCIP 2 h,buffer 4 : (A) DIG - sense RNA ; (B) RNA ; ( gx s),, PEMS

3 56 1 ATSN 111 ATS 24 h, [ ( ) mg],,48 h ( ) mg, 96 h (67132 ATS 2 h,, 15197) mg, 7 d ( ) mg,14 d ( ) mg, 96 h,,7 d [ ( ) mg], ( P < 0105 P < 0101) 112 IgG C3 ATS 2 h 24 h, IgG,, 3 +,48 h IgG2 +,96 h ,7 d ,14 d -,,48 h,(1a) 96 h, ATS 2 h 24 h,c3, ,7 d 14 d -, IgG C3 24 h 48 h,,,, 14 d,96 h,, ATSN ATS 2 h,, MC 24 h,mc, MC,7 d 14 d,mc IgG, ,48 h 1 +,96 h, (1B) Fig 1 The result of electron microscope. A : Disappear of glomerular MC at 48 h after ATS injection ( 6 000) ; B : Proliferation of glomeru2 lar MC and excessive mesangium matrix on day 7 after ATS injection( 4 000). 1 PCNA,, MC, PCNA, ATS 2 h, C5b - 9 MC, C5b PCNA 24 h 48 h,pcna,96 h,7 d 14 d,atsn PCNA,1 - SMA - 9 (2),C5b - 9 C5b - 9 A ( 1), ATS 2 h 24 h,c5b - 9 A 48 h 96 h ATS 2 h 24 h 48 h,- A,7 d 14 d,c5b - 9 A SMA 96 h,- SMA (:C5b - 9,,,7 d,a DAB ),14 d, C5b - 9 MC ( :/ (A 1) ), ATS 24 h ( ) 48 2 C5b - 9 h( ),MC 2 h ( ), ATS 2 h, C5b h

4 57 Fig 2 The marked deposits of C5b - 9 complexes in glomerular mesangium and MC surface by immunohistochemical staining at 2 h after injecting ATS( 400). 2 ATS 2 h, 3 NO - 3 ATS 24 h 48 h, NO (nmol/ 24 h), ( ), ( P < 0101) 96 h,no - 3 ( ),( P < 0105), 7 d 14 d, NO ,, ( P > 0105) 4 TNF ATS 24 h 96 h,tnf,( ) ng/ 24 h ( ) ng/ 24 h, [ ( ) ng/ 24 h ] ( P < 0101) 5 inos mrna inos mrna NO ATS 3 h,, inos mrna (3),, inos mrna ATSN Group 1 ATSN Tab 1 Comparison of several detecting items in ATSN and control group ( gx s. n = 6) Cell numbers (counts/ glomerulum) PCNA nuclear (counts/ glomerulum) Detecting items - SMA ( A value) C5b - 9 ( A value) 2 h h h h d d Control 3 P < 0. 05, 33 P < vs control group MC Thy1, T Thy1, [10 ] [2,12 ], 24 h,,, MC ( PCNA), MC Thy1,96 h (IC), ATS 2 h, IgG C3 MC,, IC,ATSN,,MC, ATS, MC C5b h,mc,,48 h 7 d,, MPGN

5 58 Fig 3 The results of in situ hybridization ( 400). The positive staining of glomerular MC inos mrna at 3 h after ATS in2 jection. 3,IgA, [2] Brandt J, Pippin J, Schulze M, et al. Role of the complement C5b - 9 [13 ] membrane attack complex (C5b - 9) in mediating experimen2 Hinglasis [14 ] tal mesangioproliferative glomerulonephritis [ J ]. Kidney Int,, IC 1996, 49 (2) : C5b - 9 IC, [3 ] Couser WG. Pathogenesis of glomerular damage in glomerular C5b - 9 nephritis[j ]. Nephrol Dial Transplant, 1998, 13 ( suppl 1) :,ATSN C5b MC, IgG [4 ] Narita I, Border WA, Kettelar M, et al. Nitric oxide mediates ATSN [3 ], immunologic injury to kidney mesangium in experimental C5b - 9 ATSN glomerulonephritis[j ]. Lab Invest, 1995, 72 (1) : C5b - 9 [5 ] Cattell V. Nitric oxide and glomerulonephritis [J ]. Kidney C5b - 9, NO - NO - 3, MC 3 h inos mrnaatsn TNF, ATSN NO TNF ATSN MC NO [4,15 ] TNF MC, [6 ] MC,C5b - 9 ATSN MC [7 ],,AT2 SN,C5b - 9 MC,C5b - 9 MC MC ( ) C5b - 9 MC [ ] [ 1 ] Johnson RJ, Pritzl P, Itda H, et al. Platelet - complement in2 teractions in mesangial proliferative nephritis in the rat [J ]. Am J Pathol, 1991, 138 (2) : Int, 2002, 61 (3) : , C5b - 9 [6 ] Ivanov AA, Gladskikh OP, Bogomazova SIu, et al. Role of, [3 ] tumor necrosis factor alpha in the regulation of extracellular matrix and proliferation of mesangial cells in nephrotoxic ATS 2 h, nephritis [J ]. Arkh Pathol, 1998, 60 (1) : , C5b - 9, [7 ],,,. C5b - 9 C5b - 9, NO [J ]. C5b - 9 MC 24, 2001, 17 (10) : h,c5b - 9 MC,48 h [8 ],,,. C5b - 9 ( ) C5b - 9 [2 ],ATSN MC [J ]., 2000, 16 (9) : C5b , C5b - 9 [9 ],,,. Thy1 MC NO TNF,ATS 24 h 48 h, [J ].,1996,12 (3) : [ 10 ] Yamamoto T, Wilson CB. Quantitative and qualitative studies

6 59 of antibody induced mesangial cell damage in the rat[j ]. Kid2 ney Int, 1987,32 (4) : [11 ],,,. (i2 NOS) crna [J ].,1999,19 (4) : [12 ] Shimizu A, Mascuda Y, Kitamura H, et al. Complement - mediated killing of mesangial cells in experimental glomeru2 lonephritis: cell death by a combination of apoptosis and necrosis [J ]. Nephron, 2000, 86 (2) : [13 ] Hinglasis N, Kazatchkine MD, Bhakdi S, et al. Immunohis2 tochemical study of the C5b - 9 complex of complement in hu2 man kidney [J ]. Kidney Int, 1986, 30 (3) : [14 ] Brune B. Nitric oxide and apoptosis in mesangial cells[j ]. Kidney Int, 2002, 61 (3) : [15 ] Nauta AJ, Daha MR, Tijsma O, et al. The membrane attack complex of complement induced caspase activation and apopto2 sis[j ]. Eur J Immunol, 2002, 32 (3) : Analysis of glomerular complement C5b - 9 deposits and synthesis of NO, TNF in the model of the rats with anti - thymocyte serum nephritis WANG Ying - wei 1, XU Jing - hua 1, TANG Ren - xian 2, GAO Feng - guang 2 ( 1 Department of Immunology, Nanjing Medical University, Nanjing , China ; 2 Department of Immunology, Xuzhou Medical College, Xuzhou , China) [ ABSTRACT] AIM : To explore the localization and semi - quantification of the glomerular complement C5b - 9 complexes and synthesis of some inflammatory mediators or cytokines such as nitric oxide (NO) and tumor necrosis factor (TNF ) in the rats with anti - thymocyte serum nephritis(atsn). METHODS : The animal model of rat ATSN was re2 produced by a single intravenous injection of anti - thymocyte serum (ATS). Then, the deposits of glomerular C5b - 9 complexes were localized and quantified by immunohistochemical staining and microscopic image scanning separately. And the glomerular mesangial cells (MC) surrounded by C5b - 9 complexes were counted under microscope. In addition, the expression of glomerular MC inducible NO synthase ( inos) mrna and excretion of urinary NO metabolite (NO - 3 ) and TNFin the rats with ATSN were detected. RESULTS : The MC in the rats with ATSN emerged necrosis followed by a rapid proliferation. In the early time of MC injury, the C5b - 9 complexes were mainly seen in glomerular mesangium and MC surface. But with the progression of ATSN, the MC enclosed by C5b - 9 appeared gradual decrease. Moreover, the expression of MC inos mrna in early stage of ATSN obviously increased and the excretion of urinary NO - 3 and TNFalso significantly increased. However, the changes of parameters mentioned above in AT2 SN proliferative stage (after 7 days) alleviated gradually. CONCL USION : The secondary lysis of MC has relation to the deposition of C5b - 9 complexes and synthesis and release of NO and TNFin rats with ATSN. [ KEY WORDS] Nephritis ; Complement membrane attack complex ; Nitric oxide ; Tumor necrosis factor ; Rats