We first discovered autoantibodies to ACE (angiotensin converting enzyme) in patients with type 2 diabetes mellitus^1^. The antibodies were positive for 64.5% of the patients with diabetes and were positive in 83.3% in the early stage of clinical diabetic nephropathy. In addition, in genetically diabetogenic OLETF (Otsuka Long-Evans Tokushima Fatty) rats^2^, one of the characteristics of which strain is spontaneous nephropathy resembling those of human type 2 diabetes, and in control LETO rats^2^, immunization with rabbit lung ACE developed glomerulopathy similar to that seen in diabetics^3^. Also, in normal New Zealand white rabbits, immunization with the rabbit lung ACE induced glomerular changes similar to those seen in diabetic nephropathy^3^. In this study, renal tissues identical to those examined in research of diabetic nephropathy by PAS staining and electron microscope in preceding study^3^, were examined by immunostaining methods, only to prove that the diabetic glomerular changes may occur by immunization with ACE, not by non-specific responses to ACE, in non-diabetogenic rats and rabbits

Michihiro Matsuki

Seikoh Nishida

Tamaki Sasaki

Tsutomu Nohno

Yasushi Hirokawa

English

Nature Precedings

 1
Letters to Nature Medicine 
 
 
 
Spontaneous glomerular deposition of immunoglobulins for ACE 
( Angiotensin Converting Enzyme ) induces spontaneous nephropathy 
in diabetogenic rats 
 
 Seikoh Nishida1, Tamaki Sasaki2, Yasushi Hirokawa1, Michihiro Matsuki1  &  
 Tsutomu Nohno3 
 
 
 Division of 1Endocrinology and 2Nephrology,  Department of Medicine, and  
 3Department of Molecular Biology, Kawasaki Medical School, Kurashiki,        
 Okayama 701-0192, Japan 
 
 
 
 Key words: OLETF rat, spontaneously glomerular deposition, immunoglobulins    
  to ACE 
 
 
 
Correspondence to the present address: Seikoh NISHIDA, Fukushima 
Rehabilitaion Academy, Yorishima 16089-31, Asakuchi, Okayama 704-0101, 
 Japan 
 
 
 
 
 2
  We first discovered autoantibodies to ACE ( angiotensin converting enzyme ) in 
patients with type 2 diabetes mellitus1. The antibodies were positive for 64.5% of 
the patients with diabetes and were positive in 83.3% in the early stage of clinical 
diabetic nephropathy. In addition, in genetically diabetogenic OLETF ( Otsuka 
Long-Evans Tokushima Fatty ) rats2, one of the characteristics of which strain is 
spontaneous nephropathy resembling those of human type 2 diabetes, and in 
control LETO rats2, immunization with rabbit lung ACE developed 
glomerulopathy similar to that seen in diabetics3. Also, in normal New Zealand 
white rabbits, immunization with the rabbit lung ACE induced glomerular 
changes similar to those seen in diabetic nephropathy3. In this study, renal 
tissues identical to those examined in research of diabetic nephropathy by PAS 
staining and electron microscope in preceding study3, were examined by 
immunostaining methods, only to prove that the diabetic glomerular changes 
may occur by immunization with ACE, not by non-specific responses to ACE, in 
non-diabetogenic rats and rabbits. 
 Studies on OLETF rats3( referred to the former study ). To be brief, 
LETO(Long-Evans Tokushima Otsuka)rats, a control strain of OLETF rat, and 
OLETF rats were monthly immunized with rabbit lung ACE. The immunization 
was terminated at the 15th injection when the kidneys were extirpated for 
histological examinations. The kidneys were fixed by 10% formaldehyde solution 
and PAS stained for light microscopic examination and were fixed by 2.5% 
glutaraldehyde in 0.1 M PBS, pH 7.4 ( 1st fixation ) and osmium ( VIII ) oxide 
solution ( 2nd fixation ) for electronmicroscopic examination.  
Immunoperoxidase staining of the kidney was performed in rats at Special 
Reference Laboratories ( Tokyo, Japan ) as reported previously4.  Cryosections 
were permeabilized with absolute ethanol, and then exposed to microwave 
irradiation for 10 min in 10 mM citricbuffer, pH 6.0, at 98℃, or treated with 
0.05% protease ( Sigma, type XXVII), at 37℃, for 30 min for activation of the 
antigen.  Next, sections were treated for 5 min with hydrogen peroxidase and 
blocked with 10 % porcine serum.  After washing, sections were incubated with 
 3
goat anti-rat IgG ( Bethyl Lab ) or goat anti-rat IgM ( Bethyl Lab ), 1 : 100, 4℃ 
overnight.  Anti-goat IgG ( SBA Inc. ) conjugated to horseradish peroxidase 
( DAKO ) was used as secondary antibody.  For staining, incubation for 7 min in 
3, 3’-diaminobenzidine containing H2O2 was used. Nuclear staining was done 
with haematoxylin.  ELISA for anti-rabbit ACE antibodies was done as reported5.   
Studies on rabbits as a control study of the OLETF rat3. NZ white female rabbits  
were immunized with the rabbit lung ACE by monthly injections. The 
immunization was terminated at the 15th injection when the kidneys were 
extirpated for histological examinations. Then, immunoperoxidase staining of the 
kidney was performed as in OLETF rats.  
 The mean body weight of the rats was 280g for LETO rats and 350g for OLETF 
rats at 10 weeks old, but after the 14th immunization the same weight of 560g 
was shown for both groups.  In immunized LETO and OLETF rats, circulating 
antibodies to ACE were detected from the 4th immunization on, and titers of the 
antibodies reached a plateau after the 6th immunization. In OLETF rats 
glomerular changes were observed from 10 months on.  Also in LETO rats 
glomerular changes were demonstrated after immunization. It is reprted, by the 
institute which established the strain, that OLETF rats were demonstrating 
proliferation of the mesangial matrix and thickening of  the basement 
membrane of glomerulus after 40 weeks of age and at 70 weeks of age a 
PAS-positive nodular lesion was observed in almost every glomerulus2. The 
immunization with ACE did not induce changes in early morning plasma glucose 
and HbA1c levels in rats and rabbits3. 
        As shown in Figs. 1 and 2, both the immunized and unimmunized LETO rats 
and OLETF rats demonstrated glomerular deposition of immunoglobulins for 
ACE.  LETO rat showed spontaneously deposited glomerular immunoglobulins 
for ACE, though glomerulus did not show nephropathy3.  However, when 
immunized with ACE, glomerular changes similar to those seen in diabetic 
nephropathy were induced in LETO rat, resulting in a decrease in deposition of 
the immunoglobulins to a similar extent observed in OLETF rat or immunized 
 4
OLETF rat or even immunized rabbit. OLETF rat demonstrated both of 
spontaneous glomerular immunodeposit and spontaneous nephropathy similar 
to those of immunized LETO rat, but when immunized with ACE, glomerulus 
showed more remarkable nephropathy than unimmunized OLETF rat3. A 
normal NZ white rabbit showed glomerular PAS-positive nodular lesions after 
immunization with ACE3 and also demonstrated glomerular deposition of 
immunoglobulins equivalent to that of OLETF rats after immunization( Fig. 3 ), 
though no deposition was seen in an unimmunized rabbit.   
        Thus, the immunoglobulins for ACE deposited in glomorulus decreased after 
immunization with ACE in unimmunized LETO rats, but not in unimmunized 
OLETF rats. Concerning the decrease in glomerular deposition of 
immunoglobulins, Mauer et al6. demonstrated complete disappearance of 
intensely stained immunoglobulin G from mesangium 2 months after 
transplantation of the kidney of a diabetic rat into a normal rat.  Mauer et al6. 
also have demonstrated progressive localization of immunoglobulins G and M, 
and complement C3 within the mesangium in rats made diabetic by alloxan or 
streptozotocin, and they stated the glomerulopathy of diabetes in rats is 
secondary to the abnormal metablolic environment in which the kiney resides or 
a consequence of the diabetic state rather than genetic disorder. In our former 
study1, autoantibodies to ACE, which were recognized most frequently in the 
pre-nephropathy and incipient-nephropathy stages, gradually decreased in 
frequency towards the final stage. This may be due to the loss of autoantibody in 
urine, and this loss was most prominent in the IgG of small molecule.           
From the results obtained in the studies for LETO and OLETF rats, though 
control strain, LETO rats appear to share some of diabetogenic genes with 
OLETF rats7, it is suggested that spontaneous, “silent” deposit of 
immunoglobulins for ACE seen in LETO rats begins to decrease when additional 
deposition by immunization with ACE was added only to create the abnormal 
metabolic environment. On the other hand, as glomerular function becomes 
impaired by immunization with ACE, this leads to further glomerular pathology 
 5
and as nephropathy progresses the immunoglobulins gradually disappear from 
glomerus. However, if diabetic state exists, nephropathy can occur only by 
spontaneous deposition of the immunoglobulins, without additional deposit, as in 
OLETF rats. Also, it is very interesting that, though diabetic state does not exist, 
nephropathy similar to that of diabetics occurs in normal rabbit by 
immunization with ACE.  A previous paper8 is suggesting for OLETF rats that 
since disturbance of renal function exists already at 6 week-old age, before 
diabetes being found, specific genes independent of diabetogenic genes must be 
working separately for process of nephropathy. In respect of immunology, we are 
easily accessible to reports showing glomerular deposition of immunoglobulin or 
complement for unidentified antigenic substance in rat nephropathy9,10. 
        From all the results combined1,3, it is suggested that the immunoglobulin for 
ACE plays an important role for the occurrence of diabetic nephropathy. 
 
      ACKNOWLEDGEMENTS. 
        This work was supported in part by Research Project Grants to S.N. from the 
Kawasaki Medical School and the Animal Research Committee of the School 
approved the studies on animals. 
      References 
      1. Nishida, S. et al. Endocrine Journal 50, 209-213(2003). 
      2. Kawano, K. et al. Diabetes 41, 1422-1428(1992). 
      3. Nishida, S. et al. Endocrine Journal 50, 801-807(2003). 
      4. Nakane, P.K. & Pierce, G.B.  J. Cell.Biol. 33, 307-318(1967).  
      5. Alhenc-Gelas, F., Weare, J.A., Johnson, R.L.,Jr. & Erdös, E.G.  J. Lab. Clin. 
Med. 101, 83-96(1983). 
      6. Mauer, S.M., Steffes, M.W. & Brown, D.M. Animal models of diabetic 
nephropathy ; in Hamburger, J., Crosnier, J., Grünfeld, J.P. & Maxwell, 
M.H.(eds) : Advances in nephrology. Chicago, Year Book Medical publishers 
vol 8, pp23-42(1979). 
      7. Kawano, K., Hirashima, T., Mori, S. & Natori, T.  Diabetes Res. Clin. Pract. 
 6
24 Suppl., 317-320(1994). 
      8. Hada, K. et al. ; in Shima, K.(ed) : Proc. OLETF Study 1 : 14, pp63-68(1995). 
(In Japanese). 
      9. Fujita, N. et al. ; in Shima, K.(ed) : Proc. OLETF Study 4 : 25, pp108-112 
(1998).(In Japanese). 
      10. Hong, K., Kinoshita, T., Miyazaki, W., Izawa, T. & Inoue, K.  J. Immunol. 
122, 2418-2423(1979). 
       
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 7
 Legends for Figures 
      Fig. 1. Glomerular deposition of immunoglobulins M(a) and G(b) for ACE in a 68 
week-old LETO rat. Also, Ig M(c) and IgG(d) in a 68 week-old LETO rat 
immunized with ACE. Original magnification × 100. Glomeruli ; 
demonstrating diffusely intense deposition of IgM and IgG in capillary 
endothelial cell and in mesangial region. 
 
      Fig. 2. Glomerular deposition of immunoglobulins M(a) and G(b) for ACE in a 68 
week-old OLETF rat. Also, Ig M(c) and IgG(d) in a 68 week-old OLETF 
rat immunized with ACE. Original magnification×100. Glomeruli ; 
demonstrating diffuse deposition of IgM and IgG in capillary endothelial 
cell and in mesangial region. 
       
Fig. 3. Glomerular deposition of immunoglobulins M(a) and G(b) for ACE in a NZ 
white rabbit after 15th monthly immunization with ACE. Original 
magnification×100. Glomeruli ; demonstrating diffuse deposition of IgM 
and IgG in capillary endothelial cell and in mesangial region. 
 
 
Addendum 
 Figures from Reference 3 as a reference. 
     Fig. 1. A 68 week-old, unimmunized LETO rat weighing 590 g demonstrates 
normal glomerulus in PAS –staining (a, ×400) and a 68 week-old LETO rat 
weighing 500 g, treated with ACE demonstrates expansion of PAS-positive 
mesangial matrix and thickening of lumen wall (b, ×400). A 68 week-old, 
unimmunized diabetic OLETF rat weighing 530 g demonstrates the 
increase in mesangial matrix and the deposit of PAS-positive materials in 
it, which perform segmental sclerosis (c, ×400). A 68 week-old diabetic 
OLETF rat weighing 575 g, immunized with ACE demonstrates a chronic 
thickening of lumen wall and expansion of the mesangial matrix where 
 8
cell infiltration and, partly, nodular lesions are seen(d, ×400). 
 
    Fig.2.   Photos a and b of a control rabbit demonstrate normal glomerulus and 
tubulointerstitium in PAS staining(a, ×100) and electron microscopic 
examination (b, ×3000).  PAS staining of immunized rabbits (×100); rabbit 
1 (c) demonstrates PAS-positive materials mainly at the mesangium 
region. Rabbit 2 (e) demonstrates PAS-positive materials mainly at the 
mesangium region. The expansion of mesangial region and a decrease in 
mesangial cells together form a nodular lesion similar to that in diabetic 
nephropathy. Electron microscopic examination( d and f, ×3000); rabbit 2 
(f) shows an increase in mesangial matrix and low electron-dense deposits 
in the mesangial region. 
 
 
 
 
 
 
 
 
 
 







Spontaneous glomerular deposition of immunoglobulins for ACE (Angiotensin Converting Enzyme) induces spontaneous nephropathy in diabetogenic rats

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Spontaneous glomerular deposition of immunoglobulins for ACE (Angiotensin Converting Enzyme) induces spontaneous nephropathy in diabetogenic rats

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