MATRIX METALLOPROTEINASES AND THEIR INHIBITORS IN CHRONIC RENAL ALLOGRAFT NEPHROPATHY (CAN)

A K Ahmed 1*, E Khedr 1, A M El Nahas 1 and T S Johnson 1.

1 Sheffield Kidney Institute (University of Sheffield), Northern General Hospital, Sheffield, United Kingdom.

Background: CAN remains the major cause of graft loss after the first year of transplantation.  A key event in renal scarring is the accumulation of extracellular matrix (ECM) proteins.  ECM turnover is regulated predominantly by matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP).  The aim of this study is to determine if changes in the MMP / TIMP system contribute to the ECM accumulation and scarring characteristic of CAN.

 

Patients and Methods: Sequential renal biopsies from 26 graft recipients (including implantation) were obtained and diagnosed by Banff 97 into acute and chronic rejection subsets.  In situ zymography was used to locate changes in proteolytic activity within the biopsies using Gelatin and Collagen I substrates.  MMPs 1, 2, 3, and 9, and TIMP 2 and 3 were localised using immunohistochemistry and evaluated by point counting.  Sections were further evaluated by Masson’s Trichrome, α smooth muscle actin (SMA) and collagen III / IV staining to assess the level of fibrosis.

 

Results: Progressive increases in Masson’s Trichrome, Collagens III, IV and α SMA staining was observed from implantation through to CAN.  In situ Zymography showed proteolytic activity decreased with disease progression, with proteolysis predominantly cytoplasmic and located mainly in the tubular epithelium.  Immunostaining for both MMPs and TIMPs was consistent with in situ zymography with staining being mainly cytoplasmic and tubular in location.  Point count analysis revealed levels of MMP 9 were reduced at implantation, with all MMPs significantly reduced in both early and CAN biopsies compared to normal.  TIMP 2 and 3 were raised significantly at implantation and remained high post transplant in both early and CAN biopsies compared to normal (see table below).

 

 

Normal

 

Implantation

 

Acute Rejection

 

CAN

 

MMP1

44.2 ± 2.6%

39.2 ± 13.9%

3.1 ± 2.0% C

5 ± 3.2% C

MMP2

17.5 ± 0.8%

11.6 ± 3.7%

7.2 ± 2% B

8.5 ± 3.1% B

MMP3

19.7 ± 4.1%

13.5 ± 4%

5.7 ± 1.6% B

6.9 ±2  % B

MMP9

28.2 ± 5%

8.5 ± 2.2% B

3.3 ± 1.2% C

5.2 ± 2.7% C

TIMP2

14.6 ± 4.48%

51.8 ± 5.2% C

29.2 ± 4.6% B

34.0 ± 4.1% B

TIMP3

23.6 ± 6.7%

44.2 ± 9.5% B

40.4 ± 5.8% B

48.3 ± 6.3% B

Data represents mean point count (% points) ± SEM.  A= P<0.05 , B= P<0.01 ,C= P<0.001 from normal.

 

Conclusions: Proteolysis was reduced post transplant due to reduced MMP and elevated TIMP expression. TIMPs were increased at implantation and remained high post transplantation. These findings are consistent with reduced ECM degradation contributing to progressive CAN.