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Collagens in chronic rejection of rat renal allografts
ELSEVIER
Collagens in Chronic Rejection of Rat Renal Allografts K. Inkinen, A. Soots, L. Krogerus,
I. Lautenschlager,
and J. Ahonen
I
NTERSTITIAL FIBROSIS is characteristic histological feature of chronic rejection of renal allograft.’ The major components of fibrosis are collagen proteins. The ratio of type I to type III collagen varies in different tissues and in some disease processes2 Increased amounts of type III collagen have been detected during repair processes.’ To establish the development of fibrosis, the role and the temporal expression of various collagen types were studied in an experimental model of renal transplantation in the rat, developing chronic rejection within 60 days under immunosuppression. MATERIALS
AND
of DNA in grafts was higher than in control kidneys at every time point, reflecting the increase of the amount of cells in the graft. There was no expression of type I and III collagen mRNA in the control kidney, whereas in the graft, type I and III collagens could be demonstrated. Chronic rejection with interstitial fibrosis was diagnosed histologically 40 to 60 days posttransplantation. CONCLUSIONS
In this study, we demonstrated the time-related development of diffuse interstitial fibrosis in chronic renal allograft rejection in a rat model. Our results show that the total collagen content increased significantly in allografts but not in control kidneys, and the increase correlated well with the histological findings of chronic rejection and graft fibrosis. In the grafts, expression of type I and III collagens was demonstrated. In control kidneys, there was no expression of either type of collagen. These results suggest that the fibrotic process of chronic allograft rejection resembles repair processes. These are preliminary results, and experiments are in progress to characterize further the development of fibrosis.
METHODS
Renal transplantations (n = 20) were performed in a strain combination of DA (RTl”)/BN (RTl”) under triple drug treatment (MP 2 mg/kg, AZA 2 mgikg, CyA 5 mg/kg daily SC) without nephrectomy of the other own kidney. The animals were killed at different times after transplantation: 4,7, 10,20,40,50, and 60 days postoperation. From the tissue homogenate (graft and own kidney as control) total collagen (as hydroxyproline) and DNA content were measured as described previously4S’,6and total RNA extracted.’ Expression of collagen was studied by Northern blot hybridization using cDNA probes specific for rat pro-alfa 1 (I) and pro-alfa 1 (III) mRNAs” by using digoxigenin labelled probes. Graft histology was evaluated according to the Banff criteria.s
From the Fourth Department of Surgery and Laboratory Department, Helsinki University Central Hospital and Department of Pathology, Maria Hospital, Helsinki, Finland. This work was supported by grants from the Sigrid Juselius Foundation, University of Helsinki, and Helsinki University Central Hospital (EVO). Address reprint requests to K. Inkinen, Fourth Department of Surgery, Research Laboratory, Helsinki University Central Hospital, Kasarmikatu 11-l 3, FIN-001 30 Helsinki, Finland.
RESULTS
Table 1 demonstrates the time-related total collagen and DNA concentrations and expression of collagen type I and III mRNAs in rat renal allografts ending up with chronic rejection. Compared with the control kidney, total collagen content of the graft was significantly (P < .02) increased from 10 days onwards, being highest on day 40. The amount Table 1. Total Collagen and DNA and Expression
of Collagen Type I and Ill mRNA in Rat Renal Allograft Days After
4
7
10
Coi Contr (Fg/mg) Col Graft (pg/mg)
7.0 rf- 1.6 8.4 + 1.3
8.9 + 0.1 12.7 t 7.1
13.1 z 1.2’
DNA Contr
(pg/mg)
3.9 f 0.9
3.8 -c 0.3
2.2 2 1.0
DNA Graft (pg/mg)
4.9 k 1 .o
4.6 k 1.2
6.2 t
+
+
+
+
mRNA Col
I
mRNA Col Ill
Col Contr: graft kidneys; ‘P < .02
Transplantation 20
6.2 t 0.0
40
50
60
6.6 -t 0.9
7.8 + 2.7
7.8 t 2.7
ND
18.1 I? 2.0
27.0 k 4.0*
20.4 ? 4.1’
I 0.8
2.6 t 0.4
4.5 -c 0.5
4.3 2 0.6
ND
7.1 k 3.7
6.6 + 2.2
5.8 ? 0.5
3.8
_
_
_
+ _
_
+
+
+
+
+
1.7
total collagen,control kidneys; mRNA
Col I: expression
Cal Graft: total collagen, graft kidneys: DNA Contr: DNA concentration, control kidneys; DNA Graft: DNA concentration, of type I collagen mRNA; mRNA Cal Ill: expression of type Ill collagen mRNA; ND: not determined.
1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0
0041-1345/97/$17.00 PII SO041 -1345(96)00669-O
1543
Transplantation
Proceedings,
29, 1543-l 544 (1997)
1544 REFERENCES 1. Tilney NL, Whitley WD, Diamond JR, et al: Transplantation 52389, 1991 2. Prockop DJ, Kivirikko KI: N Engl J Med 311:376, 1984 3. Glumoff V, MBkeIi JK, Vuorio E: Biochim Biophys Acta 1217:41, 1994
INKINEN, SOOTS, KROGERUS 4. Edwards CA, O’Brien WD Jr: Clin Chim Acta 104161, 1980 5. Schmidt G, Thannhauser SJ: J Biol Chem 161:83, 1945 6. Burton K: Biochem J 62:315, 1956 7. Chomczynski P, Sacchi N: Anal Biochem 162:156, 1987 8. Solez K, Axelsen RA, Benediktsson H, et al: Kidney Int 44:411, 1993
Collagens in Chronic Rejection of Rat Renal Allografts K. Inkinen, A. Soots, L. Krogerus,
I. Lautenschlager,
and J. Ahonen
I
NTERSTITIAL FIBROSIS is characteristic histological feature of chronic rejection of renal allograft.’ The major components of fibrosis are collagen proteins. The ratio of type I to type III collagen varies in different tissues and in some disease processes2 Increased amounts of type III collagen have been detected during repair processes.’ To establish the development of fibrosis, the role and the temporal expression of various collagen types were studied in an experimental model of renal transplantation in the rat, developing chronic rejection within 60 days under immunosuppression. MATERIALS
AND
of DNA in grafts was higher than in control kidneys at every time point, reflecting the increase of the amount of cells in the graft. There was no expression of type I and III collagen mRNA in the control kidney, whereas in the graft, type I and III collagens could be demonstrated. Chronic rejection with interstitial fibrosis was diagnosed histologically 40 to 60 days posttransplantation. CONCLUSIONS
In this study, we demonstrated the time-related development of diffuse interstitial fibrosis in chronic renal allograft rejection in a rat model. Our results show that the total collagen content increased significantly in allografts but not in control kidneys, and the increase correlated well with the histological findings of chronic rejection and graft fibrosis. In the grafts, expression of type I and III collagens was demonstrated. In control kidneys, there was no expression of either type of collagen. These results suggest that the fibrotic process of chronic allograft rejection resembles repair processes. These are preliminary results, and experiments are in progress to characterize further the development of fibrosis.
METHODS
Renal transplantations (n = 20) were performed in a strain combination of DA (RTl”)/BN (RTl”) under triple drug treatment (MP 2 mg/kg, AZA 2 mgikg, CyA 5 mg/kg daily SC) without nephrectomy of the other own kidney. The animals were killed at different times after transplantation: 4,7, 10,20,40,50, and 60 days postoperation. From the tissue homogenate (graft and own kidney as control) total collagen (as hydroxyproline) and DNA content were measured as described previously4S’,6and total RNA extracted.’ Expression of collagen was studied by Northern blot hybridization using cDNA probes specific for rat pro-alfa 1 (I) and pro-alfa 1 (III) mRNAs” by using digoxigenin labelled probes. Graft histology was evaluated according to the Banff criteria.s
From the Fourth Department of Surgery and Laboratory Department, Helsinki University Central Hospital and Department of Pathology, Maria Hospital, Helsinki, Finland. This work was supported by grants from the Sigrid Juselius Foundation, University of Helsinki, and Helsinki University Central Hospital (EVO). Address reprint requests to K. Inkinen, Fourth Department of Surgery, Research Laboratory, Helsinki University Central Hospital, Kasarmikatu 11-l 3, FIN-001 30 Helsinki, Finland.
RESULTS
Table 1 demonstrates the time-related total collagen and DNA concentrations and expression of collagen type I and III mRNAs in rat renal allografts ending up with chronic rejection. Compared with the control kidney, total collagen content of the graft was significantly (P < .02) increased from 10 days onwards, being highest on day 40. The amount Table 1. Total Collagen and DNA and Expression
of Collagen Type I and Ill mRNA in Rat Renal Allograft Days After
4
7
10
Coi Contr (Fg/mg) Col Graft (pg/mg)
7.0 rf- 1.6 8.4 + 1.3
8.9 + 0.1 12.7 t 7.1
13.1 z 1.2’
DNA Contr
(pg/mg)
3.9 f 0.9
3.8 -c 0.3
2.2 2 1.0
DNA Graft (pg/mg)
4.9 k 1 .o
4.6 k 1.2
6.2 t
+
+
+
+
mRNA Col
I
mRNA Col Ill
Col Contr: graft kidneys; ‘P < .02
Transplantation 20
6.2 t 0.0
40
50
60
6.6 -t 0.9
7.8 + 2.7
7.8 t 2.7
ND
18.1 I? 2.0
27.0 k 4.0*
20.4 ? 4.1’
I 0.8
2.6 t 0.4
4.5 -c 0.5
4.3 2 0.6
ND
7.1 k 3.7
6.6 + 2.2
5.8 ? 0.5
3.8
_
_
_
+ _
_
+
+
+
+
+
1.7
total collagen,control kidneys; mRNA
Col I: expression
Cal Graft: total collagen, graft kidneys: DNA Contr: DNA concentration, control kidneys; DNA Graft: DNA concentration, of type I collagen mRNA; mRNA Cal Ill: expression of type Ill collagen mRNA; ND: not determined.
1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0
0041-1345/97/$17.00 PII SO041 -1345(96)00669-O
1543
Transplantation
Proceedings,
29, 1543-l 544 (1997)
1544 REFERENCES 1. Tilney NL, Whitley WD, Diamond JR, et al: Transplantation 52389, 1991 2. Prockop DJ, Kivirikko KI: N Engl J Med 311:376, 1984 3. Glumoff V, MBkeIi JK, Vuorio E: Biochim Biophys Acta 1217:41, 1994
INKINEN, SOOTS, KROGERUS 4. Edwards CA, O’Brien WD Jr: Clin Chim Acta 104161, 1980 5. Schmidt G, Thannhauser SJ: J Biol Chem 161:83, 1945 6. Burton K: Biochem J 62:315, 1956 7. Chomczynski P, Sacchi N: Anal Biochem 162:156, 1987 8. Solez K, Axelsen RA, Benediktsson H, et al: Kidney Int 44:411, 1993