Immunohistochemical Analysis of Macrophages,Myofibroblasts,and Transforming Growth Factor-β Localization during Rat Renal Interstitial Fibrosis Following Long-Term Unilateral Ureteral Obstruction

Abstract

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-β (TGF-β, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of α-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-β was found in infiltrating macrophages or myofibroblasts. However, TGF-β was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-β may be renal epithelia. However, the staining intensity for TGF-β on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-β to the advanced stages of UUO-induced renal fibrosis remains to be determined.

Keywords

Fibroblast fibrogenic cytokine F344 rat immunostaining mononuclear cell renal fibrosis

References

Baur PS , Parks DH , and Hudson JD (1984). Epithelial mediated wound contraction in experimental wounds: The purse-string effect. J. Trauma 24: 713-720.

Beelen RHJ , Eestermans IL , Dopp EA , and Dijkstra CD (1987). Monoclonal antibodies ED1, ED2, and ED3 against rat macrophages: Expression of recognized antigens in different stages of differentiation. Transplant. Proc. 19: 3166-3170.

Diamond JR , Kees-Folts D. , Ding G. , Frye JE , and Restrepo NC (1994). Macrophages, monocyte chemoattractant peptide-1, and TGF-β1 in experimental hydronephrosis. Am. J. Physiol . 266: F926-F933.

Diamond JR , Pesek I. , Ruggieri S. , and Karnovsky MJ (1989). Essential fatty acid deficiency during acute puromycin nephrosis ameliorates late renal injury. Am. J. Physiol. 257: F798-F807.

Diamond JR , van Goor H. , Ding G. , and Engelmyer E. (1995). Myofibroblasts in experimental hydronephrosis . Am. J. Pathol. 146: 121-129.

Eddy AA (1994). Protein restriction reduces transforming growth factor-β and interstitial fibrosis in nephrotic syndrome. Am. J. Physiol. 266: F884-F893.

Eddy AA (1996). Expression of genes that promote renal interstitial fibrosis in rats with proteinurea. Kidney Int. 54: S49-S54.

Foo ITH , Naylor IL , Timmons MJ , and Trejdosiewicz LK (1992). Intracellular actin as a marker for myofibroblasts in vitro. Lab. Invest. 67: 727-733.

Fukatsu A. , Matsuo S. , Yuzawa Y. , Miyai H. , Futenma A. , and Kato K. (1993). Expression of interleukin 6 and major histocompatibility complex molecules in tubular epithelial cells of diseased human kidneys. Lab. Invest. 69: 58-67.

10.

Gown AM (1990). The mysteries of the myofibroblasts (partially) unmasked. Lab. Invest. 63: 1-3.

11.

Henke C. , Marineili W. , Jessurun J. , Fox J. , Harms D. , Peterson M. , Chiang L. , and Doran P. (1993). Macrophage production of basic fibroblast growth factor in the fibroproliferative disorder of alveolar fibrosis after lung injury. Am. J. Pathol. 143: 1189-1199

12.

Hines JE , Johnson SJ , and Burt AD (1993). In vivo responses of macrophages and perisinusoidal cells to cholestatic liver injury. Am. J. Pathol. 142: 511-518.

13.

Ishidoya S. , Morrissey J. , McCracken R. , and Klahr S. (1996). Delayed treatment with enalapril halts tubulointerstitial fibrosis in rats with obstructive nephropathy . Kidney Int. 49: 1110-1119.

14.

Johnson SJ , Hines JE , and Burt AD (1992). Macrophages and perisinusoidal cell kinetics in acute liver injury. J. Pathol. 166: 351-358.

15.

Kaneto H. , Morrissey J. , and Klahr S. (1993). Increased expression of TGF-β mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int. 44: 313-321.

16.

Khalil N. , Bereznay O. , Sporn M. , and Greenberg AH (1989). Macrophage production of transforming growth factor β and fibroblast collagen synthesis in chronic pulmonary inflammation. J. Exp. Med. 170: 727-737.

17.

Knecht A. , Fine LG , Kleinman KS , Rodemann HP , Muller GA , Woo DDL , and Norman JT (1991). Fibroblast of rabbit kidney in culture. II. Paracrine stimulation of papillary fibroblasts by PDGF. Am. J. Physiol . 261: F292-F299.

18.

Koike S. and Noumura T. (1993). Immunohistochemical localization of TGF-β in the developing rat gonads. Zool Sci. 10: 671-677.

19.

Kuncio GS , Neilson EG , and Haverty T. (1991). Mechanisms of tubulointerstitial fibrosis. Kidney Int. 39: 550-556.

20.

Nagle RB , Johnson ME , and Jervis HR (1976). Proliferation of renal interstitial cells following injury induced by ureteral obstruction. Lab. Invest. 35: 18-22.

21.

Nagle RB , Kneiser MR , Bulger RE , and Benditt EP (1973). Induction of smooth muscle characteristics in renal interstitial fibroblasts during obstructive nephropathy. Lab. Invest. 29: 422-427.

22.

Nakatsuji S. , Yamate J. , Kuwamura M. , Kotani T. , and Sakuma S. (1997). In vivo responses of macrophages and myofibroblasts in the healing following isoproterenol-induced myocardial injury in rats. Virchows Arch. 430: 63-69.

23.

Nath KA (1996). Reshaping the interstitium by platelet-derived growth factor. Implication for progressive renal disease. Am. J. Pathol. 148: 1031-1036.

24.

Noda S. , Masumi S. , Moriyama M. , Kannan Y. , Ohta M. , Sugano T. , and Yamate J. (1996). Population of hepatic macrophages and response of perfused liver to platelet-activating factor during production of thioacetamide-induced cirrhosis in rats. Hepatology 24: 412-418.

25.

Phillips AO , Steadman R. , Topley N. , and Williams JD (1995). E1-evated D-glucose concentrations modulate TGF-β1 synthesis by human cultured renal proximal tubular cells. The permissive role of platelet-derived growth factor. Am. J. Pathol. 147: 362-374.

26.

Rønnov-Jessen L. and Petersen OW (1993). Induction of α-smooth muscle actin by transforming growth factor-β1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab. Invest. 68: 696-707.

27.

Schreiner GF , Harris KPG , Purkerson ML , and Klahr S. (1988). Immunological aspects of acute ureteral obstruction: Immune cell infiltrate in the kidney. Kidney Int. 34: 487-493.

28.

Tamaki K. , Okuda S. , Ando T. , Iwamoto T. , Nakayama M. , and Fujishima M. (1994). TGF-β1 in glomerulosclerosis and interstitial fibrosis of adriamycin nephropathy. Kidney Int. 45: 525-536.

29.

Tang WW , Ulich TR , Lacey DL , Hill DC , Qi M. , Kaufman SA , Van GY , Tarpley JE , and Yee JS (1996). Platelet-derived growth factor-BB induces renal tubulointerstitial myofibroblast formation and tubulointerstitial fibrosis . Am. J. Pathol. 148: 1169-1180.

30.

Vande Berg JS , Rudolph R. , Poolman WL , and Disharoon DR (1989). Comparative growth dynamics and actin concentration between cultured human myofibroblasts from granulating wounds and dermal fibroblasts from normal skin. Lab. Invest . 61: 532-538.

31.

Van Goor H. , Ding G. , Kees-Folts D. , Grond J. , Schreiner GF , and Diamond JR (1994). Macrophages and renal disease. Lab. Invest. 71: 456-464.

32.

Vyalov SL , Gabbiani G. , and Kapanci Y. (1993). Rat alveolar myofibroblasts acquire α-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis. Am. J. Pathol. 143: 1754-1765.

33.

Walton G. , Buttyan R. , Garcia-Montes E. , Olsson CA , Hensle TW , and Sawczuk IS (1992). Renal growth factor expression during the early phase of experimental hydronephrosis. J. Urol. 148: 510-514.

34.

Willems IEMG , Havenith MG , De Mey JGR , and Daemen MJAP (1994). The α-smooth muscle actin-positive cells in healing human myocardial scars . Am. J. Pathol. 145: 868-875.

35.

Wright EJ , McCaffrey TA , Robertson AP , Vaughan ED , and Felsen D. (1996). Chronic unilateral ureteral obstruction is associated with interstitial fibrosis and tubular expression of transforming growth factor-β . Lab. Invest. 74: 528-537.

36.

Xing Z. , Tremblay GM , Sime PJ , and Gauldie J. (1997). Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-β1 and myofibroblast accumulation. Am. J. Pathol. 150: 59-66.

37.

Yamamoto T. , Noble NA , Miller DE , and Border WA (1994). Sustained expression of TGF-β1 underlies development of progressive kidney fibrosis. Kidney Int. 45: 916-927.

38.

Yamashiro S. , Takeya M. , Nishi T. , Kuratsu J. , Yoshimura T. , Ushio Y. , and Takahashi K. (1994). Tumor-derived monocyte chemoattractant protein-1 induces intratumoral infiltration of monocyte-derived macrophage subpopulation in transplanted rat tumors. Am. J. Pathol. 145: 856-867.

39.

Yamate J. , Ishida A. , Tsujino K. , Tatsumi M. , Nakatsuji S. , Kuwamura M. , Kotani T. , and Sakuma S. (1996). Immunohistochemical study of rat renal interstitial fibrosis induced by repeated injection of cisplatin, with special references to the kinetics of macrophages and myofibroblasts. Toxicol. Pathol. 24: 199-206.

40.

Yamate J. , Iwaki M. , Kumagai D. , Tsukamoto Y. , Kuwamura M. , Nakatsuji S. , Tsunenari I. , Kotani T. , and Sakuma S. (1997). Characteristics of rat fibrosarcoma-derived transplantable tumor line (SS) and cultured cell lines (SS-P and SS-A3-1), showing myofibroblastic and histiocytic phenotypes. Virchows Arch. 431: 431-440.

41.

Yamate J. , Tajima M. , Saitoh T. , and Shibuya K. (1994). Biological behaviour and morphological characteristics of a transplantable tumour (MM-KMY) derived from a malignant meningioma in an F344 rat. J. Comp. Pathol. 111: 243-257.

42.

Yamate J. , Tatsumi M. , Nakatsuji S. , Kuwamura M. , Kotani T. , and Sakuma S. (1993). Immunohistochemical observations of macrophages and perisinusoidal cells in carbon tetrachloride-induced rat liver injury . J. Vet. Med. Sci. 55: 973-977.

43.

Yamate J. , Tatsumi M. , Nakatsuji S. , Kuwamura M. , Kotani T. , and Sakuma S. (1995). Immunohistochemical observations on the kinetics of macrophages and myofibroblasts in rat renal interstitial fibrosis induced by cis-diamminedichloroplatinum. J. Comp. Pathol. 112: 27-39.

44.

Yamate J. , Tsujino K. , Kumagai D. , Nakatsuji S. , Kuwamura M. , Kotani T. , and Sakuma S. (1997). Morphological characteristics of a transplantable histiocytic sarcoma (HS-J) in F344 rats and appearance of renal tubular hyaline droplets in HS-J-bearing rats. J. Comp. Pathol. 116: 73-86.

45.

Young BA , Burdmann EA , Johnson RJ , Alpers CE , Giachelli CM , Eng E. , Andoh T. , Bennett WM , and Couser WG (1995). Cellular proliferation and macrophage influx precede interstitial fibrosis in cyclosporine nephrotoxicity. Kidney Int. 48: 439-448.

46.

Zhang J. , Yu ZX , Fujita S. , Yamaguchi ML , and Ferrans VJ (1993). Interstitial dendritic cells of the rat heart: Quantitative and ultrastructural changes in experimental myocardial infarction . Circulation 87: 909-920.

47.

Zhang K. , Rekhter MD , Gordon D. , and Phan SH (1994). Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study. Am. J. Pathol. 145: 114-125.