Volume 55, Issue 5, Pages 1800-1810 (May 1999) High water intake ameliorates tubulointerstitial injury in rats with subtotal nephrectomy: Possible role of TGF-β  Toshihiro Sugiura, Atsushi Yamauchi, Hiroshi Kitamura, Yasuko Matsuoka, Masaru Horio, Enyu Imai, Masatsugu Hori  Kidney International  Volume 55, Issue 5, Pages 1800-1810 (May 1999) DOI: 10.1046/j.1523-1755.1999.00443.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Renal function at two weeks following subtotal nephrectomy. Functional parameters in subtotally nephrectomized rats were measured at two weeks after the renal ablation. Abbreviations are: CONT, sham-operated control; NX, disease control (subtotal nephrectomy without treatment); HWI, subtotal nephrectomy treated with high water intake. Results are shown as mean ± sd of 10 rats. *P < 0.001 vs. CONT; **P < 0.01 vs. CONT. Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Renal function at eight weeks following subtotal nephrectomy. Functional parameters in subtotally nephrectomized rats were measured at eight weeks after the renal ablation. Abbreviations are: CONT, sham-operated control; NX, disease control (subtotal nephrectomy without treatment); HWI, subtotal nephrectomy treated with high water intake. *P < 0.001 vs. CONT; **P < 0.01 vs. CONT; #P < 0.05 vs. NX. Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Effects of a high water intake on interstitial fibrosis in rats with subtotal nephrectomy (Azan staining, original magnification ×200). Paraffin-embedded sections of the remnant kidneys at eight weeks following the ablation were used for the histologic analysis. (A) Sham-operated control (CONT). (B) Subtotal nephrectomy without treatment (disease control, NX), indicating marked interstitial fibrosis with the increase in collagen accompanied by tubular dilation. (C) Subtotal nephrectomy treated with high water intake (HWI), demonstrating marked reduction in fibrosis, whereas tubular hypertrophy remained. Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 Northern blot analysis of TGF-β and fibronectin mRNA expression. (A) Total RNA was isolated from remnant kidneys at eight weeks after subtotal nephrectomy. Northern blot analysis was performed using 32P-labeled TGF-β and fibronectin (FN) cDNA. Abbreviations are: C, sham-operated control; N, disease control (subtotal nephrectomy without treatment); H, subtotal nephrectomy treated with high water intake. (B) The radioactivity of the hybridized probe in hybridized membrane was measured by computing densitometer. Abbreviations are as follows: CONT, sham-operated control; NX, disease control (subtotal nephrectomy without treatment); HWI, subtotal nephrectomy treated with high water intake. Results are mean ± sd of four experiments. *P < 0.01 vs. CONT; #P < 0.01 vs. NX. Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 Northern blot analysis of TGF-β and fibronectin mRNA expression. (A) Total RNA was isolated from remnant kidneys at eight weeks after subtotal nephrectomy. Northern blot analysis was performed using 32P-labeled TGF-β and fibronectin (FN) cDNA. Abbreviations are: C, sham-operated control; N, disease control (subtotal nephrectomy without treatment); H, subtotal nephrectomy treated with high water intake. (B) The radioactivity of the hybridized probe in hybridized membrane was measured by computing densitometer. Abbreviations are as follows: CONT, sham-operated control; NX, disease control (subtotal nephrectomy without treatment); HWI, subtotal nephrectomy treated with high water intake. Results are mean ± sd of four experiments. *P < 0.01 vs. CONT; #P < 0.01 vs. NX. Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 In situ hybridization of TGF-β, fibronectin, and SMIT mRNA in remnant kidneys. Cryosections of the remnant kidneys at eight weeks after the nephrectomy were used. In situ hybridization was performed using 35S-labeled rat TGF-β, fibronectin (FN), and Na+/myo-inositol cotransporter (SMIT) antisense cRNA probes, and autoradiography of the sections is shown. Abbreviations are: (A) CONT, sham-operated control; NX, subtotal nephrectomy without treatment (disease control); HWI, subtotal nephrectomy treated with high water intake. Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 6 In situ hybridization of TGF-β mRNA in the tubulointerstitium of an outer medulla (original magnification ×200). Cryosections of the remnant kidneys at eight weeks after the nephrectomy were used. In situ hybridization was performed using 35S-labeled rat TGF-β antisense cRNA probe. (A) Sham-operated control (CONT). (B) Subtotal nephrectomy without treatment (disease control, NX). (C) Subtotal nephrectomy treated with high water intake (HWI). Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 7 In situ hybridization of TGF-β mRNA in glomeruli (original magnification ×200). Cryosections of the remnant kidneys at eight weeks after the nephrectomy were used. (A) Sham-operated control (CONT). (B) Subtotal nephrectomy without treatment (disease control, NX). (C) Subtotal nephrectomy treated with high water intake (HWI). Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 8 Immunohistochemical staining for TGF-β and fibronectin in the tubulointerstitium of an outer medulla (original magnification ×400). Paraffin-embedded sections of the remnant kidneys at eight weeks following the ablation were used for the immunostaining of TGF-β (A, C, E) and fibronectin (B, D, E). (A, B) Sham-operated control (CONT). (C, D) Subtotal nephrectomy without treatment (disease control, NX). (E, F) Subtotal nephrectomy treated with high water intake (HWI). Kidney International 1999 55, 1800-1810DOI: (10.1046/j.1523-1755.1999.00443.x) Copyright © 1999 International Society of Nephrology Terms and Conditions