Ruth McCracken

Role of TNFR1 and TNFR2 receptors in tubulointerstitial fibrosis of obstructive nephropathy

Unilateral ureteral obstruction (UUO) results in tubulointerstitial fibrosis of the obstructed kidney. In this study, we report the contribution of tumor necrosis factor-α (TNF-α) to the fibrosis that develops after ureteral obstruction. Mice in which individual TNF-α receptors TNFR1 or TNFR2 had been genetically knocked out were used, and results were compared with mice of C57Bl/6 background after 5 days UUO. Both kidneys were removed and examined histologically for changes in interstitial volume (Vvint), collagen IV deposition, α-smooth muscle actin (α-SMA) matrix score, nuclear factor-κB (NF-κB) activity, and TNF-α mRNA levels. We found that the Vvint of contralateral unobstructed kidneys averaged ∼7% and was indistinguishable among the three genotypes of mice. Vvintof ureteral obstructed kidney of C57Bl/6 mice averaged 33 ± 3.9% after 5 days of UUO. Vvint of obstructed kidneys of TNFR1 mice was significantly reduced to 19.4 ± 3.1%, whereas that of TNFR2 mice was significantly decreased to 25.4% ± 4.8%. There was a modest but significant difference between Vvint of TNFR1 and TNFR2 ( P < 0.047). Both collagen IV and α-SMA matrix scores were decreased significantly in obstructed kidney of TNFR1 mouse compared with that of C57Bl/6 and TNFR2 mice. Nuclear extracts prepared from kidney cortex were found to have a significant increase in NF-κB binding activity in obstructed kidney compared with contralateral kidney. Individual knockout of the TNFR1 or TNFR2 genes resulted in significantly less NF-κB activation compared with the wild type, with TNFR1 being less than TNFR2 knockout. There was a significant increase in TNF-α mRNA in the kidney with ureteral obstruction in all three genotypes. TNFR1 knockout displayed a significant reduction in amount of TNF-α mRNA induced compared with wild-type or TNFR2 knockout mice. Treatment of TNFR1 knockout mice with an angiotensin converting enzyme inhibitor further decreased Vvint and TNF-α mRNA induction, suggesting an interaction of ANG II and TNF-α systems. These results suggest that TNF-α contributes, in part, to changes in interstitial volume, myofibroblast differentiation, and NF-κB activation in the kidney during ureteral obstruction. These changes appear to be mediated through both TNFR1 and TNFR2 gene products with effects through the TNFR1 receptor predominating. Furthermore, ANG II appears to stimulate TNF-α pathophysiological events leading to renal fibrosis.Unilateral ureteral obstruction (UUO) results in tubulointerstitial fibrosis of the obstructed kidney. In this study, we report the contribution of tumor necrosis factor-alpha (TNF-alpha) to the fibrosis that develops after ureteral obstruction. Mice in which individual TNF-alpha receptors TNFR1 or TNFR2 had been genetically knocked out were used, and results were compared with mice of C57Bl/6 background after 5 days UUO. Both kidneys were removed and examined histologically for changes in interstitial volume (Vv(int)), collagen IV deposition, alpha-smooth muscle actin (alpha-SMA) matrix score, nuclear factor-kappaB (NF-kappaB) activity, and TNF-alpha mRNA levels. We found that the Vv(int) of contralateral unobstructed kidneys averaged approximately 7% and was indistinguishable among the three genotypes of mice. Vv(int) of ureteral obstructed kidney of C57Bl/6 mice averaged 33 +/- 3.9% after 5 days of UUO. Vv(int) of obstructed kidneys of TNFR1 mice was significantly reduced to 19.4 +/- 3.1%, whereas that of TNFR2 mice was significantly decreased to 25.4% +/- 4.8%. There was a modest but significant difference between Vv(int) of TNFR1 and TNFR2 (P < 0. 047). Both collagen IV and alpha-SMA matrix scores were decreased significantly in obstructed kidney of TNFR1 mouse compared with that of C57Bl/6 and TNFR2 mice. Nuclear extracts prepared from kidney cortex were found to have a significant increase in NF-kappaB binding activity in obstructed kidney compared with contralateral kidney. Individual knockout of the TNFR1 or TNFR2 genes resulted in significantly less NF-kappaB activation compared with the wild type, with TNFR1 being less than TNFR2 knockout. There was a significant increase in TNF-alpha mRNA in the kidney with ureteral obstruction in all three genotypes. TNFR1 knockout displayed a significant reduction in amount of TNF-alpha mRNA induced compared with wild-type or TNFR2 knockout mice. Treatment of TNFR1 knockout mice with an angiotensin converting enzyme inhibitor further decreased Vv(int) and TNF-alpha mRNA induction, suggesting an interaction of ANG II and TNF-alpha systems. These results suggest that TNF-alpha contributes, in part, to changes in interstitial volume, myofibroblast differentiation, and NF-kappaB activation in the kidney during ureteral obstruction. These changes appear to be mediated through both TNFR1 and TNFR2 gene products with effects through the TNFR1 receptor predominating. Furthermore, ANG II appears to stimulate TNF-alpha pathophysiological events leading to renal fibrosis.

The expression of mRNA for tumour necrosis factor-α increases in the obstructed kidney of rats soon after unilateral ureteral ligation

Summary: Cytokines, including transforming growth factor (TGF)‐β1, contribute to the tubulointerstitial fibrosis of ureteral obstruction. Tumour necrosis factor (TNF)‐α, a proinflammatory cytokine produced by multiple cells including macrophages and resident renal cells, has a role in inflammatory cell recruitment in glomerular injury. We measured TNF‐α mRNA in the renal cortex of rats at different times after the onset of unilateral ureteral obstruction (UUO) and determined whether angiotensin II (AngII) inhibition or total body irradiation affects the mRNA levels of TNF‐α. Rats were killed at 1, 2, 4, 24, 72 and 120h after UUO. Levels of TNF‐α mRNA increased significantly in the obstructed kidney at 1h (X 2), 2h (X 2.7), 4h (X 3.6), 24h (X 2.7), 72h (X 1.8) and 120h (X 2.8) after ureteral ligation when compared to the contralateral kidney of the same animals or to control (normal) kidneys. Tumour necrosis factor‐α mRNA increased in renal cortical tubules but not in glomeruli. Treatment with enalapril, an angiotensin‐converting enzyme (ACE) inhibitor, before and after UUO decreased TNF‐α mRNA levels in the obstructed kidney by about 40% at 4h after the onset of UUO, but at 120h there was no difference in TNF‐α levels in the obstructed kidney of treated and untreated animals. Total body irradiation, which depletes macrophages in the obstructed kidney, did not prevent the upregulation of TNF‐α mRNA expression at 4 h after UUO. Thus, TNF‐α may have a role in initiating tubulointerstitial injury in the obstructed kidney. Leucocytes infiltrating the renal interstitium of the obstructed kidney do not appear to contribute to the increased mRNA expression of TNF‐α. Angiotensin II may contribute, at least in part, to the early increased expression of TNF‐α mRNA in the obstructed kidney.

Osteopontin Expression in the Kidney during Unilateral Ureteral Obstruction

Osteopontin is a bone protein also expressed in other tissues. Increased osteopontin is thought to be associated with tissue inflammation. We used immunocytochemical analyses and polymerase chain reaction amplification of mRNA to examine osteopontin expression and regulation in unilateral ureteral obstruction (UUO) in rats, a model of inflammatory kidney disease. In the obstructed kidney, osteopontin mRNA and protein were significantly increased. The increase reached 4-fold after 1 day of UUO and persisted at this level for the 5-day duration of UUO. Immunocytochemical analyses showed increased osteopontin protein in tubular cells of the obstructed kidney cortex from days 1 through 5 of UUO. No such significant increase was apparent in the glomerulus or interstitium. Increased osteopontin mRNA and protein likewise occurred in the tubular cells of the obstructed kidney of rats that had undergone whole-body irradiation to eliminate macrophage infiltration into the experimental kidney. Purified osteopontin was found to be a chemoattractant for macrophages isolated from the rat peritoneum. Enalapril treatment, which decreases macrophage infiltration of the obstructed kidney, had no effect on the increase in osteopontin mRNA but significantly attenuated the increase in protein in tubular cells. Western blot analysis of whole cortical homogenates revealed that the osteopontin antibody recognized one protein of 67 kD. The amount of this protein was substantially decreased in kidney homogenates obtained from enalapril-treated compared to untreated animals. Increased osteopontin synthesis may, therefore, contribute in part to the inflammatory response that characterizes obstructive nephropathy.