Tumor necrosis factor-alpha induces renal cyclooxygenase-2 expression in response to hypercalcemia

Abstract

The effect of tumor necrosis factor-alpha (TNF) on cyclooxygenase-2 (COX-2) expression in the renal outer medulla (OM) was determined in a model of dihydrotachysterol (DHT)-induced hypercalcemia. Increases in serum calcium and water intake were observed during ingestion of a DHT-containing diet in both wild type (WT) and TNF deficient mice (TNF(-/-)). Polyuria and a decrease in body weight were observed in response to DHT treatment in WT and TNF(-/-) mice. A transient elevation in urinary TNF was observed in WT mice treated with DHT. Moreover, increased urinary levels of prostaglandin E(2) (PGE(2)) and a corresponding increase in COX-2 expression in the OM were observed in WT mice fed DHT. Increased COX-2 expression was not observed in TNF(-/-) mice fed DHT, and the characteristics of PGE(2) synthesis were distinct from those in WT mice. This study demonstrates that COX-2 expression in the OM, secondary to hypercalemia, is TNF-dependent.

Fig 1. Food intake and body weight in WT and TNF^−/− mice ingesting DHT

WT and TNF^−/− mice were maintained in metabolic cages and fed control diet for 4 days before being placed on DHT-containing diet for 7 days. A. Food consumption was monitored on a daily basis. The data represent means ± SEM, * p<0.05, ** p<0.01, *** p<0.001 vs. control (day 0) vs treatment day. B. Body weight was monitored on a daily basis. The data are shown as mean ± SEM. ** p<0.01, *** p<0.001 vs. control (day 0) vs treatment day.

Fig 1. Food intake and body weight in WT and TNF^−/− mice ingesting DHT

WT and TNF^−/− mice were maintained in metabolic cages and fed control diet for 4 days before being placed on DHT-containing diet for 7 days. A. Food consumption was monitored on a daily basis. The data represent means ± SEM, * p<0.05, ** p<0.01, *** p<0.001 vs. control (day 0) vs treatment day. B. Body weight was monitored on a daily basis. The data are shown as mean ± SEM. ** p<0.01, *** p<0.001 vs. control (day 0) vs treatment day.

Fig 2. DHT increases total serum Ca²⁺ levels and urinary Ca²⁺ excretion in WT and TNF^−/− mice

WT and TNF^−/− mice were maintained in metabolic cages and fed the control diet for 4 days before being placed on the DHT diet for 7 days. A. Serum Ca²⁺ levels, and B. urinary Ca²⁺ excretion was measured in mice fed control or DHT-containing diet for 7 days. The data are shown as mean ± SEM; n=3–6. * p<0.05, ** p<0.01, *** p<0.001 control (day 0) vs treatment day.

Fig 2. DHT increases total serum Ca²⁺ levels and urinary Ca²⁺ excretion in WT and TNF^−/− mice

WT and TNF^−/− mice were maintained in metabolic cages and fed the control diet for 4 days before being placed on the DHT diet for 7 days. A. Serum Ca²⁺ levels, and B. urinary Ca²⁺ excretion was measured in mice fed control or DHT-containing diet for 7 days. The data are shown as mean ± SEM; n=3–6. * p<0.05, ** p<0.01, *** p<0.001 control (day 0) vs treatment day.

Fig 3. DHT increases urine volume in WT and TNF^−/− mice

WT and TNF^−/− mice were maintained in metabolic cages, fed control diet for 4 days and then placed on DHT diet for 7 days. Urine volume per cage was measured on a daily basis. The data are shown as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 control (day 0) vs treatment day.

Fig 4. Water intake increased with DHT treatment

WT and TNF^−/− mice were maintained in metabolic cages, fed control diet for 4 days and placed on DHT containing diet for a period of 7 days, food and water intake were monitored on a daily basis. The data represent mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 control (day 0) vs treatment day.

Fig 5. DHT increases urinary TNF levels

WT mice were maintained in metabolic cages, fed control diet for 4 days, and then placed on DHT-containing diet for 7 days; urine was collected daily and analyzed for TNF levels. The data are shown as mean ± SEM; n=9 cages. * p<0.05, ** p<0.01 control (day 0) vs treatment day.

Fig 6. COX-2 expression in outer medulla of WT mice is elevated in response to DHT treatment

WT mice were fed control diet for 4 days then given a DHT-containing diet for 1, 3, or 7 days. Expression of COX-2 in the OM was analyzed by Western blot. The top panel shows a representative Western blot and the bottom panel shows densitometry analysis after COX-2 expression was normalized to β-actin levels. The data are shown as mean ± SEM, n=4. * p<0.05 control (day 0) vs treatment day.

Fig 7. DHT increases COX-2 expression in WT but not TNF^−/− mice

WT mice (A) and TNF^−/− mice (B) were fed control or DHT-containing diet for 7 days. Expression of COX-2 in the OM was analyzed by Western blot. The top panels are representative Western blots and the bottom panels show densitometry analysis of COX-2 expression normalized to β-actin levels. The data are shown as mean ± SEM, n=5. * p<0.05.

Fig 8. Effects of DHT on urinary PGE₂ levels in WT and TNF^−/− mice

A) WT mice (n=9 cages) and B) TNF^−/− mice (n=3 cages) were maintained in metabolic cages, fed control diet for 4 days, and then placed on DHT-containing diet for 7 days. Urine was collected daily and analyzed for PGE₂ levels. The data are shown as mean ± SEM; * p<0.05, ** p<0.01 control (day 0) vs treatment day.