Troglitazone induces a cellular acidosis by inhibiting acid extrusion in cultured rat mesangial cells.

We studied the effect of troglitazone on cellular acid-base balance and alanine formation in isolated rat mesangial cells. Mesangial cells were grown to confluency in RPMI 1640 media on 30-mm chambers used to monitor both cellular pH using the pH-sensitive dye 2'7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein and metabolic acid production as well as glutamine metabolism. Troglitazone (10 microM) induced a spontaneous cellular acidosis (6.95 +/- 0.02 vs. 7.47 +/- 0.04, respectively; P < 0.0001) but without an increase in lactic acid production. Alanine production was reduced 64% (P < 0.01) consistent with inhibition of the glutamate transamination. These findings pointed to a decrease in acid extrusion rather than an increase in acid production as the underlying mechanism leading to the cellular acidosis. To test their acid extrusion capabilities, mesangial cells were acid loaded with NH and then allowed to recover in Krebs-Henseleit media or in Krebs-Henseleit media minus bicarbonate (HEPES substituted), and the recovery response (Delta pH(i)/min) was monitored. In the presence of 10 microM troglitazone, the recovery response to the NH acid load was virtually eliminated in the bicarbonate-buffered media (0.00 +/- 0.001 vs. 0.06 +/- 0.02 pH(i)/min, P < 0.0001 vs. control) and reduced 75% in HEPES-buffered media (0.01 +/- 0.01 vs. 0.04 +/- 0.02 pH(i)/min, P < 0.002 vs. control). These results show that troglitazone induces a spontaneous cellular acidosis resulting from a reduction in cellular acid extrusion.

Troglitazone suppresses the secretion of type I collagen by mesangial cells in vitro.

BACKGROUND: Our laboratory has shown that troglitazone, a thiazolidinedione and peroxisomal proliferator activated receptor gamma (PPAR-gamma) agonist, prevents mesangial expansion and glomerulosclerosis in diabetic rats. We investigated and compared the action of two PPAR agonists at the level of the mesangial cell. METHODS: Rat mesangial cells were grown in medium containing 5 mmol/L glucose, 30 mmol/L glucose, or 5 mmol/L glucose plus 25 mmol/L mannitol. The cultures were either left untreated, treated with 10 micromol/L troglitazone (PPAR-gamma), or 100 micromol/L clofibrate (PPAR-alpha). The following parameters were used to assess mesangial cell responses: detection of PPAR-gamma and -alpha mRNA, the degree of PPAR-gamma and -alpha activation, spread cell area, total protein production, and laminin and type I collagen production. RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) showed the presence of PPAR-gamma and -alpha mRNA in rat mesangial cells. PPAR-gamma and -alpha proteins are active in mesangial cells and the extent of activation is affected by different glycemic conditions. Troglitazone and clofibrate treatment corrected in part the increase in spread cell area seen in mesangial cells in hyperglycemic conditions. However, neither agonist corrected the increase in total protein production induced by hyperglycemia. Treatment with troglitazone resulted in a significant, specific decrease in type I collagen along with a slight decrease in laminin production in both medium conditions. Clofibrate had no effect on laminin synthesis in either medium condition but did decrease type I collagen synthesis in cells grown in hyperglycemic conditions. CONCLUSION: PPAR-alpha and -gamma mRNA signaling pathways are in place and active in mesangial cells. Both agonists affect the phenotypic behavior of mesangial cells and ameliorate changes resulting from hyperglycemia. The data indicate that the correction of mesangial cell phenotype by troglitazone may influence production/deposition of a pathological fibrotic connective tissue matrix (that is, type I collagen) by these cells.

Troglitazone inhibits glutamine metabolism in rat mesangial cells.

Troglitazone is a peroxisome proliferator-activated receptor-gamma agonist that has been shown to halt mesangium expansion in experimental models of type 2 diabetes mellitus and to act directly on rat mesangial cells. Because glutamine serves as the precursor for cellular biosynthetic processes, we asked whether troglitazone would inhibit mesangial cell glutamine metabolism under these conditions. Confluent monolayers of rat mesangial cells were incubated in RPMI medium in the presence of troglitazone or vehicle (DMSO). Troglitazone effected a dose-dependent reduction in glutamine utilization and in alanine formation, associated with a decrease in monolayer collagen-glycosaminoglycan content. Despite the reduced glutamine uptake, ammonium formation did not decrease, consistent with increased glutamate flux through the deamination pathway. Assayable activity of the alanine aminotransferase decreased by 63%, whereas assayable glutamate dehydrogenase remained unchanged. In control monolayers, the sum of ammonium plus alanine plus glutamate nitrogen released accounted for <75% of the glutamine nitrogen uptake. In troglitazone-treated monolayers, all of the glutamine nitrogen taken up could be accounted for as ammonium nitrogen released into the medium. These results are consonant with troglitazone reducing glutamine metabolism and specifically the transamination pathway in rat mesangial cells associated with a reduction in collagen-glycosaminoglycan content.