During systemic acidosis, renal proximal tubular cells exhibit enhanced rates of bicarbonate and ammonium ion synthesis and undergo extensive hypertrophy. The former adaptations are accomplished, in part, by increased expression of glutaminase (GA). LLC-PK₁-FBPase⁺ cells, a gluconeogenic line of porcine kidney cells, exhibit a rapid activation of the ERK1/2 and p38 MAPK pathways and a 2- to 3-fold increase in GA mRNA when transferred to acidic medium (pH 6.9). Transforming growth factor- (TGF-), a potent activator of MAPK and Smad signaling cascades, also causes extensive renal hypertrophy. Thus, the potential role of TGF- in the renal response to metabolic acidosis was investigated. Western blot analyses established that in LLC-PK₁-FBPase⁺ cells, TGF- activated the ERK1/2, p38 MAPK and Smad1/5/8 pathways, but not the JNK and Smad2/3 pathways. Addition of TGF- to cells cultured in normal medium (pH 7.4) produced a steady increase in GA mRNA, resulting in a 2-fold induction after 18 h. Western blot analysis indicated that treatment with either TGF- or acidic medium resulted in an increased level of fibronectin. However, the effects of the two treatments on both GA mRNA and fibronectin levels occurred with different time courses and were additive. In addition, the rates of ammonia production were decreased slightly by addition of TGF-. Finally, a GA-luciferase reporter construct, that is activated 3.5-fold by treatment with acidic medium, is not affected by TGF-. Therefore, TGF- and metabolic acidosis activate some of the same signaling pathways in LLC-PK₁-FBPase⁺ cells, but produce separate effects on GA expression.