ZDF rats have a constellation of metabolic abnormalities, and TZD treatment may improve renal acidification and NHE3 function by acting on mechanisms independent of renal lipid content. We used FFA-incubated OKP cells to investigate the effect of intracellular lipid reduction in the absence of other potential confounding factors. MEK162 OKP cells avidly take up FFA carried on albumin from the culture medium and store them predominantly as triglycerides in intracellular lipid droplets (46). OKP cells also appear to be rather susceptible to lipotoxicity. While a high dose of FFA (1.5 mM) causes irreversible cellular damage by promoting apoptosis, a low dose of FFA (0.75 mM), with no significant effect on apoptosis or baseline NHE3 activity, is sufficient to abolish the insulin stimulation of NHE3 function (6).

We examined the reversibility of this phenomenon in lipid-loaded OKP cells maintained in a nutrient-poor medium to promote lipid catabolism, mitochondrial beta-oxidation of intracellular FFA, and hence recovery from lipotoxicity. Compared with cells studied immediately after lipid loading (Fig. 4A), intracellular lipid droplets were markedly reduced in cells allowed to recover for 36 h (Fig. 4B) and the regulation of NHE3 by insulin was partly restored (Fig. 4C). Cells maintained in nutrient-poor medium continued to reduce their intracellular lipid stores and remained viable in culture for up to 6 days, but NHE3 activity and insulin response gradually decreased with prolonged serum and nutrient deprivation in both vehicle- and FFA-incubated cells (not shown).

These cell culture artifacts seen with prolonged serum starvation make these later time points unsuitable for the study of NHE3 regulation. Fig. 4. Recovery of insulin-stimulated NHE3 activity in opossum kidney (OKP) cells after intracellular lipid reduction. OKP cells were incubated with 0.75 mM nonesterified fatty acids (FFA) or vehicle (albumin) for 12 h, with or without incubation with FFA-free … Effect of rosiglitazone on NHE3 activity in OKP cells. Our findings in lean control rats do not support a direct effect of TZD on urinary acidification in the absence of renal steatosis (Fig. 2). However, the presence of intracellular lipid deposits may alter the transcriptional and metabolic makeup of the proximal tubule, and it is theoretically possible that TZD may have a direct effect on urinary acidification in the altered environment of the steatotic kidney.

This effect, if Anacetrapib present, would not be related to the reduction of steatosis but rather to TZD, which would drastically alter the interpretation of our data. To examine this possibility, we incubated control and lipid-loaded OKP cells with different concentrations of rosiglitazone for 24 h. Rosiglitazone treatment had no effect on intracellular lipid content in OKP cells, as estimated by Oil Red O staining (not shown).