TNF-alpha modulates the Na+/ K+ ATPase and the Na+K+2Cl- symporter in LLC-PK cells

Tumour necrosis factor alpha (TNF-alpha) has been implicated in the development of diabetic nephropathy and the accompanying increase in sodium retention. Inhibition of renal Na(+)/K(+) ATPase was reported to accompany cell death. As TNF is known to induce both apoptosis and cell survival, this work investigated the effect and mechanism of action of TNF-alpha on the Na(+)/K(+) ATPase and the Na(+)K(+)2Cl(-) symporter using LLC-PK(1) cells, a porcine renal proximal tubules cell line.

Cells were incubated for 2 h with TNF-alpha in presence and absence of pyrrolidinedithiocarbamate, SP600125 and FK009, respective inhibitors of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), c-Jun N-terminal kinase (JNK) and caspases. The activity of the pump was assayed by measuring the ouabain-inhibitable release of inorganic phosphate. Changes in its expression and the expression of the symporter were monitored by western blot analysis.

TNF-alpha up-regulated both transporters. NF-kappaB, JNK and the caspases were all mediators of the cytokine action. TNF up-regulated the Na(+)/K(+) pump by stimulating JNK which in turn, activated NF-kappaB and inhibited the caspases. TNF effect on the cotransporter was also mediated via activation of JNK which however inhibited NF-kappaB and by so doing prevented activation of caspases. As caspases were demonstrated to down-regulate the two transporters, their inhibition by TNF is responsible for the observed up-regulatory effect.

It was concluded that the Na(+)/K(+) ATPase and Na(+)K(+)2Cl(-) are both targets of TNF-alpha and the effect of the cytokine favours cell survival over cell death.