TNF-alpha reduces the Na+/K+ ATPase activity in LLC-PK1 cells by activating caspases and JNK and inhibiting NF-kappaB.

TNF-alpha has recently been implicated in diabetic nephropathy, which is usually accompanied by higher sodium retention. The kidneys play a major role in sodium homeostasis by regulating tubular sodium reabsorption, a process geared by the sodium gradient established by the Na(+)/K(+) ATPase. The aim of this work was to investigate the effect of TNF on the ATPase, and consequently its implication in kidney malfunction, using LLC-PK1 cells. The cytokine reduced the Na(+)/K(+)ATPase activity significantly. In an attempt to elucidate the signalling pathway involved, PDTC (pyrrolidinedithiocarbamate), SP600125 and FK009 respectively inhibitors of NF-kappaB, c-JNK and caspases, were added to the cells in the presence and absence of TNF, and changes in the activities of JNK and PDTC were determined. The activity of the pump was assayed by measuring the ouabain-inhibitable release of inorganic phosphate. The effect of the cytokine was abrogated completely when JNK and caspases were inhibited but was unaffected by NF-kappaB inhibition. The role of each mediator in the signalling cascade was studied further by applying different combinations of the inhibitors. TNF-alpha was found to act at 1 h by activating caspases, which in turn activate JNK; the latter exerts an inhibitory effect on NF-kappaB, a transcription factor that stimulates the Na(+)/K(+) ATPase when active. It was concluded that TNF-alpha exerts opposite effects on the Na(+)/K(+)ATPase at different times, though the effects are always mediated via cJNK, NF-kappaB and caspases.