Question: 

Acute renal failure (ARF), accompanied with reduced urinary concentrating ability, is a frequent complication of sepsis. Efficient urinary concentration depends, amongst other factors, on the expression of the kidney specific Cl^- channel (ClC-K1), the urea transporter A1 (UT-A1), and the water channel aquaporin-2 (AQP-2), which are regulated by the transcription factor tonicity enhancer binding protein/nuclear factor of activated T-cells (TonEBP/NFAT5). Since the development of reduced urinary concentration during ARF is incompletely understood, we addressed the effect of endotoxemia on functional and molecular mechanisms that determine urinary concentrating ability.

Methods: 

Rats were injected with lipopolysaccharide (LPS) (5 mg/kg bw i.p.) or only vehicle PBS as control. After 24 h, urine and kidney tissue samples were taken for analysis. Urine osmolality and urinary concentration of NO degradation poducts nitrite/nitrate were measured. Expression of ClC-K1, UT-A1, AQP-2, inducible NO synthase (iNOS) and TonEBP/NFAT5 was assessed by Western blot analysis and/or qRT-PCR. S-nitrosylation of TonEBP/NFAT5 was monitored by biotin switch assay and immunoprecipitation. TonEBP/NFAT5 DNA binding activity was determined by analytical TonEBP/NFAT5-DNA immunoprecipitation. The effect of the NO donor SNAP on UT-A reporter activity was investigated in MDCK cells.

Results: 

In rats injected with LPS, urine osmolality was reduced by 40%, along with medullary induction of iNOS and a dramatic increase in urinary NO degradation products nitrite and nitrate. This was associated with an increased S-nitrosylation of TonEBP/NFAT5 and reduced TonEBP/NFAT5 DNA binding activity in the renal inner medulla. Concomitantly, mRNA abundance of the TonEBP/NFAT5 regulated genes ClC-K1, UT-A1, and AQP-2 was diminished substantially. These results were confirmed in MDCK cells transfected with a reporter construct driven by the UT-A promoter, demonstrating that the NO donor SNAP significantly reduces UT-A reporter activity under isotonic and hypertonic conditions.

Conclusions: 

The present data demonstrate that LPS increases medullary NO production in view of iNOS induction, leading to S-nitrosylation of TonEBP/NFAT5, which impairs the transcriptional activity of TonEBP/NFAT5. These events entail reduced expression of TonEBP/NFAT5 target genes required for urinary concentration, and may provide further insights into the molecular mechanisms underlying the urinary concentration defect in sepsis.