Molecular aspects in renal acid-base adaptation

Systemic metabolic acidosis induces an array of adaptive responses in renal proximal tubular cells. Metabolically, enzyme activities of phosphate-dependent glutaminase (PDG) and cytosolic PEPCK are increased, resulting in increased glutamine extraction, and enhanced rates of ammonium excretion and bicarbonate reabsorption. The increase in PEPCK results from an acid-induced enhanced rate of transcription, whereas the acid induction of PDG occurs by stabilization of its mRNA. LLC-PK1 -FBPase+ is a gluconeogenic and pH-responsive renal proximal tubule-like cell line. Upon incubation with acidic medium (pH 6.9), LLC-PK1-FBPase+ cells respond with increased rates of ammonia production, accompanied by 2- to 3-fold increases in PDG and PEPCK mRNA levels and enzyme activities, respectively. Previous evidence suggests that an intracellular acidosis (pHi) triggers these adaptive responses and that induction of PEPCK may be mediated via the p38 mitogen-activated protein kinase (MAPK) pathway, while neither ERK nor JNK MAPKs play a significant role. Therefore, the role of p38 and its downstream pathways in mediating the acid response of LLC-PK1-FBPase+ cells were explored in detail. Incubation of LLC-PK1-FBPase+ cultures with acidic medium resulted in a biphasic phosphorylation, and thus activation, of p38, with peaks at 1 h and 9 h after onset of acidosis, as proved by Western blotting. Phospborylation of the transcription factor ATF-2, a known substrate of the p38 kinase, occured with a slight lag. The protein synthesis inhibitor anisomycin (AI), and hyperosmotic sorbitol, both strong p38 activators, induced time-dependently the phosphorylation of p38 and of ATF-2, but with different time courses. Cycloheximide (CHX), however, did not induce p38 or ATF-2 phosphorylation. In Northern blot analysis, AI increased PEPCK mRNA levels, comparable to acid stimulation, while CHX again showed no effect on mRNA induction, confirming the Western blot experiments. SB203580 (SB), a specific p38 inhibitor, dose-dependently blocked both acid- and AI-induced PEPCK mRNA levels, with maximum inhibition at 10 micro-M. AI-induced phosphorylation of ATF-2 was also completely blocked by SB. Thus, acid- as well as AI-induced transcription of PEPCK could be transmitted via SB-sensitive p38 isoforms. A Western blotting survey revealed that the SB- sensitive p38alpha, one of the four p38 isoforms, is strongly expressed in LLC-PK1-FBPase+ cells, and only the SB-insensitive p38delta is found in minor levels. The octanucleotide sequence TTACGTCA in the CRE-1 site of the PEPCK promoter is a perfect match to the consensus element for binding ATF-2. Gel-shift analysis using a labeled oligonucleotide containing the CRE-1 element produced a band that was partially supershifted with antibodies specific for ATF-2. Thus, it is most likely that the SB-sensitive p38alpha/ATF-2 signaling pathway is the mediator of the pH-responsive induction of PEPCK mRNA transcription in renal proximal tubule-like LLC-PK1-FBPase+ cells. In the present grant proposal, the work on acid-induced p38 MAPK signaling shall be continued, with special emphasis on the role of upstream MAP kinase kinases (MKK3 and MKK6), by transfecting cells with dominant- negative constructs, and with a detailed analysis of further downstream effector substrates of SB-sensitive p38alpha, as described above. In addition, a broad search for acid-base regulated renal genes shall be initiated, using the novel technology of cDNA microarrays. This innovative approach should provide new insights into the effects of acidosis on the genetic regulation and the induction of the appropriate transcriptional program of renal proximal tubular cell metabolism and function.