Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB136

Poster Communications

Contribution of the proton sensors OGR1 and GPR4 to renal disease progression in mice

J. Sprenger1,5, E. M. Pastor Arroyo1,5, T. Knöpfel1, G. Pellegrini2, N. Olsen Camara3,4, C. A. Wagner1,5, P. Imenez Silva1,5

1. Institute of Physiology, University of Zurich, Zurich, Switzerland. 2. Institute of Veterinary Pathology, University of Zurich, Zurich, Switzerland. 3. Division of Nephrology, Federal University of Sao Paulo, Sao Paulo, Brazil. 4. Institute of Biomedical Sciences - Department of Immunology, University of Sao Paulo, Sao Paulo, Brazil. 5. National Center for Competence in Research NCCR Kidney.CH, Zurich, Switzerland.

Chronic kidney disease (CKD) represents a major health problem and contributes to high economical costs. Local tissue inflammation and acidification already occur in early stages of CKD. Both local conditions may act synergistically, accelerating disease progression. Therefore, elucidating mechanisms linking extracellular pH, pH-sensing, and immune responses may provide relevant insights to better understand renal disease progression. Moreover, it may provide relevant therapeutic targets. This study aims to investigate the contribution of the pH sensing G protein coupled receptors (GPCRs) OGR1 and GPR4 in renal inflammation and fibrosis. A ten-day high-oxalate diet (0.67% oxalate in calcium-free diet) was used to induce crystalline nephropathy in litter-mate wild-type (WT), OGR1 knock-out (KO) and GPR4 KO male C57BL/6 mice. Age, sex and genotype matched littermates received control diet (calcium-free). There was a three days adaptation period with calcium-free diet prior to treatment and a three days recovery period with standard diet (1.05% calcium) afterwards. At the end, blood and kidneys were collected from isoflurane-anesthetized mice. Plasma parameters were measured and RNA was extracted from kidneys to perform qPCR. In another set of experiments, the anesthetized mice were perfused with 4% PFA/PBS and kidneys were collected for histological evaluation. Data are presented as mean ± SD, compared by ANOVA. Plasma urea and creatinine values were elevated upon oxalate diet in all three genotypes. However GPR4 KO displayed significantly less elevated values (71.3 mg/dL ±13.8 and 0.2 mg/dL ±0.05, n=4) compared to WT (173.7 mg/dL ±64.7 and 0.5 mg/dL ±0.2, n=7). In situ RNAscope mRNA hybridization and qPCR showed that OGR1 and GPR4 mRNA expression were highly increased in WT mice subjected to the oxalate nephropathy model. Proinflammatory factors and cytokines including Nalp3, Tnf, Tgfβ, and Ccl5 were similarly upregulated in all oxalate treated mice. Only Il-1β values of OGR1 KO and GPR4 KO mice were significantly less elevated upon oxalate diet compared to WT (both GPR4 and OGR1 showed ~40% reduced increase compared to WT, n=4-7). Fibrosis markers Mmp9, Timp1, and Acta2 were attenuated in GPR4 KO. Taken together, GPR4 KO mice showed evidence of lower fibrosis and had better preserved kidney function when compared to WT mice challenged with high oxalate diet. Preliminary histological assessment of kidney tissue showed similar oxalate crystal count, collagen staining by van Gieson and epithelial damage between WT and OGR1 KO mice. Histological assessment of renal tissue from GPR4 mice is ongoing. GPR4 may be involved in the progression of oxalate nephropathy.

Where applicable, experiments conform with Society ethical requirements