Correlations Between Urate Transporters And Hyperuricemia In Chronic Kidney Diseases And Its Mechanisms

Background Hyperuricemia is found in many chronic kidney diseases. It is an independent risk factor for cardiovascular and renal diseases demorstrated by many studies. The excretion of urate/uric acid (UA) is mainly performed by kidney. Therefore, hyperuricemia is susceptible in patients with renal disease. UA is eliminated from the body by kidney through filtering freely by gromerulars, then majority of them is reabsorbed and some of them is secreted again into the lumen, finally only about 10% of the filtered UA is eliminated. The reabsorption and secretion of UA in tubular cells are fulfilled mainly by three transporters, a urate-anion exchanger (URAT1) which localizes at apical membrane and reabsorbs UA from lumen, two organic anion transporters (OAT1 and OAT3) which express at base membrane and responsible for UA secretion. Thus, the levels of these transporters' expression are likely responsible for hyperuricemia in patients with CKD. But we do not find any reports about the correlations between these transporters and hyperuricemia yet. Objective We tested urinary UA levels and observed renal OAT1, OAT3 and URAT1 protein levels from CKD patients with or without hyperuricemia, and found the correlations of them. Then, we stimulated human proximal tubular (HKC) cells with angiotensin II or aldosterone to mimic RAAS activation in CDK, so as to clarify the mechanism of UA elevation in patients with CKD result from the altering of these transporters' expression. Methods Mouse anti-hURAT1, mouse anti-hOAT1 and mouse anti-hOAT3 antibodies were generated by genetic immunization. Using these antibodies, we investigated the three UA transporters' localization in human kidney;CKD patients with normal renal function were divided into two groups: hyperuricemia group (20 patients, blood uric acid 522. 22±63.39 μmol/L) and normal uric acid group (20 patients, blood uric acid 293.82±65.73 μmol/L). Their urinary UA and renal OAT1, OAT3 and URAT1 protein levels were detected;HKC cells were stimulated with angiotensin II or aldosterone to mimic RAAS activation in CDK and OAT1, OAT3 and URAT1 protein levels were detected with Western blot analysis. To clarify whether these effects were mediated by MAPK pathway, the MAPK phosphorylations were also detected by Western blot analysis in HKC cells before and after stimulation with aldosterone. Results Mouse anti-URAT1, mouse anti-OAT1 and mouse anti-OAT3 polyclonal antibodies were generated successfullyby genetic immunization. Mouse anti-URATl antibody recognized two bands of 58 kD URATl and 64 kD glycosylated URATl, mouse anti-OATl antibody recognized two bands of 55 kD OATl and 90 kD glycosylated OATl, and mouse anti-OAT3 antibody recognized two bands of 57 kD OAT3 and 92 kD glycosylated OAT3. Immunohistochemistry showed that URATl was located on the brush border membrane of renal proximal tubular cells, while OATl and OAT3 were located on the base membrane. The urinary UA excretion in hyperuricemia group were significantly lower than in normal uric acid group (1.765 + 0.744 mmol/24hours vs 4.251 + 2.814 mmol/24hours, p<0.05), while the URATl expression were significantly higher than in normal uric acid group (p<0.05). But OATl and OAT3 expression indicated no difference between two groups respectively. Stimulation with angiotensin II could not altered OATl, OAT3 and URATl protein levels. Stimulation with aldosterone could signicantly induce URATl upregulation, while could not effect OATl and OAT3 protein level in HKC cells. At the same time, stimulated with aldosterone led to potent activation of ERKl/2, but not JNK or p38. These effects were prevented by pretreatment with ERK kinase inhibitor U0126, while were not prevented by mineralocorticoid receptor antagonist spironolactone. Conclusions Our studies demonstrate, we believe for the first time, that upregulation of renal URATl is responsible for hyperuricemia in patients with CKD. Aldosterone is a key stimulator on upregulation of URATl. And this process is mediated by a nongenomic signaling pathway, independent of the mineralocorticoid receptor, then by ERK 1/2 activation. These results suggest us that ERK may be a molecular target for drug development to prevent serum UA elevation in patients with CKD.