The Possible Role Of Tubuloglomerular Feedback In Hyperuricemia Induced Hypertension

BACKGROUNDHypertension is a severe threat to human health, with increasing prevalence in China in recent years. A growing body of experimental and clinical evidence has related hyperuricemia with hypertension. The hypothesized mechanisms involved include renin-angiotensin system (RAS) activation, endothelial dysfunction and vascular impairment. But the exact mechanism remains unclear and controversial. Tubuloglomerular feedback (TGF) in kidney is of great importance in regulating water-salt balance and systemic blood pressure. Initiated by signals of sodium concentration, macula densa (MD) produces factors to influence renin release and afferent arteriole constriction through paracrine manner, directly resulting in alteration of glomerular filtration rate and reabsorbtion of salt. RAS activation which is involved in vessel tone regulation, also has an effect in regulating systemic blood pressure. Therefore, to explore hyperuricemia related imbalance of TGF and altered excretion of vaso-active substances is quite meaningful to the understanding of mechanism and potential management of hypertension.OBJECTIVESIn this study, we aimed1) to observe the relationship of hyperuricemia with blood pressure or renin expression in normal blood pressure or hypertensive patients,2) to investigate the effects of hyperuricemia on kidney local renin expression, afferent arteriole lesion and the3pathways of MD paracrine in newly established hyperuricemic mice model with early hypertension,3) to study the direct effects of uric acid on MD cell in expression of Cox-2, nNOS and enzymes in ATP metabolism in vitro, and4) to explore whether hyperuricemia induced hypertension still exists and how vascular lesions change in TGF abolished mice (A1AR-/-mice).METHODS AND RESULTS1. Initial observation of the relationship between hyperuricemia and blood pressure or kidney renin expression.17patients diagnosed as benign hypertensive nephrosclerosis and19patients as glomerular minimal lesion by renal biopsy from2004to2011in Peking Union Medical College Hospital were involved. Their clinical data were collected and renal local renin expression was demonstrated by immunohistochemistry. We observed positive correlation between uric acid and renin expression in glomerular minimal lesion patients (P<0.05), but no such results in benign hypertensive nephrosclerosis.2. Blood pressure elevated in hyperuricemic mice1) Establishment of hyperuricemic mice modelWe compared three methods to induce hyperuricemia in C57BL/6J mice, including feeding with2%oxonic acid food and gavage with oxonic acid at dose of250mg/kg/d or500mg/kg/d plus0.1%uric acid in drinking water.250mg/kg/d oxonic acid gavage succeeded in inducing hyperuricemia after lw which lasted for more than8weeks. This model had their average serum uric acid level elevated by59.8±24.3μmol/L, without negative effects on mice physical condition, renal function or renal pathology.2) Relationship of serum uric acid and blood pressureEvaluated by non-invasive blood pressure analyzer, systolic blood pressure (SBP) prominently rose from base level of106.6±7.4mmHg to114.2±10.9mmHg at2w in hyperuricemic mice (P=0.017), which was also significantly higher than normal control and allopurinol treated control. Positive correlation existed between serum uric acid level and SBP(r=0.333,P<0.001).3. The effects of hyperuricemia on renin expression and afferent arterioleSignificantly higher positive renin juxtaglomerular apparatus/glomeruli rate in hyperuricemic mice than in normal control or allopurinol treated control was observed by immunohistochemistry (31.0±6.3%vs.16.8±6.3%and19.1±4.7%, P=0.008and0.013, respectively). Similar results were showed by immunofluorescence. No significant difference showed in serum renin concentration tested by ELISA. Immunohistochemistry demonstrated decreased nNOS expression in cortex. No significant differences in Cox-2, CD39, CD73were observed by real-time PCR. a-SMA immunohistochemistry showed smooth muscle area of afferent arteriole in hyperuricemic mice was significantly larger than normal or allopurinol treated control (115.25±20.78μm2vs.81.46±18.44μm2and79.12±9.79μm2, P=0.020and0.006, respectively), together with prominently decreased lumen area (39.03±4.64μm2vs.51.07±8.45μm2and65.25±11.78μm2, P=0.030and0.005, respectively)4. The effects of uric acid on MD cell paracrine propertiesThe positive expression of Cox-2and nNOS showed by real-time PCR and immunofluorescence testified MMDD1cell identification. MTS test and trypan blue exclusion test demonstrated that3mg/dl to18mg/dl uric acid had no influence in MD cell proliferation or death. Real-time PCR and western blot showed6mg/dl and12mg/dl uric acid upregulated Cox-2and downregulated nNOS expression in both48h and72h. Uric acid at the dose of6mg/dl promoted CD39and CD73mRNA expression, but12mg/dl did not. Uric acid transporter URATl was identified in MMDDl cells with western blot. And blocking URATl with Benzbromarone significantly inversed the upregulation of CD73by6mg/dl uric acid.5. The effect of adenosine and A1AR in hyperuricemia induced hypertensionA1AR-/-C57BL/6J mice were brought in for our experiment, and breeding as well as gene identification was accomplished. By250mg/kg/d gavage of oxonic acid A1AR-/-mice had their serum uric acid elevated to the same level as wildtype (WT) mice, but without SBP elevation (P<0.05vs. hyperuricemic WT). No significant difference was showed between hyperuricemic A1AR-/-mice and normal WT mice. However, SBP of A1AR-/-mice rose from99.2±13.8mmHg to113.7±19.4mmHg (P=0.005) at1hour after introperitoneal furosemide injection, while SBP of other mice showed no change.2weeks of low salt diet did not alter SBP of hyperuricemic A1AR-/-mice. a-SMA immunohistochemistry showed hyperuricemic AlAR-/-mice did not develop thickening of afferent arteriole smooth muscle.CONCLUSIONSIn our experimental conditions, we observed:1. Significant correlation between serum uric acid and kidney local renin expression exists in normal blood pressure patients with glomerular minimal lesion.2. Gavage with oxonic acid successfully induced hyperuricemia in C57BL/6J mice with elevated SBP, stimulated renin expression and thickened afferent arteriole smooth muscle.3. In vitro, uric acid directly stimulated Cox-2, CD39and CD73expression and inhibited nNOS in MMDDl cells. URATl may take part in the regulation of CD73by uric acid.4. Hyperuricemic A1AR-/-mice did not show hypertension or afferent arteriole smooth muscle thickening. Acute renin stimulation caused SBP elevation in hyperuricemic A1AR-/-mice. These results indicated the importance of adenosine and A1AR in hyperuricemia induced hypertension.