MO448: Progressive Tubular Injury Caused by High Phosphate Intake is Associated With Activation of STAT3/KIM-1 Signalling and Macrophage Recruitment in Mice

Abstract BACKGROUND AND AIMS Dietary phosphate intake greatly exceeds the recommended daily allowance in the Western population. Elevated phosphate levels are linked to increased cardiovascular and all-cause mortality, impaired bone health and premature ageing. The renal effects of chronic high dietary phosphate intake for healthy individuals are still not clear. METHOD Male C57BL/6 mice received a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD) for 1 to 6 months. We collected blood, urine and kidneys to investigate phosphate metabolism and progression of kidney injury. In vitro analysis for the toxic effects of phosphate or the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor (FGF) 23 was done in HK-2 humane proximal tubule (PT) cells. RESULTS HPD in mice caused hyperphosphataemia despite increased phosphaturia. Both intact FGF23 and PTH plasma levels were significantly increased in mice on HPD at 1 month onward. Mice on HPD showed significantly increased creatinine levels and albuminuria. Histopathological analysis of the kidneys revealed increased PT injury in mice receiving HPD compared to NPD controls from 2 months onward followed by a rapid progression of organ damage from month 5 to 6. Picrosirius red staining of kidney sections showed a progressively increased accumulation of collagen fibers in renal cortex of mice on HPD, which was significant from 4 months onward and accompanied by enhanced expression of transforming growth factor-β1, collagen 1 and fibronectin. The significant induction of the tubular injury marker Havcr1, encodes for Kim-1, started already after 1 month of feeding HPD, progressed over time and was strongest after 6 months. Immunofluorescence staining revealed Kim-1 localization to damaged PT, which was most prominent after 3 and 6 months of HPD. Renal Kim-1 protein levels were positively associated with tubular injury score and tubulointerstitial fibrosis. Histological staining and quantification revealed significantly increased number of pStat3+ cells in PTs of HPD-fed mice at months 1, 2, 5 and 6, indicating activated Stat3 signaling. The number of pStat3+ cells was positively associated with Kim-1 synthesis. Inflammation cytokine array with kidney tissue lysates showed, among others, enhanced expression of monocyte chemoattractant protein (MCP)-1 and macrophage colony-stimulating factor (MCSF) in HPD-fed mice. The mRNA expression of Csf1 (encodes for MCSF) was upregulated in HPD-fed mice and most pronounced after 6 months. Ccl2 mRNA expression (encodes for MCP-1) was significantly enhanced after 3, 5 and 6 months of HPD compared to NPD. Immunofluorescence staining showed a strong and specific induction of MCP-1 in PT epithelial cells due to HPD. The HPD-induced Ccl2 expression was positively associated with Kim-1 expression indicating an interaction between pStat3/Kim-1 signalling pathway and MCP-1. Immunohistochemical staining and flow cytometry analysis revealed a significantly increased recruitment of F4/80+ macrophages in renal tissue of HPD-fed mice after 3 and 6 months. The enhanced renal macrophage recruitment in the entire HPD cohort was associated with increased MCP-1 protein synthesis and increased tubular injury score. In vitro experiments using HK-2 cells showed that stimulation with FGF23 or high phosphate, but not PTH, induced the phosphorylation of STAT3. Interestingly, only treatment with high phosphate significantly upregulated HAVCR1 and CCL2 expression. CONCLUSION Chronic oral phosphate load in mice induced progressive inflammatory kidney injury dominated by macrophages. The direct toxic effects of phosphate are mediated by Stat3/Kim-1 signalling activation in PT cells. Our data support the hypothesis that kidney injury induced by widespread high dietary phosphate intake may cause a global health problem and underline the urgent need for clinical studies on this issue.