Abstract P161: Effect of Rapamycin on the Expression of T Regulatory Cells, Foxp3, and Toll-Like Receptors in the Kidneys of Npr1 Gene-Knockout Mice

Abstract

The alterations in the inflammatory system contribute to the pathogenesis of hypertension leading to end-organ damage. Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) disruption elevates blood pressure and activates the immunogenic responses in mice. The goal of this study was to determine the effect of rapamycin on the expression of T regulatory (Treg) cells, Foxp3, and Toll-like receptors (TLRs) in Npr1 gene-disrupted and -duplicated mice. The adult 0-copy ( Npr1 -/- ), 2-copy ( Npr1 +/+ ), and 4-copy ( Npr1 ++/++ ) female mice were pre-treated with rapamycin (1mg/kg/day). Kidneys, spleen, and blood were collected, qRT-PCR, Western blot, immunofluorescence, and flow cytometry analyses were done to assess the expression of Tregs, Foxp3, and TLRs. The disruption of Npr1 led to significant reductions in Foxp3 + mRNA expression in 0-copy (77.5%) and 1-copy (71.5%) mice but not in 2-copy and 4-copy mice. Similarly, CD25 + expression was reduced in 0-copy mice by 75% and in 1-copy by 60% compared to 2-copy and 4-copy mice. In contrast, the total CD4 + count was up-regulated by 40% in 0-copy and 31% in 1-copy mice. Treatment with rapamycin up-regulated Foxp3 + cells in 0-copy (17.38%, P <0.001) and 1-copy (8%, P <0.05) mice, and CD25 + T cell levels were increased by 62.4% ( P <0.001) in 0-copy mice and 38.6% ( P <0.05) in 1-copy mice compared to vehicle-treated control mice. In contrast, CD4 + cell expression was significantly reduced by 15.4% ( P <0.001) in 0-copy and 12.2% ( P <0.05) in 1-copy mice. Interestingly, rapamycin significantly ( P < 0.001) increased renal Foxp3 + protein expression to 85% in 0-copy mice and 68% in 1-copy mice compared to untreated controls. The expression of TLR2/TLR4 was significantly increased by 3.4- to 4.5-fold in 1-copy and 0-copy mice than 2-copy and 4-copy mice; however, rapamycin greatly decreased the expression of both TLRs in both 1-copy and 0-copy mice. In summary, rapamycin treatment upregulated the expression of Foxp3 + , CD25 + cells, and TLRs while suppressing pathogenic CD4 + T cell expression in 0-copy and 1-copy mice kidneys. The present results implicate that Npr1 and immune cell interactions could provide new therapeutic targets for the treatment and prevention of hypertension and kidney dysfunction in humans.