Functional and Molecular Studies of Renal Sphingosine 1 Phosphate Signaling Pathway in IUGR Benjamin Bhunu1 and Suttira Intapad1 Tulane University School of Medicine

Abstract

Fetal Growth Restriction (FGR) is now regarded as a risk factor for adult renal diseases, however, the precise underlying mechanism that is responsible for this observation remains vague. Sphingosine 1 Phosphate (S1P) is a lysophospholipid with important functions in the regulation of renal physiology and the cardiovascular system. Previously, we demonstrated that in FGR but not control animals, acute activation of Sphingosine 1 Phosphate Receptor 1 (S1PR1) transiently reduces Blood Pressure (BP) in an endothelial nitric oxide synthase dependent manner. We have also shown that both male and female FGR mice have lower nephron number in the kidneys, however, unlike females, male FGR offspring have reduced renal function suggesting sex specific differences in fetal programing of kidney diseases. We have also shown that within 5 minutes of pharmacologically activating S1PR1 with SEW2871, GFR significantly reduces in male control but not IUGR animals. The objective of this study was to test the effect of longer stimulation of S1PR1 agonist on renal function and to determine the distribution of renal S1P signaling molecules. On their 13th day of gestation, pregnant C57BL mice went through a Reduced Uterine Pressure Perfusion (RUPP) or sham surgery to generate FGR and control offspring respectively. At 6 months, measurement of Glomerular Filtration Rate (GFR) and expression of proteins that are involved in S1P signaling was performed using plasma FITC‐Anulin decay, PCR, and immunohistochemistry. For immunohistochemistry, the Integrated Optical Densities (IDO) values from (Diaminobenzidine) DAB‐stained kidney sections were used to statistically quantify protein expression. GFR was lower in male FGR offspring than control (8.85 ±0.55 vs 13.86±1.13 µL/min/kg BW, P˂0.05, FGR vs control males) while in females there was no statistical difference (12.33±0.87 vs 15.92 ±1.94 L/min/kg BW, P˃ 0.05 control females vs FGR). Administration of SEW2871 and measurement of GFR after one hour confirmed that GFR decreases only in control males but not IUGR while in females both FGR and control groups had similar reduction response (6.00±0.46 vs 8.41±0.79 µL/min/kg BW, P˃ 0.05 control males vs FGR) (5. 97±0.48 vs 5.72 ±1.01 µL/min/kg BW, P˃ 0.05 control females vs FGR). Whole kidney transcriptomics showed no statistical differences in the expression profile of S1PR 1‐3 and Sphingosine 1 Phosphate Kinases 1 and 2 (SPHK1‐2) between FGR and control male and female animals. Immunohistochemistry analysis of the kidney medulla and cortex of male animals revealed no difference in the expression profile of S1PR1 in both FGR and control and increased expression of S1PR2 in both the medulla and cortex of FGR animals. S1PR3 was decreased in both the medulla and cortex of FGR mice. SPHK 1 and 2 were not differentially expressed in the medulla of control and FGR animal whereas in the cortex both the two receptors were downregulated in FGR. Our results indicate that acute activation of S1PR1 differentially regulates GFR between male and female FGR. Consequences of the upregulation of S1PR2 and the down regulation of S1PR 3 and SPHK 1‐2 proteins in the kidney of male FGR warrants further investigations.