Abstract: PO0161
Sphingolipid Transporter 2 (Spns2) Expression, Localization, and Role in AKI
Session Information
- AKI Mechanisms - 1
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Skrypnyk, Nataliya, Division of Nephrology and Center for Immunity, Inflammation, and Regenerative Medicine, University of Virginia, Charlottesville, Virginia, United States
- Zheng, Shuqiu, Division of Nephrology and Center for Immunity, Inflammation, and Regenerative Medicine, University of Virginia, Charlottesville, Virginia, United States
- Cechova, Sylvia, Division of Nephrology and Center for Immunity, Inflammation, and Regenerative Medicine, University of Virginia, Charlottesville, Virginia, United States
- Poudel, Nabin, Division of Nephrology and Center for Immunity, Inflammation, and Regenerative Medicine, University of Virginia, Charlottesville, Virginia, United States
- Lynch, Kevin, Department of Pharmacology, University of Virginia, Charlottesville, Virginia, United States
- Okusa, Mark D., Division of Nephrology and Center for Immunity, Inflammation, and Regenerative Medicine, University of Virginia, Charlottesville, Virginia, United States
Background
The sphingosine 1-phosphate (S1P) transporter Spns2 exports S1P from the cell and regulates the gradient of high circulating S1P levels to low tissue levels. Activation of S1P receptors can protect kidneys from acute injury, but little is known of the role of Spns2 in kidney. In these studies, we investigated the expression, localization, and role of Spns2 in the kidney after ischemia-reperfusion injury (IRI).
Methods
In vivo. Expression of Spns2 mRNA and protein in the kidneys and urine of C57BL/6 mice was analyzed 24 hours after sham surgery or IRI. Spns2 protein was assessed by western blot (WB) of membrane-enriched fractions of kidney. Renal function was evaluated by measurement of plasma creatinine. Spns2 localization and its co-localization with tubular markers in kidney (lens culinaris agglutinin, marker of brush border of S1-S3 segment of proximal tubules (PT) and wheat germ agglutinin a marker of brush border of S3 segment and cytosol of distal nephron) was by immunofluorescence (IF). To study the role of Spns2 after kidney injury, we pretreated mice 24h before IRI with Spns2 inhibitor (10 and 30 mg/kg, ip). In vitro. We generated clone with the highest Spns2 expression by the method of limited dilutions in TKPTS; Spns2 expression was validated by WB, quantitative real time PCR and IF.
Results
We demonstrated that the kidney has the highest mRNA expression of Spns2 when compared to other organs (liver, lung, spleen, heart, thymus). We determined that Spns2 is localized to the brush border of S1-S2 segment of PT and its expression is reduced 24h after IRI, while levels of Spns2 protein increased in urine. Pretreatment of mice with Spns2 inhibitor produced dose dependent protection against AKI, as judged by plasma creatinine and Kim1 mRNA expression at 24h after IRI and also induced dose dependent lymphopenia in mice. Spns2-overexpressing cells had increased TGFβ1 mRNA expression.
Conclusion
Spns2 is expressed in high abundance in kidney and is localized to the brush border of S1-S2 segments of the PT. Following IRI, PT Spns2 expression is reduced and appears in the urine. SPNS2 can serve as a potential target to prevent AKI, however further studies are needed to determine whether the protective effect of Spns2 inhibitor was due to lymphopenia or to a direct effect of Spns2 expressed in PT.
Funding
- NIDDK Support