Background Proliferative lupus nephritis (LN) is characterized by robust glomerular and tubulo- interstitial inflammation, sub-endothelial deposits of immunoglobulin, and increased endothelial cell permeability. Sphingosine 1- Phosphate Receptor 1 (S1PR1) has multiple protective effects on endothelial cells (ECs): it maintains barrier function thereby protecting against vascular leakage, it limits the number of leukocytes adhering to and transmigrating across ECs, and it protects ECs against apoptosis in response to inflammatory cytokines. Despite these important protective effects, the role of S1PR1 signaling in endothelial cells in lupus nephritis (LN) has yet to be elucidated. In prior work, we showed that S1PR1 modulators attenuated immune complex mediated vascular injury in skin and lung, leading to our hypothesis that EC S1PR1 signaling limits inflammatory injury in lupus nephritis. In current studies, we assessed whether patients with LN have decreased EC S1PR1 expression, and we performed in vitro mechanistic studies to determine whether S1PR1 maintains barrier function, at least in part, by restraining the metalloproteinase cleavage of VE-cadherin, a key protector against vascular permeability that is highly expressed on renal ECs and is shed during inflammatory states. To further support the hypothesis that S1PR1 signaling is protective in immune complex mediated disease, we also examined the effects of a S1PR1 antagonist on serum induced arthritis, a relatively acute model of immune complex mediated arthritis to determine whether it increased disease severity.
Methods (1) Assessment of S1PR1 expression in human LN: renal biopsy samples from 15 SLE patients with class IV LN and kidney samples from age, sex and race matched controls were evaluated by immunohistochemistry and scored for the expression of S1PR1 in a blinded fashion. Staining of S1PR1 was assigned an arbitrary value of 0-4 with 4 corresponding to maximal expression and 0 corresponding to no signal above the isotype control antibody. (2) In vitro studies to test the hypothesis that S1PR1 antagonism induced VE-cadherin shedding: HUVEC were treated with an S1PR1 antagonist NIBR-0213 (1 uM) for 30, 60, or 180 min +/- the presence of a pan-metalloproteinase inhibitor marimastat (MM) and VE-cadherin shedding was assessed by western blotting. (3) EC resistance, a measure of barrier function, was measured in NIBR treated ECs +/- MM by Electric cell- substrate impedance sensing (ECIS). (4) In vivo studies to determine whether NIBR-0213 exacerbated injury in serum induced arthritis, a model of immune complex mediated injury: NIBR 30 mg/kg was administered once daily to WT C57BL/6 mice receiving 75 ul of K/BxN serum on days 0 and 2. Clinical scores were assessed daily and histological scoring was assessed on H&E stained paraffin embedded sections.
Results Assessment of S1PR1 staining in human renal tissues: 5/15 patients with lupus nephritis had EC S1PR1 expression scores of 0 in renal microvasculature compared to 0/10 controls and 10/15 patients had EC S1PR1 expression scores of 0-1 compared to 2/10 controls (figure 1).
In vitro mechanistic studies: acute blockade of S1PR1 signaling with the antagonist NIBR-0213 induced shedding of VE-cadherin in a metalloproteinase - dependent manner in as measured by increased C- terminal (remaining transmembrane fragment) and N-terminal VE-cadherin (cleaved extracellular domain) in HUVEC lysates and supernatants, respectively (Fig. 2). ECIS demonstrated that NIBR induced a drop in resistance (a measure of barrier function), as expected, but that metalloproteinase inhibition attenuated this drop in resistance suggesting that the increase in permeability induced by S1PR1 blockade requires shedding of VE-cadherin and/or other molecules that contribute to functional adherens junctions.
In vivo S1PR1 blockade in serum induced arthritis: NIBR-0213 treatment of mice subjected to serum induced arthritis exacerbated injury - based on clinical and histological assessments (Fig 3), suggesting S1PR1 signaling contributes to maintenance of EC barrier function and inhibition of S1PR1 signaling leads to vascular escape of mediators that contribute to tissue damage.
Conclusion Our studies indicate that EC S1PR1 signaling maintains barrier function, in part by restraining MMP-dependent cleavage of VE-cadherin, and thereby may protect against immune complex mediated injury in experimental models. In some patients with LN, markedly decreased EC S1PR1 expression may result in loss of barrier integrity and increased vulnerability to glomerular injury. We identify a potential new approach to attenuate renal immune complex driven glomerular injury – enhancement of EC barrier function through S1PR1 signaling. Future studies will test the role of EC S1PR1 signaling in a mouse model of lupus nephritis.
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