Basic Research

Endothelial Sphingosine 1‑Phosphate Receptor‑1 Mediates Protection and Recovery from Acute Kidney Injury

Heather M. Perry, Liping Huang, Hong Ye, Chong Liu, Sun-sang J. Sung, Kevin R. Lynch, Diane L. Rosin, Amandeep Bajwa and Mark D. Okusa
JASN November 2016, 27 (11) 3383-3393; DOI: https://doi.org/10.1681/ASN.2015080922

Abstract

Epithelial and endothelial injury and a cascade of immune and interstitial cell activation in the kidney lead to AKI. After mild to moderate AKI, the epithelium can regenerate and restore kidney function, yet little is known about the endothelium during these repair processes. Sphingosine 1-phosphate receptor 1 (S1P1), a G protein–coupled receptor, is necessary for vascular homeostasis. Here, we used an inducible genetic approach in a mouse model of AKI, ischemia–reperfusion injury (IRI), to determine the temporal effects of endothelial S1P1 during AKI. Deletion of endothelial S1P1 before IRI exacerbated kidney injury and inflammation, and the delayed deletion of S1P1 after IRI prevented kidney recovery, resulting in chronic inflammation and progressive fibrosis. Specifically, S1P1 directly suppressed endothelial activation of leukocyte adhesion molecule expression and inflammation. Altogether, the data indicate activation of endothelial S1P1 is necessary to protect from IRI and permit recovery from AKI. Endothelial S1P1 may be a therapeutic target for the prevention of early injury as well as prevention of progressive kidney fibrosis after AKI.

Epidemiologic and mechanistic studies have demonstrated that AKI and CKD are interconnected syndromes. Following an acute insult, kidneys may either recover function or progressively decline.1 Early endothelial injury and activation after AKI is characterized by increased microvascular permeability, recruitment of immune cells, vasoconstriction, and procoagulation2; prevention of endothelial dysfunction reduces AKI.3 Thus, preserving endothelial function might be key to attenuating progressive kidney damage and subsequent CKD. However, the mechanisms restoring endothelial function during adaptive recovery of the kidney are not well understood.4

The pathophysiologic role of the renal endothelium in the initiation and progression of fibrosis is largely thought to be regulation of tissue hypoxia and inflammation. There is growing evidence that the renal endothelium lacks the capacity to proliferate after injury and thereby undergoes microvascular dropout, thus leading to local tissue hypoxia and progressive fibrosis.5 Endothelial activation of chemokines and leukocyte adhesion molecules recruits immune cells, in particular monocytes, to the injured tissue.6 Monocytes can then differentiate into phenotypically distinct macrophages, depending on the environmental milieu, and promote repair and/or fibrosis.7 Chronic inflammation and progressive fibrosis are part of the maladaptive repair processes after AKI, and this dysregulation can lead to CKD.1

Sphingosine 1‑phosphate (S1P), a bioactive lysophospholipid, can be released into the extracellular environment, especially by endothelial cells, and signal through its G protein–coupled receptors (S1P1–5). S1P1 is highly expressed on the endothelium and is critical for vascular development and endothelial barrier integrity.8 We sought to understand the role of endothelial S1P1 prior to and during recovery from AKI using a unique mouse model to temporally delete S1P1 specifically in the endothelium. Our findings demonstrate that deletion of endothelial S1P1 prior to ischemia–reperfusion injury (IRI), a model of reversible AKI, exacerbated IRI and inflammation. Additionally, delayed deletion of endothelial S1P1 after IRI during the recovery period resulted in continued endothelial activation, inflammation, reduced kidney function, and the development of fibrosis. These results indicate that endothelial S1P1 can prevent IRI and is necessary during recovery from IRI to suppress endothelial activation and prevent progressive fibrosis.

Results

Endothelial S1P1 Protects Kidneys from IRI and Inflammation

The endothelial-specific functions of S1P1 at different stages of kidney IRI have not been investigated. S1pr1 levels in the kidney increased 24 hours after injury, and returned to control levels during the repair phase after IRI (Supplemental Figure IA). To induce conditional deletion of S1P1 in endothelial cells in a time-dependent manner, we generated an inducible, endothelial-specific, S1P1 knockout mouse by crossing an iTie2CreERT2 mouse (tamoxifen-inducible Cre under the control of the Tie2 promoter) with the S1pr1fl/fl mouse to produce littermate iTie2CreERT2 S1pr1fl/fl, iTie2CreERT2 S1pr1wt/fl, and iTie2CreERT2 S1pr1wt/wt mice. Genomic PCR analysis of tail, kidney, liver, and aorta (Supplemental Figure IB) confirmed the anticipated PCR products, which have been previously described in Tie2Cre and S1pr1fl/fl mice.9 To determine the extent of kidney S1pr1 deletion in iTie2CreERT2 S1pr1fl/fl mice, tamoxifen was administered daily intraperitoneally for 5 days, as published10 (see Concise Methods). As shown in Figure 1A, S1pr1 transcript levels were reduced in the kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with control mice. Under physiologic conditions, endothelial deletion of S1pr1 did not result in apparent tissue morphology abnormalities or increased vascular permeability in various organs (Supplemental Figure I, C and D). Additionally, creatinine clearance was similar between iTie2CreERT2 S1pr1wt/wt (0.24±0.019 ml/min, n=3) and iTie2CreERT2 S1pr1fl/fl mice (0.21±0.037 ml/min, n=4) with values approximating published values11 and there was no evidence of albuminuria12 (<10 μg albumin/24 hours in all mice). However, total leukocyte content was decreased in the liver, lung, and spleen in iTie2CreERT2 S1pr1fl/fl mice compared with control mice (Supplemental Figure IE).

Figure 1.
Figure 1.

Generation of tamoxifen-inducible endothelial-specific S1pr1 knockout mice. Tamoxifen was administered intraperitoneally for 5 days and mice were euthanized on day 6. (A) S1pr1 normalized to Gapdh transcript levels in iTie2CreERT2 S1pr1wt/wt and iTie2CreERT2 S1pr1fl/fl kidneys by quantitative PCR, n=6. (B) tdTomato (red; native fluorescence) and CD31 (green) expression with the nuclear stain, 4′,6-diamidino-2-phenylindole (dapi) (blue), in peritubular (a) and glomerular (b) capillaries in iTie2CreERT2 tdTomato kidneys quantified (c) as the percentage of tdTomato+ cells associated with CD31 expression (pink/yellow) or any nucleated cell lacking CD31 by immunofluorescence. n=5. ***P<0.001. Scale bar, 20 µm.

To assess the efficiency and location of active Cre-mediated loxp site recombination in the kidney, the iTie2CreERT2 mouse was crossed to a Rosa26-floxed stop tdTomato Cre-reporter mouse. After tamoxifen administration, expression of TdTomato, a red fluorescent protein mutant that freely diffuses throughout the cell,13 was observed in 14.55±3.49% of the total kidney endothelium in two subpopulations of endothelial cells: the glomerular and peritubular capillaries, but not in artery or arteriole endothelial cells. Furthermore, nearly 100% of tdTomato+ cells were also CD31+ (Figure 1B). Consistent with previous reports, we also found that unlike the constitutive Tie2Cre mouse, the inducible Tie2CreERT2 is not expressed in cell types other than endothelial cells in the adult mouse.14

In the constitutive Tie2Cre mouse, Tie2 is expressed on endothelial cells18 and early in mural26 and myeloid cell14,15 development, however, when tamoxifen is provided to the adult iTie2CreERT2 mouse, Cre activation is largely restricted to endothelial cells.14 To extend our findings with the iTie2CreERT2 tdTomato reporter mouse (Figure 1), we performed immunofluorescent labeling in kidneys of iTie2CreERT2 S1pr1fl/fl and control mice. We observed colocalization of Tie2 with CD31 immunoreactivity, but not CD45 or PDGF receptor beta (PDGFRβ), in kidneys of iTie2CreERT2 S1pr1fl/fl and control mice (Supplemental Figure IV, a–d). Additionally, although CD31 can be transiently expressed on leukocytes,16 CD31+ cells were not CD45+ in kidneys in our mice. In our model, Tie2 expression appears to be specific to CD31+ cells and not CD45+ or PDGFRβ+ cells, and likely S1P1 is only deleted in endothelial cells, not leukocytes or fibroblasts/pericytes.

We first sought to determine if endothelial S1P1 confers protection against kidney IRI. Tamoxifen was administered daily for 5 days to littermate iTie2CreERT2 S1pr1fl/fl and iTie2CreERT2 S1pr1wt/wt mice prior to sham or 24 minutes of bilateral kidney ischemia, and renal function and tissue injury were examined 24 hours later (Figure 2A). Plasma creatinine (PCr) levels were significantly increased and substantial acute tubular necrosis was observed in iTie2CreERT2 S1pr1fl/fl mice compared with sham control and IRI in iTie2CreERT2 S1pr1wt/wt mice (Figure 2B, C). Inflammation is a key characteristic of AKI.6 The number of CD11b+Ly6G+ neutrophils was increased in kidneys of iTie2CreERT2 S1pr1fl/fl mice that underwent IRI compared with controls (Figure 2D), as quantified by flow cytometry. Taken together, our data are consistent with previous findings that S1P1 deletion in endothelial cells prior to IRI exacerbates kidney injury and inflammation.17

Figure 2.
Figure 2.

Endothelial deletion of S1P1 exacerbates IRI. iTie2CreERT2 S1pr1wt/wt and iTie2CreERT2 S1pr1fl/fl mice were treated with tamoxifen once daily for 5 days, then randomized to bilateral IRI or sham operation, and euthanized after 24 hours of reperfusion. (A) Protocol for experimental setup. (B) PCr. (C) Renal histology in iTie2CreERT2 S1pr1wt/wt (a) and iTie2CreERT2 S1pr1fl/fl (b) mice that underwent IRI. n=2–6 for sham groups; n=17 for IRI groups. Scale bar =100 µm. (D) Quantitation of renal neutrophils (CD11b+Ly6G+) by flow cytometry; n=2 for sham groups; n=4–6 for IRI groups. *P<0.05; **P<0.01; ****P<0.0001.

To determine the relative contribution of endothelial S1P1 signaling in protection from IRI, FTY720, a S1P1 agonist, or vehicle was administered to iTie2CreERT2 S1pr1fl/fl and iTie2CreERT2 S1pr1wt/wt mice 1 hour prior to IRI. As shown in Supplemental Figure II, FTY720 afforded partial protection to iTie2CreERT2 S1pr1fl/fl mice, which may be due to incomplete deletion of endothelial S1pr1 in this model, or the protective effect of FTY720 on proximal tubule S1P1.18

Endothelial S1P1 Is Necessary for Recovery from IRI and Prevents Early Onset Fibrosis

Recovery of endothelial function after AKI is not well understood. We hypothesized that S1P1 in endothelial cells is necessary for tissue recovery in a model of reversible IRI. To test this hypothesis, iTie2CreERT2 S1pr1fl/fl and age-matched control iTie2CreERT2 S1pr1wt/wt mice were randomized to sham or 24 minutes of unilateral kidney ischemia and assessments were made 1, 9, and 14 days later (Figure 3A). Unilateral IRI with simultaneous contralateral nephrectomy followed by reperfusion for 24 hours confirmed that iTie2CreERT2 S1pr1fl/fl and control mice were equally susceptible to the initial injury prior to tamoxifen administration (Figure 3B). In separate groups of mice, and in contrast to the protocol described in Figure 2A, initiation of tamoxifen administration was delayed until the beginning of the recovery phase at the peak of tubule proliferation19 when acute inflammation wanes20 at day 3 of reperfusion (Figure 3A). PCr returned to sham levels in iTie2CreERT2 S1pr1wt/wt control mice by day 9, however, the delayed deletion of endothelial S1P1 prevented recovery of kidney function, and PCr remained elevated in iTie2CreERT2 S1pr1fl/fl mice on day 9 and day 14 (Figure 3C). S1pr1 transcript levels were reduced by 45% (0.54 arbitrary units ±0.068 n=3 versus 1.0±0.140, n=2) in isolated CD31+ cells from kidneys that underwent IRI in iTie2CreERT2 S1pr1fl/fl compared with control mice on day 9. Expression of Kim‑1, a marker of tubular injury (Figure 3D), and renal histology (Figure 3, E and F) mirrored PCr in mice that were euthanized on day 9. Additionally, kidneys from iTie2CreERT2 S1pr1fl/fl mice were unable to recover when tamoxifen was initiated either 1 or 2 days after IRI (Supplemental Figure III, A and B). These data provide evidence that whereas kidneys from control mice are able to undergo adaptive renal repair within 9 days after unilateral kidney ischemia, endothelial deletion of S1P1 delayed by 1–3 days after IRI may result in maladaptive repair processes, thereby preventing renal recovery.

Figure 3.
Figure 3.

Endothelial S1P1 is necessary for recovery from IRI. (A) iTie2CreERT2 S1pr1wt/wt and iTie2CreERT2 S1pr1fl/fl mice were treated with tamoxifen once daily for 5 days beginning on day 3 after unilateral IRI, nephrectomized (unoperated kidney) on day 8 or 13, and euthanized on day 9 or 14 for PCr and other endpoints (for panels C–E; day 14 in panel C only). (B) In separate groups of mice, unilateral IRI and simultaneous nephrectomy of contralateral kidneys were performed on day 0, and equivalent initial injury in both groups of mice (prior to tamoxifen administration) is shown by PCr 24 hours later. (C) PCr at day 9 or in a separate group of mice, day 14. (D) Kidney Kim1 relative to Gapdh transcript levels and (E) renal histology on day 9 (n=4–6). (F) Stereological quantification of renal injury in (E) expressed as a percentage of the total surface area of kidney section. ***P<0.001 and ****P<0.0001 compared with all other groups. Con, contralateral control; unoperated kidney. IRI, kidney exposed to unilateral ischemia–reperfusion; operated kidney. Kim1, kidney injury molecule‑1. Scale bar, 50 µm.

Progressive fibrosis is one consequence of maladaptive repair processes, and the endothelium is an important regulator of renal fibrosis.5 We next sought to determine if delayed deletion of S1P1 in endothelial cells affected progression to fibrosis after ischemic injury. As shown in Figure 4, A and B, IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice displayed an increased area of large collagen fibers in the cortex and medulla, as assessed by picrosirius red morphometry compared with sham controls, and to contralateral control and IRI kidneys of iTie2CreERT2 S1pr1wt/wt mice subject to the experimental protocol in Figure 3A. Although morphologic differences may not be apparent among sham and contralateral control kidneys of iTie2CreERT2 S1pr1fl/fl and control iTie2CreERT2 S1pr1wt/wt mice, transcript levels may be different in sham mice, potentially resulting in the profibrotic phenotype of IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice. Therefore, we compared transcript markers of fibrosis in sham and IRI kidneys of iTie2CreERT2 S1pr1fl/fl and control mice. Transcript levels of Acta2 (encoding the protein αSMA), Col1a1, Col3a1, Fn1, and Vim were also increased in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with controls (Figure 4C), suggesting that there is active cellular production of extracellular matrix; however, at this early time point, accumulation may not yet be sufficient to produce substantial large collagen fiber deposition identified by picrosirius red (Figure 4A). Col1a1 and Col3a1 levels were also increased in IRI kidneys of iTie2CreERT2 S1pr1wt/wt mice compared with shams, indicating that there may be activation of some fibrotic pathways even in mice that have recovered from IRI (Figure 4C). Myofibroblasts are key effector cells in the development of fibrosis. To determine whether increased fibrosis was associated with myofibroblast formation in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice, we first measured transcript levels of the cytokines Tgfb, Ctgf, Igf1, Fgf2, Pdgfa, and Pdgfb, which are important growth factors driving myofibroblast activation and differentiation. Although transcript levels in IRI kidneys of control mice remained similar to sham, levels of these cytokines were elevated in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with all controls (Figure 4D). We next detected renal myofibroblasts by immunofluorescent colocalization of αSMA, COL1, or COL3 with PDGFRβ in kidney sections. Here, the contralateral control kidney was used for comparison to detect any potential systemic contributions to phenotype in mice that underwent unilateral IRI. As shown in Figure 4E, there was a marked increase in the density of PDGFRβ+αSMA+, PDGFRβ+COL1+, and PDGFRβ+COL3+ cells in the cortex of IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with controls. Together, these data demonstrate that the delayed deletion of S1P1 in endothelial cells after IRI promotes myofibroblast activation and differentiation, and initiation of fibrosis.

Figure 4.
Figure 4.

Endothelial S1P1 is necessary for the prevention of fibrosis after IRI. Kidneys from iTie2CreERT2 S1pr1wt/wt and iTie2CreERT2 S1pr1fl/fl mice as in Figure 3A. (A) Picrosirius red staining of kidney using brightfield (left) and polarized (right) microscopy. (B) Quantification of red/yellow birefringence of mature collagen fibers in (A) expressed as a percentage of the total surface area of kidney section. Transcript levels of (C) fibrosis markers and (D) growth factors in sham or unilateral IRI-operated kidneys by quantitative PCR (relative to Gapdh) at day 9, n=7–8. (E) Detection of myofibroblasts (orange) by colocalization of αSMA (yellow) and PDGFRβ (red) (a–d) or collagen (yellow) and PDGFRβ (red) (e–h) in contralateral control (a, b) or IRI (c–h) kidneys by immunofluorescence microscopy. n=4–5. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 compared with all other groups. Acta2/αSMA, actin, alpha 2, smooth muscle, aorta; auto, intensity of green autofluorescence of kidney tissue adjusted to reveal tubule architecture; Col1a1/COL1, collagen, type I, alpha 1; Col3a1/COL3, collagen, type III, alpha 1; Con: contralateral control kidney; Ctgf, connective tissue growth factor; Fgf2, fibroblast growth factor 2; Fn1, fibronectin; Igf1, insulin-like growth factor 1; IRI, kidney exposed to unilateral ischemia–reperfusion; Pdgfa, PDGF, alpha; Pdgfb, PDGF, beta; PDGFRβ, PDGF receptor beta; Tgfb, TGF, beta 1; Vim, vimentin. Scale bar, 50 µm.

PDGFRβ, a marker for fibroblasts/pericytes, is also expressed by myofibroblasts.21 Likely, in our studies myofibroblasts are not derived from endothelial cells or leukocytes, because PDGFRβ did not colocalize with Tie2, CD31, or CD45 (Supplemental Figure IV, a–f).

The Delayed Deletion of Endothelial S1P1 After IRI Does Not Result in Reduced Renal VE‑Cadherin Expression or Capillary Rarefaction

A major function of S1P1 in the endothelium is to maintain endothelial barrier integrity.22 To determine if altered endothelial integrity might be contributing to the inability of the kidney to recover from IRI in iTie2CreERT2 S1pr1fl/fl mice, we assessed VE‑cadherin expression of mice subject to the protocol in Figure 3A. On day 9 after IRI, there was increased VE‑cadherin expression (Supplemental Figure V, A and B) in IRI kidneys from iTie2CreERT2 S1pr1fl/fl mice compared with controls, suggesting that there may be compensation by the endothelium to enhance barrier integrity at this time point.

Capillary rarefaction, a hallmark of fibrosis, is defined as large areas of kidney tissue that exhibit a loss of the endothelium.5 Representative images at low magnification clearly demonstrate a uniform endothelium across a large area of IRI kidneys of iTie2CreERT2 S1pr1fl/fl and iTie2CreERT2 S1pr1wt/wt mice (Supplemental Figure V, C). Indeed, capillary density was similar in IRI kidneys of iTie2CreERT2 S1pr1fl/fl and iTie2CreERT2 S1pr1wt/wt mice (1.41±0.050% and 1.34±0.263%, n=6 and 4, respectively), which argues against capillary rarefaction at this early time point of 9 days reperfusion. Consistent with these data, there was no difference in transcript levels of Vegfa (data not shown) in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with contralateral control kidneys, or to contralateral control or IRI kidneys of iTie2CreERT2 S1pr1wt/wt mice. Although the renal endothelium does not exhibit vascular dropout in iTie2CreERT2 S1pr1fl/fl mice, local areas of nonperfused (latent) capillaries might result in regional reductions in blood flow3 and contribute to fibrosis.5 Representative images demonstrate that all CD31+ cells were perfused in IRI kidneys and contralateral controls in iTie2CreERT2 S1pr1fl/fl mice (Supplemental Figure V, D) and in iTie2CreERT2 S1pr1wt/wt mice (not shown). Additionally, there were increased transcript levels of the gene encoding nitric oxide synthase 3, endothelial cell (Supplemental Figure V, E), an enzyme necessary for vasorelaxation and maintenance of blood flow, in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with controls. These data suggest that there may be sustained vasorelaxation to ensure blood flow to IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice. Altogether, our data suggest that loss of endothelial S1P1 during recovery from IRI does not result in altered capillary perfusion or rarefaction at day 9 of reperfusion, and that the early development or enhanced progression of fibrosis in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice may be due to other processes.

S1P1 Is Necessary to Suppress Endothelial Activation of Leukocyte Adhesion Molecule Expression and Inflammation during Recovery from IRI

Inflammation is a key regulator of fibrosis, and the endothelium is the gatekeeper for inflammation.5 Therefore, we hypothesized that the delayed deletion of endothelial S1P1 after IRI results in chronic inflammation. Recruitment of innate immune cells to injured tissue is directed by the production of chemoattractants such as CXCL1 and CCL2 (also known as KC and MCP1, respectively). Indeed, there were increased transcript levels of Cxcl1 and Ccl2 (Figure 5A) in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with controls. Consistent with our hypothesis, there was also an increased density of neutrophils and macrophages detected by immunofluorescence in the cortex of IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with controls (Figure 5B). Furthermore, total CD11b+Ly6G+ neutrophils and CD11b+Ly6G– F4/80loLy6Clo monocytes/macrophages were increased in IRI kidneys from iTie2CreERT2 S1pr1fl/fl compared with controls (Figure 5C), as assessed by flow cytometry, using a previously described gating strategy23 and shown in Supplemental Figure VI. There was no difference in the total number of CD11b+F4/80hiMHChiCD11c+ dendritic cells, CD3+ T cells or CD19+ B cells among all groups in the kidney (data not shown).

Figure 5.
Figure 5.

Delayed deletion of endothelial S1pr1 results in kidney inflammation. Kidneys from iTie2CreERT2 S1pr1wt/wt and iTie2CreERT2 S1pr1fl/fl mice as in Figure 3A. (A) Chemokine transcript expression by quantitative PCR (relative to Gapdh), n=7–8, (B) neutrophils (7/4) and monoctyes (F4/80) by immunofluorescence and (C) quantification of neutrophils (Ly6G+CD11b+Ly6C+) and Ly6Chi and Ly6Clo macrophages (Ly6G-CD11b+F4/80lo) by flow cytometry at day 9. Statistics in macrophage graph are shown for the Ly6Clo subset, n=5. *P<0.05, **P<0.01, ****P<0.001 compared with all other groups. Ccl2, chemokine (C-C motif) ligand 2; Cxcl1, chemokine (C-X-C motif) ligand 1. Scale bar, 50 µm.

To gain mechanistic insight on how S1P1 expression by endothelial cells may be regulating inflammation, we first measured transcript levels for leukocyte adhesion molecules: intercellular adhesion molecule‑1 (Icam1) and vascular cell adhesion molecule‑1 (Vcam1), key regulators of leukocyte adhesion and transendothelial migration, and E‑selectin (Sele) and P‑selectin (Selp), important molecules regulating leukocyte rolling (Table 1). All four adhesion molecule transcripts were increased in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with controls (Figure 6A). Although transcript levels for the adhesion molecule PECAM1 (or CD31) were not increased (data not shown), protein expression appeared to be increased (Supplemental Figure V, C, Figure 6B). Leukocyte adhesion molecule expression by endothelial cells can be stimulated by the proinflammatory, profibrotic cytokines IL1β, TNFα, and IL6. These cytokines were also increased in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice compared with controls (Figure 6C). To determine if S1P1 can directly regulate adhesion molecule expression, S1P1 was stably overexpressed in a mouse renal endothelial cell line. S1pr1 transcript levels were increased 10‑fold compared with cells stably overexpressing a control vector (data not shown). The overexpression of S1P1 attenuated IL1β- and TNFα-induced Vcam1 transcript and protein (CD106) levels in renal endothelial cells (Figure 6D, E), suggesting that S1P1 directly regulates cytokine induction of leukocyte adhesion molecule expression in endothelial cells. Altogether, our data suggest that S1P1 is necessary to suppress endothelial cell activation of leukocyte adhesion molecule expression to prevent excessive inflammation during recovery from IRI, and that this process is crucial for adaptive repair processes and prevention of progressive fibrosis (Figure 7).

View this table:
Table 1.

Primer sequences for real-time quantitative PCR

Figure 6.
Figure 6.

Endothelial S1P1 suppresses activation of leukocyte adhesion molecule expression after IRI. (A–C) Kidneys from iTie2CreERT2 S1pr1wt/wt and iTie2CreERT2 S1pr1fl/fl mice as in Figure 3A. (A) Leukocyte adhesion molecule transcript expression by quantitative PCR, (B) PECAM1 detected by immunoperoxidase labeling (brown), and (C) cytokine transcript expression detected by quantitative PCR (relative to Gapdh), at day 9. n=4–6. (D) Vcam1 transcript levels were measured by quantitative PCR and (E) vascular cell adhesion molecule 1 (CD106) protein expression was measured by flow cytometry in S1pr1 stably overexpressing (S1P1) or control (Ctrl) mouse glomerular endothelial cells treated with vehicle, 1 ng/ml IL1β or 10 ng/ml TNFα for 48 hours. Data (D, E) are a summary of four or five independent experiments. Experimental conditions were performed in duplicate and averaged, and all data are normalized to vehicle-treated control cells. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 compared with all other groups. Icam1, intercellular adhesion molecule‑1; Vcam1, vascular cell adhesion molecule‑1; Sele, selectin, endothelial cell; Selp, selectin, platelet; Il1b, IL1β; Tnfa, TNFα; Il6, IL6; FMO, fluorescence minus one; veh, vehicle. Scale bar, 25 µm.

Figure 7.
Figure 7.

Model of the role of endothelial S1P1 during kidney recovery from IRI. Deletion of S1P1 results in increased leukocyte adhesion molecule expression on the endothelium and inflammation, resulting in a proinflammatory, profibrotic microenvironment and progressive fibrosis.

Discussion

In this study, we used an inducible genetic approach to determine the contribution of endothelial S1P1 at different stages of IRI. We provide evidence that S1P1 is necessary in both the prevention of and recovery from IRI. Moreover, the S1P1 signaling pathway in the endothelium is crucial to suppress leukocyte adhesion molecule expression in modulating the renal microenvironment for adaptive repair processes necessary for recovery of kidney function.

Sutton and colleagues proposed clinical and cellular phases of AKI,2 yet the endothelial functions important during the maintenance and repair phases remain largely elusive. Additionally, the mechanisms regulating restoration of endothelial function during recovery from AKI are largely unknown. Novel findings from our study suggest that S1P1 is necessary for the recovery of endothelial function during these phases and that S1P1 directly regulates endothelial leukocyte adhesion molecule expression to suppress inflammation after IRI. As such, we have identified suppression of inflammation induced by neutrophils and macrophages as a major process necessary for kidney recovery; endothelial expression of S1P1 is crucial for this process.

Macrophages have emerged as important inflammatory cells in the recovery of kidney function, as well as fibrosis, and can dictate the balance between wound healing and progressive fibrosis. The functional and phenotypic definition of monocytes/macrophages in kidney pathophysiology is complex due to the intrinsic plasticity of the cells, different gating strategies used to define populations, different model of fibrosis, and attempts to relate in vitro studies to in vivo findings.7 Nonetheless, Ly6Chi monocyte/macrophages are thought to be proinflammatory and contribute to initial kidney damage, whereas Ly6Clo monocyte/macrophages appear later and have a genetic signature associated with wound healing and/or fibrosis.24 We demonstrate that the delayed deletion of endothelial S1P1 results in increased numbers of kidney Ly6Clo monocyte/macrophages at day 9 after IRI. Ly6Clo monocyte/macrophages can produce growth factors that can promote fibrosis including PDGF, IGF1 and FGF2 and potentially TGFβ.7 The increased number of kidney Ly6Clo monocyte/macrophages is associated with increased production of these growth factors in IRI kidneys of iTie2CreERT2 S1pr1fl/fl mice, suggesting that these monocytes/macrophages may be driving the profibrotic phenotype in kidneys with the delayed deletion of endothelial S1P1. Finally, the cytokine profile in IRI kidneys with the delayed deletion of S1P1 is both profibrotic and proinflammatory (Il1b, Tnfa, Il6, Cxcl1, Ccl2), which may be due to a feed forward loop to promote increased inflammation. Future studies would be needed to determine the cellular origin of these cytokines and further define the specific autocrine and/or paracrine pathway inducing inflammation and fibrosis.

Activation of the S1P1 signaling pathway can increase VE‑cadherin expression/function resulting in adherens junctional stability, and maintenance or enhancement of barrier integrity.22 Injury to the renal microvascular endothelium alters barrier function, and the postnatal deletion of S1P1 in endothelial cells prior to IRI increases vascular permeability in sham mice and after 24 hours of reperfusion.17 Alterations in barrier function are thought to return to normal within 48 hours of AKI.25 In this study, tamoxifen was administered at day 3 after IRI, likely circumventing early disruptions in barrier integrity. Under physiologic conditions, there was no difference in vascular leak in the kidney, liver, and intestine. There was a slight decrease in vascular leak in the lung with the deletion of S1pr1 in endothelial cells, and a trend toward increased permeability in the heart, unlike the previous report.17 Likely, there was not increased kidney vascular leak due the incomplete loss of S1pr1 in our iTie2CreERT2 S1pr1fl/fl mice. Interestingly, we observed an increase in kidney VE‑cadherin transcript and protein expression with the delayed deletion of endothelial S1P1 at day 9 after IRI. One possible explanation for this observation is that endothelial barrier integrity might have been altered with the loss of S1P1 earlier than day 9 and there may be compensatory mechanisms by neighboring cells without S1P1 deleted to strengthen barrier function by day 9. Alternatively, the endothelium may strengthen barrier function to preserve integrity during inflammation and leukocyte transmigration in IRI kidneys of mice with the delayed deletion of S1P1.

Recent studies have begun to elucidate the importance of the endothelium in the initiation and progression of fibrosis. Studies on human biopsies have associated microvascular rarefaction with progressive renal failure.5 Renal endothelial injury after AKI can result in a reduction in capillary density, called capillary rarefaction, which may be due to a lack of upregulation of vascular endothelial growth factor in response to injury.26,27 However, we did not observe a reduction in capillary density, a difference in Vegfa transcript levels, or a latent endothelium 9 days after IRI in kidneys of mice with the delayed deletion of endothelial S1P1 after IRI. Capillary rarefaction may require a more severe ischemic injury than 24 minutes of clamp time and/or require a longer time than 9 days to manifest. There were also increased transcript levels of Nos3 in kidneys of mice with the delayed deletion of endothelial S1P1. Taken together, these data suggest that the increased fibrosis in kidneys of mice with the delayed deletion of endothelial S1P1 is not due to capillary rarefaction and that there may be sustained vasorelaxation to maintain capillary perfusion.

Work from our lab and others have demonstrated that S1P1 signaling is necessary for protection from AKI in various cell types. Administration of the selective S1P1 agonist, SEW2871, or the nonselective agonist that is approved for treating patients with relapsing/remitting multiple sclerosis, FTY720, protects from IRI and acute inflammation.28,29 The protective effects of FTY720 or SEW2871 are due to S1PR expression on proximal tubule cells, and S1P1 protects tubule cells from apoptosis during AKI.18,30 The findings from our current study and others17 demonstrate that S1P1 activation in endothelial cells is also protective from IRI-induced AKI. These data suggest that patients at high risk for AKI may benefit from a prophylactic approach to prevent AKI.31 Moreover, our findings suggest that activation of S1P1 could also be a therapeutic approach to suppress endothelial activation and inflammation after AKI.

In summary, we identify endothelial S1P1 as a protective factor against kidney injury and necessary for recovery of kidney function. S1P1 orchestrates the kidney microenvironment by suppressing endothelial cell activation of leukocyte adhesion molecules and subsequent inflammation to prevent maladaptive repair and tubular atrophy in protection against fibrosis.

Concise Methods

Mice and Tamoxifen Administration

Experiments were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and all procedures were approved by the University of Virginia Animal Care and Use Committee. Twelve to fourteen–week-old male mice on a pure C57BL6/J background (at least 10 generations of backcrossing) were used for all experiments. iTie2CreERT2 mice were generously provided by the German Cancer Research Center (DKFZ) and were bred with S1pr1fl/fl mice (generously provided by Dr. Richard L. Proia, NIH) to establish iTie2CreERT2 S1pr1fl/fl mice and pedigreed colony controls (iTie2CreERT2 S1pr1wt/wt mice), or were bred with (Gt)Rosa26Sor floxed-stop tdTomato reporter mice (Jackson ImmunoResearch Laboratories; Ai9 mice). Mice were maintained in standard vivarium housing on a chow diet and water was freely available. Tamoxifen (Sigma-Aldrich) was dissolved in 5% ethanol in corn oil and administered intraperitoneally to all mice at 40 mg/kg per day, as described elsewhere.10 Nonperfused, necrotic kidneys were observed in ~ 10% of iTie2CreERT2 S1pr1fl/fl mice that underwent IRI. These mice were excluded from all analysis. Genotyping primers and strategy have been previously described.9,18

IRI

Mice were anesthetized with an intraperitoneal injection of a ketamine (120 mg/kg), xylazine (12 mg/kg), and atropine (0.324 mg/kg) mixture, and placed on a warm pad to maintain body temperature at 34–35°C. Mice were then randomized to sham or IRI operation. Bilateral or unilateral flank incision was performed and either both renal pedicles or only the left pedicle was cross clamped for 24 minutes. A 24—minute clamp time was selected to produce a subthreshold, moderate ischemic injury with little to no rise in PCr, and we hypothesized that endothelial loss of S1P1 would result in exacerbated IRI. We chose unilateral IRI for recovery experiments based on the observed severity of injury in bilateral IR experiments and anticipated that the iTie2CreERT2 S1pr1fl/fl mice would not survive without a healthy kidney. Additionally, unilateral ischemia is a characterized model for tubule repair and fibrosis.32–34 In our laboratory, kidneys that underwent 24 minutes of unilateral ischemia were able to recover kidney function within 9 days. Sham-operated mice underwent the same procedure with the exception of pedicle clamping. Surgical wounds were closed and buprenorphine (0.15 mg/kg, intraperitoneally) was administered as an analgesic. In unilateral IRI experiments, mice were reanesthetized and subjected to right kidney nephrectomy 24 hours prior to euthanizing to reveal changes in kidney function of the operated, ischemic (left) kidney assessed by measuring PCr. Mice were euthanized by isoflurane overdose and cervical dislocation for confirmation of death. Both the ischemic (IRI) and unoperated (nephrectomized; contralateral control) kidneys were saved for analysis.

Assessment of Kidney Function and Histology

Blood was collected under isoflurane anesthesia from the retro-orbital sinus, and PCr (mg/dl) was determined by Jaffe reaction (Figure 2B; Supplemental Figure II) following the manufacturer’s protocol (Sigma-Aldrich), or by using an enzymatic method with minor modifications from the manufacturer’s protocol (twice the volume of sample; Diazyme Laboratories). For creatinine clearance in mice under physiologic conditions, mice were placed in metabolic cages on day 5 of tamoxifen injection, and urine was collected over a 24-hour period. Animals were then euthanized on day 6. Hematoxylin and eosin staining and stereological analysis were performed as previously described.35 For picrosirius red staining, kidneys were fixed in 4% PLP (4% paraformaldehyde, 1.4% DL-lysine, 0.2% sodium periodate in 0.1 M phosphate buffer, pH 7.4) for 5 hours, changed to 70% ethanol, and subsequently embedded in paraffin and sectioned at 4‑μm thick. Sections were dewaxed, rehydrated, and stained in picrosirius red solution (Electron Microscopy Services) for 1 hour. After washing in acidified water, sections were dehydrated in three changes of 100% ethanol, cleared with xylene, and mounted in resinous medium. Photomicrographs of picrosirius red birefringence were captured using a Zeiss AxioImager microscope with polarizing filter (Carl Zeiss GmbH) and Stereo Investigator software (version 9; MBF Bioscience). Quantification of the fibrotic area was done by measuring total red/yellow birefringent pixels per total kidney area with ImageJ software (http://imagej.nih.gov/ij/).

Immunofluorescence

Kidneys were fixed in 1% PLP for 5 hours, incubated in 5% sucrose overnight, 15% sucrose for 3 hours, and 30% sucrose for 2 hours at 4°C, embedded and frozen in Optimal Cutting Temperature compound (Ted Pella, Inc.). Tissues were sectioned 5-μm thick and permeabilized with 0.3% Triton X‑100. Nonspecific binding was blocked with 10% horse serum and anti-mouse CD16/32 (clone 2.4G2; eBioscience). Sections were then labeled with primary antibodies at 2 μg/ml in 10% horse serum in PBS: rat anti-CD31 A647 or A555 (clone MEC 13.3, BioLegend), rabbit anti-αSMA A647 (polyclonal, Thermo Fisher Scientific), goat anti-collagen I and anti-collagen III A647 (Southern Biotech), rat anti-PDGFRβ A555 or A647 (clone APB, BioLegend), rat anti-Tie2 PE (clone TEK4, eBioscience), rat anti-CD45 FITC (clone 30F11, eBioscience), rat anti-CD144 A555 (BV13, BioLegend), rat anti-F4/80 A647 (clone BM8, Invitrogen), and rat anti-neutrophil PE (clone 7/4, Cedarlane). For assessment of latent versus patent vasculature experiments, mice were injected with 100 µl of 1 mg/ml Lycopersicon esculentum lectin conjugated with Texas-Red (Vector Laboratories, Burlingame, CA), which will specifically bind via glycoprotein interactions to any endothelium in perfused blood vessels,36 via tail vein 5 minutes prior to euthanasia. Coverslips were applied with ProLong Gold Antifade with 4′,6-diamidino-2-phenylindole to detect nuclei (Life Technologies), and individual channel images were acquired using the Carl Zeiss Axiovert 200 microscopy system with Apotome imaging and Axiovision software (version 4.8.2; Carl Zeiss Microscopy, LLC). For quantitation, four to five representative images per kidney were acquired, and the CD144+ or CD31+ pixel area was divided by total area to determine the percentage of total CD144+ or CD31+ immunoreactivity per field of view. To determine capillary density, CD31 labeling was skeletonized using the binary function and quantified.37 For lectin staining, the percentage of CD31+ area that was also lectin+ was quantified. Color was applied to grayscale monochrome images, and total brightness and contrast were adjusted equally across the entire image and among images of the same channel. All image quantification and adjustments were performed with NIH ImageJ (http://imagej.nih.gov/ij).

Flow Cytometry

Kidney single-cell suspensions were prepared as previously described.23 Single-cell suspensions were blocked for nonspecific Fc binding with anti-mouse CD16/32 (eBiosciences, clone 2.4G2) and labeled with the following primary antibodies directly conjugated to fluorophores: anti-CD106 (1 µg/ml, clone 429), Ly6G (2 µg/ml, clone 1A8), CD11b (2 µg/ml, clone M1/70), F4/80 (2 µg/ml, clone Cl:A3-1), CD11c (2 µg/ml, clone N418), MHCII (2 µg/ml, clone M5/114.15.2), CD3 (2 µg/ml, clone 145-2C11), CD19 (1 µg/ml, clone 1D3) (BioLegend), CD45 (1 µg/ml, clone 30F11) (eBioscience), and Ly6C (0.7 µg/ml, clone AL-21) (BD Biosciences). 7AAD (Life Technologies) was used to discriminate live/dead cells and quantitation of cell number was determined using counting beads as per the manufacturer’s protocol (CountBright, Life Technologies). Data were acquired on a FACS Calibur cytometer (Becton Dickinson [BD]) with Cytek 8 color flow cytometry upgrade (Cytek Development, Inc.) and analyzed with FlowJo software 9.0 (TreeStar, Inc.). The gating strategy for myeloid subsetting has been previously described23 and is shown in Supplemental Figure VI. Gates were determined using fluorescence minus one control samples.

Vascular Permeability

Vascular permeability by Evan’s Blue extravasation was assessed in kidney, liver, lung, and spleen using a modified protocol.38 A 1.0% Evans Blue Dye (Sigma-Aldrich E-2129) solution was made in formamide and 30 mg/kg was administered into the vasculature via retro-orbital injection. One hour later, the mouse was perfused with PBS containing 1 U/ml heparin. Organs were removed and dried at 60°C for 18 hours. Dried tissue was chopped in formamide and incubated for 18 hours at 60°C. After centrifugation at 10,000×g for 20 minutes, supernatants were measured at an absorbance of 620 nm and at 740 nm for reference.

Immunoperoxidase Staining

For CD31 detection, paraffin-embedded kidney tissue sections were deparaffinized, rehydrated, and unmasked. Sections were blocked with 4% horse serum and avidin block (Vector Laboratories) for 1 hour, and subsequently incubated with CD31 (goat polyclonal, sc-1506, Santa Cruz Biotechnology) at 1:200 overnight with biotin block (Vector Laboratories). Sections were then incubated with biotinylated horse anti-goat IgG (5 µg/ml, Vector Laboratories) for 1 hour, and a streptavidin–peroxidase complex (Vectastain ABC kit, Vector Laboratories) and peroxidase reaction product were revealed according to the manufacturer’s instructions (Vector Laboratories DAB Peroxidase substrate kit). Sections were then counterstained with hematoxylin QS (Vector Laboratories H-3404), dehydrated, and mounted with Cytoseal 60.

Cell Culture

Mouse glomerular endothelial cells, a gift from Dr. Michael Madaio (Medical College of Georgia, Augusta, Georgia), were cultured as previously described.39,40 Cells with stable overexpression of S1pr1 were generated as previously described.30 Confluent cells were treated with vehicle (PBS+0.1% BSA), 1 ng/ml murine recombinant IL1β (Peprotech), or 10 ng/ml murine recombinant TNFα (Sigma-Aldrich, T1539) for 48 hours, then cells were washed and either harvested for RNA extraction or trypsinized for single-cell suspensions for flow cytometry.

Statistical Analyses

First, a normality test was used to determine if the data fit a Gaussian distribution. When comparing two normally distributed data sets with equal variance, a t test was used. For data with unequal variance, a t test with a Welch correction was used. For non-Gaussian distributed data, a Mann–Whitney test was performed to determine statistical significance. A two-way ANOVA was used to compare means between groups with two independent variables, and a Tukey’s multiple comparisons test post hoc analysis was used to correct for multiple comparisons. If data had unequal variance, a natural log transformation was applied prior to two-way ANOVA. Data were analyzed using Prism 6.0b (GraphPad Software Inc.). Results are displayed containing all replicated experiments, and values shown are mean only or mean ± SEM P-values <0.05 were considered statistically significant.

Disclosures

None.

Acknowledgments

We thank the University of Virginia Research Histology Core for their assistance in preparation of histology slides.

Research conducted for this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award Nos. R01DK085259, R01DK062324 (M.D.O.), T32DK 72922 (H.M.P.), K01DK091444 (A.B.), by the American Society of Nephrology Ben Lipps Fellowship Program (H.M.P.), by a National Kidney Foundation Fellowship (A.B.) and by an American Heart Association career development grant under award No. 11SDG7000007 (A.B.). The stereology data described here was gathered on an “MBF Bioscience and Zeiss microscope system for stereology and tissue morphology” funded by National Institutes of Health grant 1S10RR026799-01 (M.D.O).

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

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Endothelial Sphingosine 1‑Phosphate Receptor‑1 Mediates Protection and Recovery from Acute Kidney Injury
Heather M. Perry, Liping Huang, Hong Ye, Chong Liu, Sun-sang J. Sung, Kevin R. Lynch, Diane L. Rosin, Amandeep Bajwa, Mark D. Okusa
JASN Nov 2016, 27 (11) 3383-3393; DOI: 10.1681/ASN.2015080922
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