缺血再灌注損傷與s1p腎2013kia

1、basic r esearch & 2013 International Society of Nephrologysee commentary on page 733Selective deletion of the endothelialsphingosine-1-phosphate 1 receptor exacerbates kidney ischemiareperfusion injuryAhrom Ham1, Mihwa Kim1, Joo Yun Kim1, Kevin M. Brown1, Marcus Fruttiger2, Vivette D. DAgati3 an

2、dH. Thomas Lee11Department of Anesthesiology, Anesthesiology Research Laboratories, College of Physicians and Surgeons of Columbia University, Columbia University, New York, New York, USA; 2UCL Institute of Ophthalmology, University College London, London, UK and 3Department of Pathology, College of

3、 Physicians and Surgeons of Columbia University, New York, New York, USAThe role for the endothelial sphingosine-1-phosphate 1receptor (S1P1R) in acute kidney injury (AKI) remains unclear as germline endothelial S1P1R deletion is embryonically lethal. Here, we generated conditional endothelial S1P1R

4、 deficiency by crossing mice with floxed S1P1R with mice expressing a tamoxifen-inducible form of Cre recombinase under the transcriptional control of the platelet-derived growth factor-b (Pdgfb) gene. Mice with tamoxifen-induced deletion of endothelial S1P1R had increased renal tubular necrosis, in

5、flammation, and impaired vascular permeability, as well as exacerbated renal tubular apoptosis after ischemic AKI compared with tamoxifen-treated wild-type mice.Moreover, endothelial S1P1R deletion resulted in increased hepatic injury after ischemic AKI. As a potential mechanism for exacerbated rena

6、l injury, conditional endothelial S1P1Rnull mice had markedly reduced endothelial HSP27 expression compared with wild-type mice. Cultured glomerular endothelial cells treated with a specific S1P1R antagonist (W146) for 3 days also showed reduced HSP27 expression compared with vehicle-treated cells.

7、Finally, mice treated with W146 for 3 days also showed reduced endothelial HSP27 expression as well as exacerbated renal and hepatic injury after ischemic AKI. Thus, our studies demonstrate a protective role for endothelial S1P1R ischemic AKI most likely by regulating endothelial barrier integrity a

8、nd endothelial HSP27 expression.Kidney International (2014) 85, 807823; doi:10.1038/ki.2013.345;published online 11 September 2013KEYWORDS: apoptosis; Cre recombinase; inflammation; necrosis; tamoxifenRenal ischemia and reperfusion (IR) injury is a major cause of perioperative acute kidney injury (A

9、KI) for patients under- going major vascular, cardiac, or transplantation surgery and is directly associated with high mortality, morbidity, and cost.1,2 Furthermore, patients who suffer from AKI also frequently develop extrarenal organ dysfunction including hepatic dysfunction, intestinal barrier d

10、isruption, respiratory failure, and the systemic inflammatory response syndrome with the eventual development of sepsis and multiorgan failure.3,4 These extrarenal systemic complications secondary to AKI are the leading causes of mortality in the intensive care unit.5 Unfortunately, the severity and

11、 incidence of AKI have been increasing without effective therapy or improvements in patient survival for the past 60 years.6Sphingolipids are integral components of the plasma membrane and modulate diverse pathways of cell death.7,8including necrosis, apoptosis, inflammation, and immuIn particular,

12、sphingosine-1-phosphate (S1P), produced by phosphorylation of sphingosine by sphingosine kinases, is the natural ligand for a family of five lysophospholipid-targeted G-protein-coupled receptors (S1P15Rs) and is a powerful modulator of cell death and stress.8 Encouraging preclinical studies suggest

13、that modulation of S1PRs in renal proximal tubule cells reduces ischemic AKI. Specifically, we and others showed previously that activation of S1P1R or inhibition of S1P2R in renal proximal tubules provided powerful renalischemic AKI in mice.9,10However, unlike the well-characterized role for proxim

14、altubule S1P1R modulationischemic AKI, the exact roleof endothelial S1P1R in ischemic AKI remains unclear as germline global endothelial S1P1R deletion results in embryo- nic lethality. Furthermore, the role of S1P in modulating endothelial cell function and survival remains controversial. Some stud

15、ies suggest that endothelial S1P serves to strengthen the vascular endothelial cell barrier, whereas other studies suggest detrimental, proinflammatory roles for S1P in endo- thelial cells.1113In this study, we aimed to test whether endothelial S1P1Rs Correspondence: H. Thomas Lee, Department of Ane

16、sthesiology, Anesthe-siology Research Laboratories, College of Physicians and Surgeons of Columbia University, Columbia University, P&S Box 46 (PH-5), 630 West 168thStreet, New York, New York 10032-3784, USA.: Received 25 May 2013; revised 8 July 2013; accepted 12 July 2013;publ

17、ished online 11 September 2013have a protective roleAKI because of renal IR injury.807Kidney International (2014) 85, 807823bas ic r es earch A Ham et al.: Endothelial S1P1R protectsischemic AKIInducible endothelial S1P1R-null micePdgfbiCreER (450 bp) S1P1R flox (250 bp)S1P1R WT (200 bp)Wild typeGFP

18、Nuclear stainMerge50 mm50 mm50 mm50 mm50 mm50 mmFigure 1 | Generation of mice with conditional endothelial-specific sphingosine-1-phosphate 1 receptor (S1P1R) deletion. (a) Tailotypes of conditional endothelial-specific S1P1R-null (S1P1Rf/f PdgfbiCreER or S1P1Rf/ polymerase chain reaction (PCR) conf

19、irtPdgfbiCreER) mice and the wild-type (S1P1Rf/f or S1P1Rf/ ) mice. (b) As PdgfbiCreER mice express an internal ribosomal entry site element and a sequence coding for enhanced green fluorescent protein (EGFP), we detected the expression of EGFP in kidney endothelial cells from in S1P1Rf/fPdgfbiCreER

20、 mice with fluorescent microscopy. EGFP expression was strong in endothelial cells from kidneys of S1P1Rf/f PdgfbiCreER mice (arrow). Besides faint autofluorescence from external elastic membrane, EGFP expression was not detected in S1P1Rf/f mice kidney endothelial cells. We also performed 40,6-diam

21、idino-2-phenylindole (DAPI) nuclear staining in both S1P1Rf/f PdgfbiCreER mice and in S1P1Rf/f mice (blue).lines14,15To do this, we generated mice with an inducible deletion of endothelial S1P1R by crossing mice with floxed S1P1R (S1P1Rf/f) with mice that express a tamoxifen-inducible form of Cre re

22、combinase (iCreERT2) under the transcriptional control of the platelet-derived growth factor-b (Pdgfb) gene. We subjected these mice as well as wild-type (S1P1Rf/f) mice to mild (20 min) renal IR injury. We also explored the potential mechanisms for exacerbated renal injury in endothelial S1P1Rnull

23、mice subjected to renal IR injury. As S1P1R modulates heat-shock protein 27 (HSP27) expressionin some celland as HSP27 is a well-known cyto-protective protein in endothelial cells,16 we also tested thehypothesis that genetic deletion or chronic blockade of S1P1R decreases endothelial HSP27 expressio

24、n.RESULTSGeneration of conditional endothelial S1P1Rdeficient mice To conditionally delete S1P1R in endothelial cells of adult mice, we crossed floxed S1P1R (S1P1Rf/f) mice with mice that express a platelet-derived growth factor-b (Pdgfb) promoter808Kidney International (2014) 85, 807823S1P1Rf/f Pdg

25、fbiCireER miceS1P1Rf/f miceS1P1Rf/fS1P1Rf/S1P1Rf/f PdgfbiCreERS1P1Rf/PdgfbiCreERNaive mice´ 400Genotyof inducible endothelial specific S1P1R-null micebasi c r es earch A Ham et al.: Endothelial S1P1R protectsischemic AKIfrom our breeding (Figure 1a). As the PdgfbiCreER mice also express an inte

26、rnal ribosomal entry site element and a sequence coding for enhanced green fluorescent protein (EGFP), we detected kidney endothelial cell EGFP expressionin S1P1Rf/fPdgfbiCreERmice with fluorescent microscopyGAPDH(Figure 1b).17 EGFP expression was strong in kidney endo-thelial cells from S1P1Rf/f Pd

27、gfbiCreER mice but was not visible (beyond faint background autofluorescence from external elastic membrane) in kidney sections from S1P1Rf/f mice.S1P1Rf/f PdgfbiCreER mice were treated daily with 5 mg/kg tamoxifen (intraperitoneally) for 3 days for Cre-recombina- semediated deletion of endothelial

28、S1P1R. S1Pf/f mice were also treated with tamoxifen for 3 days and used as controls. We confirmed deletion of endothelial S1P1R in S1P1Rf/f PdgfbiCreERS1P R1S1P R2S1P3R S1P4RS1P5RProximal tubule cellsEndothelial cellsmice with RT-PCR. S1P1 5R mRNA levels infreshly isolated endothelial cells (prepare

29、d with magneticbeadcoupledbody capture) and proximal tubules(prepared with percoll gradient separation) were measured with conventional and real-time RT-PCR. A representativeProx S1P1Rf/fProx S1P1Rf/f PdgfbiCreERgel image of PCR products and the qufied mRNA levels(Figure 2) show a markedly decreased

30、 S1P1R mRNA (B90%)End S1P Rf/f1in endothelial cells isolated from kidneys of S1P Rf/f1f/fEndo S1P Rf/f PdgfbiCreERiCreER1Pdgfbmice compared with the wild-type (S1P1R )1.25mice endothelial cells. Other S1PR subtypes (S1P25R) did not change with conditional endothelial S1P1R deletion(Figure 2). Figure

31、 2 also shows that renal proximal tubular S1P15R mRNA expression was similar between tamoxifen- treated S1P1Rf/f PdgfbiCreER mice and S1P1Rf/f mice.We also performed immunohistochemistry for kidney S1P1R and for CD34 (an endothelial cell marker) in1.000.750.50tamoxifen-treated S1P1Rf/fPdgfbiCreERand

32、 S1P1Rf/f mice.Figure 3 shows representative images for S1P1R and CD34 immunoreactivity in the kidney. Figure 3a shows images0.25*from kidney blood vessels and Figure 3b shows images of immunohistochemistry images for peritubular capillaries and glomeruli. As shown in Figure 3a, S1P1R immunoreactivi

33、ty was markedly attenuated in kidney blood vessels from tamoxifen-treated S1P1R Pdgfb mice when compared with S1P1Rf/f mice. CD34 immunoreactivity was similar0.00S1P1RS1P2RS1P3RS1P4RS1P5RFigure 2 | Conditional deletion of renal endothelial sphingosine- 1-phosphate 1 receptor (S1P1R) in S1P1Rf/f Pdgf

34、biCreER mice.S1P15R mRNA levels measured with reverse transcription-polymerase chain reaction (RT-PCR) in freshly isolated renal endothelial cellsf/fiCreERbetween tamoxifen-treated S1P1Rf/fPdgfbiCreER(with Dynabeads coupled to CD144body) and renal proximalmice andtubules (with Percoll density gradie

35、nt separation) from conditional S1P1R-null mice (S1P1Rf/f PdgfbiCreER) and wild-type mice (S1P1Rf/f).(a) Representative gel image of PCR products and (b) densitometricS1P Rf/f mice in kidney blood vessels. Furthermore, Figure 3b1shows that S1P1R is robustly expressed in peritubular capillaries and g

36、lomeruli from S1P1Rf/f mice but not from S1P1Rf/f PdgfbiCreER mice. Equivalent CD34 immunoreactiv- ity was detected in peritubular capillaries and glomeruli fromqufication of relative band intensities normalized toglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (N ¼ 4).Endothelial cells from c

37、onditional endothelial S1P1R-null mice show significantly (B90%) reduced S1P1R mRNA expression comparedS1P Rf/f mice and S1P Rf/f PdgfbiCreER mice. These findingswith wild-type mice without any changes in other S1PR subtype11are consistent with previous studies that pericytes and smooth muscle cells

38、 do not produce Pdgfb in vivo.17 S1P1R immunoreactivity was positive in kidney leukocytes from conditional endothelial S1P1Rnull mice (Figure 3).(S1P25R) mRNA expression (N ¼ 4). Furthermore, proximal tubuleS1P1R mRNA levels were similar between conditional endothelialS1P1Rnull mice and wild-ty

39、pe mice. *Po0.05 versus S1P1Rf/f mice.driven by a tamoxifen-inducible form of Cre-recombinaseEndothelial S1P1R deletion exacerbates renal and hepaticinjury after ischemic AKI in miceNext, we tested whether endothelial S1P1R protects ischemic AKI and remote hepatic injury in mice. We pre- viously sho

40、wed that ischemic AKI also results in hepatic(iCreERT2)invascularendothelialcells(PdgfbiCreERmice).17,18 Tail polymerase chain reaction (PCR) provided genotypes of conditional endothelial S1P1Rnull (S1P1Rf/fPdgfbiCreER)mice and wild-type (S1P1Rf/f) mice generated809Kidney International (2014) 85, 80

41、7823Fold S1PR mRNA/GAPDHover S1P1Rf/f miceMice were injected with 5 mg/kg tamoxifen daily for 3 daysbas ic r es earch A Ham et al.: Endothelial S1P1R protectsischemic AKInecrosis, vacuolization, and elevated plasma transaminase levels in mice.19 Plasma creatinine and alanine amino- transferase (ALT)

42、 values were similar between sham- operated (anesthesia, laparotomy, right nephrectomy, andby significantly higher plasma creatinine and ALT levels compared with S1P1Rf/f mice subjected to renal IR.We also assessed renal function by estimating glomerular filtration rate 24 h after renal IR injury in

43、 mice with the fluorescein isothiocyanate-inulin clearance technique as described by Qi et al.20 The glomerular filtration rate inrecovery) tamoxifen-treated S1P1Rf/fPdgfbiCreERmice andS1P1Rf/f mice (Figure 4a). As expected, plasma creatininetamoxifen-treated S1P1Rf/fPdgfbiCreERand ALT levels increa

44、sed mildly but significantly in tamoxi- fen-treated wild-type (S1P1Rf/f) mice 24 h after mild (20 min) renal IR. However, conditional endothelial S1P1Rdeficientmice subjected torenal IR was lower (0.22±0.08 ml/min per 100 g bodyweight, N ¼ 3) than the glomerular filtration rate in(S1P1Rf/f

45、PdgfbiCreER)tamoxifen-treated S1P1Rf/f mice subjected to renal IR (0.66±0.21 ml/min per 100 g body weight, N ¼ 3).Chronic S1P1R blockade exacerbates renal and hepatic injury after ischemic AKI in miceWe then tested the effects of chronic S1P1R blockade (W146 treatment daily for 3 days) in

46、C57BL/6 mice subjected to sham operation or to 20 min renal IR injury. Again, sham- operated vehicle-treated and W146-treated mice had similar baseline renal and hepatic markers of injury (Figure 4b). Vehicle-treated mice showed increased renal and hepatic injury after 20 min renal IR. However, chro

47、nic W146-treated mice had significantly elevated plasma markers of renal and hepatic injury.mice subjected to renal IR hadsignificantly increased renal and hepatic injury as evidenced50 mm50 mmEndothelial S1P1Rdeficient mice have increased kidneynecrosis, neutrophil infiltration, and apoptosis and l

48、iver necrosis and vacuolization after renal IRFigure 5a shows representative hemtoxylin and eosin (H&E) images (from six experiments) of tamoxifen-treated mice subjected to sham operation or to 20 min renal ischemia and24 h of reperfusion (original magnification, 200). Sham-operated tamoxifen-tr

49、eated wild-type (S1P1Rf/f) mice and tamoxifen-treated endothelial S1P1Rdeficient mice had normal renal histology (Figure 5a). Compared with sham- operated S1P1Rf/f mice, the kidneys of tamoxifen-treated S1P1Rf/f mice subjected to renal IR showed moderate tubular 50 mm 50 mm S1P Rf/f miceS1P Rf/f Pdg

50、fbiCreER mice11Figure 3 | Reduced kidney endothelial sphingosine-1-phosphate 1 receptor (S1P1R) protein expression in conditional endothelial S1P1Rnull (S1P1Rf/f PdgfbiCreER) mice. (a) Representative images(original magnification, 600) for S1P1R and CD34 immunoreactivity(brown stain, arrows) in kidn

51、ey blood vessels from tamoxifen-treated S1P1Rf/f PdgfbiCreER and S1P1Rf/f mice. S1P1R immunoreactivity was markedly reduced (near absent) in tamoxifen-treated S1P1Rf/f PdgfbiCreER mice when compared with S1P1Rf/f mice. S1P1R immunoreactivity was still visible from intravascular leukocytes in conditi

52、onal endothelial S1P1Rnull mice. CD34 immunoreactivity was similar between tamoxifen-treated S1P1Rf/f PdgfbiCreER mice and S1P1Rf/f mice. Representative of four experiments. (b) Representative50 mm50 mmimages (original magnification, 600) for S1P1R and CD34immunoreactivity (brown stain, arrows) in p

53、eritubular capillaries and glomeruli from tamoxifen-treated S1P1Rf/f PdgfbiCreER and S1P1Rf/fmice. S1P1R immunoreactivity was absent in peritubular capillaries and glomeruli from tamoxifen-treated S1P1Rf/f PdgfbiCreER mice when compared with S1P1Rf/f mice. Again, leukocyte S1P1R immunoreactivity was

54、 visible in kidneys from conditional endothelial S1P1Rnull mice. CD34 immunoreactivity was similar between tamoxifen-treated S1P1Rf/f PdgfbiCreER mice and S1P1Rf/f mice.Representative of four experiments.50 mm50 mmS1P1Rf/f miceS1P1Rf/f PdgfbiCreER mice810Kidney International (2014) 85, 807823CD34S1P

55、1RCD34S1P1RMice injected with 5 mg/kg tamoxifen 72 h prior´ 600Mice injected with 5 mg/kg tamoxifen 72 h prior´ 600basi cr es earch A Ham et al.: Endothelial S1P1R protectsischemic AKI3#*3#*22* 1100#*150#*200150100*100505000Figure 4 | Endothelial sphingosine-1-phosphate 1 receptor (S1P1R)

56、deficiency or chronic S1P1R antagonist treatment exacerbates renal and hepatic injury after renal ischemiareperfusion (IR). (a) Plasma markers for renal and hepatic injury from tamoxifen-treated endothelialS1P1Rnull (S1P1Rf/f PdgfbiCreER) mice or wild-type (S1P1Rf/f) mice subjected to sham operation

57、 or to 20 min of renal ischemia and 24 hreperfusion (N ¼ 57). Endothelial S1P1R deletion significantly increased renal and hepatic injury in mice. (b) Plasma markers for renal andhepatic injury from vehicle- or W146-treated (0.1 mg/kg daily for 3 days) C57BL/6 mice subjected to sham operation or to 20 min of renalischemia and 24 h of reperfusion (N ¼ 6). Chronic S1P1R antagonist treatment increased renal and hepatic injury after renal IR injury. *Po0.05versus respective sham-operated mice. #Po0.05 versus wild-