Loss of diacylglycerol kinase ε causes thrombotic microangiopathy by impairing endothelial VEGFA signaling

Loss of function of the lipid kinase diacylglycerol kinase ε (DGKε), encoded by the gene DGKE, causes a form of atypical hemolytic uremic syndrome that is not related to abnormalities of the alternative pathway of the complement, by mechanisms that are not understood. By generating a potentially novel endothelial specific Dgke-knockout mouse, we demonstrate that loss of Dgke in the endothelium results in impaired signaling downstream of VEGFR2 due to cellular shortage of phosphatidylinositol 4,5-biphosphate. Mechanistically, we found that, in the absence of DGKε in the endothelium, Akt fails to be activated upon VEGFR2 stimulation, resulting in defective induction of the enzyme cyclooxygenase 2 and production of prostaglandin E2 (PGE2). Treating the endothelial specific Dgke-knockout mice with a stable PGE2 analog was sufficient to reverse the clinical manifestations of thrombotic microangiopathy and proteinuria, possibly by suppressing the expression of matrix metalloproteinase 2 through PGE2-dependent upregulation of the chemokine receptor CXCR4. Our study reveals a complex array of autocrine signaling events downstream of VEGFR2 that are mediated by PGE2, that control endothelial activation and thrombogenic state, and that result in abnormalities of the glomerular filtration barrier.


Introduction
Atypical hemolytic uremic syndrome (aHUS) is a form of thrombotic microangiopathy (TMA) characterized by hemolytic anemia, thrombocytopenia, and acute renal failure not associated with enteric infections caused by Shiga toxin-producing bacteria. TMA in aHUS affects invariably the microvasculature in the renal glomeruli, causing renal insufficiency. Mutations in complement and complement-regulating genes have been identified in about 50% of idiopathic aHUS cases, pointing at the central role of the complement cascade in this disease (1). Notwithstanding, we and others described a genetic form of aHUS that is caused by mutations in the gene DGKE that encodes the lipid kinase diacylglycerol kinase ε (DGKε), revealing that factors unrelated to the complement system can cause aHUS by mechanisms that are not understood (2,3).
Here, based on in vitro studies on primary endothelial cells and on the use of an endothelial specific Dgke-knockout mouse, we show that the signaling downstream of VEGFR2 is compromised in the absence of DGKε in the endothelium because of impaired activation of Akt and that the latter is restored by increasing cellular levels of PIP2. Mechanistically, we found that defective Akt activation in endothelial cells results in lack of expression of the gene prostaglandin-endoperoxide synthase 2, which encodes the enzyme cyclooxygenase 2 (Cox2) and induces the synthesis of its main product prostaglandin E 2 (PGE 2 ).
Loss of function of the lipid kinase diacylglycerol kinase ε (DGKε), encoded by the gene DGKE, causes a form of atypical hemolytic uremic syndrome that is not related to abnormalities of the alternative pathway of the complement, by mechanisms that are not understood. By generating a potentially novel endothelial specific Dgke-knockout mouse, we demonstrate that loss of Dgke in the endothelium results in impaired signaling downstream of VEGFR2 due to cellular shortage of phosphatidylinositol 4,5-biphosphate. Mechanistically, we found that, in the absence of DGKε in the endothelium, Akt fails to be activated upon VEGFR2 stimulation, resulting in defective induction of the enzyme cyclooxygenase 2 and production of prostaglandin E 2 (PGE 2 ). Treating the endothelial specific Dgke-knockout mice with a stable PGE 2 analog was sufficient to reverse the clinical manifestations of thrombotic microangiopathy and proteinuria, possibly by suppressing the expression of matrix metalloproteinase 2 through PGE 2 -dependent upregulation of the chemokine receptor CXCR4. Our study reveals a complex array of autocrine signaling events downstream of VEGFR2 that are mediated by PGE 2 , that control endothelial activation and thrombogenic state, and that result in abnormalities of the glomerular filtration barrier.
JCI Insight 2021;6(9):e146959 https://doi.org/10.1172/jci.insight.146959 Treating endothelial specific Dgke-knockout mice with a stable PGE 2 analog reversed not only the aHUS, but also the proteinuria, possibly through CXCR4-mediated downregulation of matrix metalloproteinase 2 (MMP-2). Our data indicate that the endothelium is the tissue that is primarily affected by loss of Dgke and reveal multiple complex autocrine signaling events downstream of VEGFR2 that result in endothelial activation, thrombogenic state, and abnormalities of the glomerular filtration barrier. These signaling steps may be manipulated to modify the clinical course of the DGKE-related aHUS and conceivably of other diseases caused by endothelial activation or abnormal angiogenesis.

VEGFR2-dependent Akt activation is compromised in DGKE-knockdown human umbilical vein endothelial cells and in human microvascular endothelial cells.
VEGFR2 is expressed in endothelial cells and exerts proproliferative, prosurvival, and promigratory functions, promoting angiogenesis upon stimulation by VEGFA (13)(14)(15)(16)(17). VEGFR2 signals downstream by autophosphorylating multiple tyrosine residues, where effector proteins are recruited (14). This results in, among other effects, activation of PLCγ, which then hydrolyzes PIP2 into inositol 1,4,5-triphosphate [Ins(1,4,5)P 3 , hereafter IP3] and DAG, and activation of phosphatidylinositol-3-kinase (PI3K), which leads to the generation of PIP3 from its precursor, PIP2, and ultimately to the activation of Akt ( Figure 1A) (18)(19)(20)(21)(22). Since VEGF signaling is crucial to the maintenance of the glomerular microvasculature (23,24), and because the disruption of VEGF signaling in humans and mice results in glomerular lesions that resemble those observed in humans with loss-of-function mutations in DGKE (2,25), we hypothesized that the loss of DGKE may impair VEGF signaling in endothelial cells because of the shortage of PIP2 ( Figure 1A). To test this hypothesis, we knocked down DGKE in human umbilical vein endothelial cells (HUVECs) by shRNA interference, using a nontargeting construct (sh-GFP) as a control  Figure 1B), and tested the activation of Akt by Western blot (phosphorylation of threonine 308 and serine 473 of Akt) upon VEGFA stimulation. Unlike untransfected and sh-GFP-transfected cells, DGKE-knockdown cells failed to activate Akt when exposed to VEGFA ( Figure 1C and Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.146959DS1). Consistent with the impaired Akt activation, the expression of Cox2, which in endothelial cells is induced by VEGFA (26)(27)(28)(29), was also compromised ( Figure 1D). However, Akt activation was partially rescued when cells were exposed to PIP2 and, as expected, to PIP3 ( Figure 1E and Supplemental Figure 1, C and D). Likewise, intracellular calcium levels that are mobilized from the endoplasmic reticulum by increased intracellular concentrations of IP3 did not rise after VEGFA stimulation, but they did increase after supplementing the culture medium with PIP2 ( Figure 1F). We obtained overlapping results using human microvascular endothelial cells (HMECs) (Supplemental Figure 2, A and B). These results indicate that Akt activation downstream of VEGFR2 is defective in DGKE-knockdown endothelial cells because of decreased availability of PIP2.
Endothelial specific Dgke-knockout mice fully recapitulate the human disease. To test if VEGFA signaling in endothelial cells lacking Dgke is also compromised in vivo, we generated a conditional knockout mouse in which exon 2 of Dgke was deleted in a tissue-specific manner upon Cre recombination, using knockout first embryonic stem (ES) cells from the Knockout Mouse Project (KOMP) repository (30,31). C57BL/6 Dgke-floxed mice are viable and fertile, and they produce progeny at the expected Mendelian ratio. We confirmed that exon 2 of Dgke is excised upon Cre recombination, by using the CMV-Cre mice to delete Dgke in all tissues (32) (Figure 2A). We also verified that the expression of the transgene is tissue specific, by generating Nphs2Cre Dgke LacZ knockin mice and showing that the LacZ expression was present exclusively in the glomeruli (30, 33) ( Figure 2B). To generate endothelial specific Dgke-knockout mice, Dgke-floxed mice were then crossed to mice in which the Cre recombinase is driven by the angiopoietin receptor Tie2 promoter in endothelial cells and in 2% to 7% of blood mononuclear cells (34). We assessed the knockout efficiency by real-time PCR (RT-PCR) on RNA extracted from Tie2Cre Dgke fl/fl lungs, which is one of the most vascularized tissues ( Figure 2C), given that no antibody is available that reliably detects Dgke. We excluded the possibility that abnormal platelet function due to expression of Tie2 in megakaryocytes could contribute to the observed phenotype by performing in vitro studies of platelet aggregation (Supplemental Figure 3). Unlike the constitutive Dgke-knockout mice that do not have spontaneous phenotype, endothelial specific Tie2Cre Dgke fl/fl conditional knockout mice developed occlusion of the glomerular capillaries (  Figure 2I), and renal insufficiency as early as 2 months of age ( Figure 2J). Remarkably, these mice also developed albuminuria ( Figure 2K), which is observed in all patients affected by DGKE disease, by 3 months of age. The presence of albuminuria in endothelial specific Tie2Cre Dgke fl/fl knockout mice and its appearance at a later time, compared with the renal insufficiency, suggest that the endothelium is the cellular compartment responsible for the DGKE disease and that the impairment of the glomerular barrier may be a later and secondary event. Interestingly, we did not detect obvious signs of TMA in liver, small intestine, and spleen arterioles of Tie2Cre Dgke fl/fl knockout mice (Supplemental Figure 5), which may indicate a particular susceptibility of the glomerular microvasculature to this disease.
A phosphatase and tensin homolog inhibitor rescues the phenotype of endothelial specific Dgke-knockout mice. As observed in the DGKE-knockdown cells, Akt phosphorylation was lower in kidney cortexes of Tie2Cre Dgke fl/ fl mice, compared with controls ( Figure 3A, lanes 1 through 6; and Supplemental Figure 1, E and F). Hence, we tested whether increasing the endothelial levels of PIP3 would rescue the phenotype in vivo. To this end, we crossed Tie2Cre Dgke fl/fl mice to Pten-floxed mice (35), to obtain Tie2Cre Dgke fl/fl Pten +/fl in which only 1 of the 2 Pten alleles is present in endothelial cells; however, we were not able to obtain pups of the desired genotype, likely because of in utero mortality. For this reason, we used a potent small molecule inhibitor of PTEN (VO-OHpic) (36), and injected 8-week-old Tie2Cre Dgke fl/fl mice i.p. daily for 4 weeks. Although schistocytosis was clearly evident in peripheral blood smears of vehicle-treated mice ( Figure 3, E and F, and Supplemental Figure 4C), the serum hemoglobin concentrations did not significantly differ from those of VO-OHpic-treated mice ( Figure 3G). These results indicate that Akt activation is impaired in kidneys of endothelial specific knockout mice and that increasing PIP3 cellular levels is sufficient to rescue the symptoms in Tie2Cre Dgke fl/fl knockout mice.
Overexpression of Cox2 in endothelial cells rescues the phenotype of Tie2Cre Dgke fl/fl mice. We previously reported that the expression of the gene prostaglandin-endoperoxide synthase 2, also known as cyclooxygenase 2 (Cox2), and the synthesis of its main product PGE 2 were impaired in Dgke constitutive knockout mice (37). However, still undetermined is if impaired Cox2 induction is only associated with or determines the phenotype in Dgke-knockout mice. To answer this question, we crossed Tie2Cre Dgke fl/fl mice with B6.129S4-Tg(CAG-EGFP,-Ptgs2,-hrluc) (Cox2-COE) mice to obtain Tie2Cre Dgke fl/fl Cox2-COE double transgenics. Cox2-COE mice were engineered to carry a transgene that contains a CAG promoter that drives the expression of Cox2. The promoter is followed by a loxP-flanked sequence containing enhanced GFP and a transcriptional/translational STOP sequence that allows tissue-specific overexpression of Cox2 in the presence of Cre recombinase (38)  These results show that the lack of endothelial expression of Cox2 is causative of the Dgke phenotype. They also denote that the failed induction of Cox2 in endothelial cells and not the lack of its substrate AA causes aHUS and proteinuria in Tie2Cre Dgke fl/fl mice.
A stable PGE 2 analog rescues the phenotype of endothelial specific Dgke-knockout mice. Cox2 is the rate-limiting enzyme for the generation of PGE 2 , a potent promoter of endothelial cell migration, survival, and angiogenesis (39,40). VEGFA induces Cox2 and PGE 2 in endothelial cells (26,28,29,41), which suggests that its proangiogenic effect might be mediated at least partly by the production of PGE 2 by mechanisms that are not completely understood (27,42,43). In a precedent work we showed that urinary concentration of PGE 2 , but not of the stable PGI 2 metabolite 6-keto PGF 1α , was reduced in the Dgke constitutive knockouts compared with control mice. Dgke-knockout mice showed defective vascularization of surgical sponges implanted subcutaneously, and this defect was rescued by injecting the sponges with PGE 2 (44), which pointed to the existence of a defect of PGE 2 -mediated angiogenesis in these mice. To clarify the role of PGE 2 in determining the clinical manifestations in Tie2Cre Dgke fl/fl mice, we tested if increasing circulating levels of PGE 2 would rescue the hematologic and renal manifestations in our mouse model. To this end, we implanted subcutaneous osmotic pumps in 8-week-old Tie2Cre Dgke fl/fl mice to continuously deliver the stable PGE 2 analog 16,16 dimethyl-PGE 2 (dmPGE 2 ) and looked for changes in the phenotype after 4 weeks. Infusion of dmPGE 2 was sufficient to normalize all the clinical manifestations in Tie2Cre Dgke fl/fl mice ( Figure 5, A-H, and Supplemental Figure 4E). These results indicate that impaired PGE 2 production is a key factor in determining the presence of disease in the absence of Dgke and that the anemia observed in these mice is hemolytic and not caused by bone marrow failure.
Treatment with a PGE 2 stable analog normalizes endothelial cell activation and prothrombotic diathesis in  and the initiator of coagulation tissue factor (TF) in vitro (45). We tested the expression of P selectin and TF in kidney cortexes of Tie2Cre Dgke fl/fl mice at 3 months of age and of plasma thrombin-antithrombin complexes (TAT) and found that all these parameters were significantly increased, compared with control mice (Figure 6, A-C), confirming in vivo in Tie2Cre Dgke fl/fl mice that lack Dgke have activation and prothrombotic state of the endothelium. In line with this observation, we also observed increased endothelial susceptibility to procoagulant inflammatory stimuli, by treating Tie2Cre Dgke fl/fl mice and controls at 2 months of age with the synthetic double-strand DNA polyinosinic:polycytidylic acid [poly(I:C)], a potent inducer of interferon-α (46) ( Figure 6D). Of note, these results suggest that VEGFA signaling controls the activation and the thrombogenic state of the endothelium indirectly through the regulation of PGE 2 . PGE 2 controls the endothelial expression of CXCR4 in the kidney. Stromal-derived factor 1 (SDF-1, also known as CXCL12) is a soluble chemokine expressed in the stromal component of several organs, and CXCR4 is its cognate receptor expressed on cells of the hematopoietic linage and on endothelial cells. In the kidney, like VEGFA, SDF-1 is expressed in podocytes (47)(48)(49). The activation of the SDF-1/ CXCR4 signaling in endothelial cells exerts promigratory and proangiogenic effects, and it is required for the development of the renal vasculature (50). Hypoxia is a strong inducer of VEGFA, SDF-1, and CXCR4 (51,52). In addition, high levels of SDF-1 are known to act in a feed-forward loop to further increase CXCR4 expression in endothelial cells (53). Because endothelial disfunction is expected to result in reduced tissue oxygenation, we tested the expression of hypoxia-induced genes in cortex extracts of Tie2Cre Dgke fl/fl and control mice and found that the expression of HIF-1 and HIF-2 was higher in Tie2Cre Dgke fl/fl kidney cortexes, compared with controls ( Figure 7, A and B), as well as finding higher expression of HIF-1 and HIF-2 transcriptional targets SDF-1 and VEGFA (Figure 7, C and D). However, in spite of the hypoxic milieu of the Tie2Cre Dgke fl/fl kidney cortexes, the expression of CXCR4 was surprisingly low, compared with controls ( Figure 7E). To confirm that the lower levels of CXCR4 in Tie2Cre Dgke fl/fl kidneys were caused by differences in CXCR4 expression in endothelial cells and not in other cell populations, we performed flow cytometry analysis of single-cell suspensions of kidney cortexes of Tie2-Cre Dgke fl/fl and control mice double-stained with antibodies against CXCR4 and against the endothelial cell marker VE-Cadherin. Consistent with other reports (54), VE-Cadherin + endothelial cells represented about 3%-4% of the cells in our samples (Supplemental Figure 6B, Q2 + Q3). This analysis confirmed that, compared with controls, the proportion of CXCR4-expressing endothelial cells was reduced in Tie2-Cre Dgke fl/fl mice  Figure 6, A and C). Of note, because CXCR4 expression can be induced by PGE 2 through the activation of adenylate cyclase (55-57), we tested the expression of CXCR4 in DGKE-knockdown HUVECs and HMECs by Q-PCR and found that CXCR4 expression was strongly downregulated in these cells, but it was rescued to normal levels after supplementation with PGE 2 ( Figure  7H and Supplemental Figure 2C). Remarkably, treatment with dmPGE 2 increased the number of endothelial cells expressing CXCR4 in kidney cortexes of Tie2-Cre Dgke fl/fl mice (Figure 7, I and J). These results show that in the kidneys, PGE 2 controls the endothelial expression of CXCR4 and thus the activity of the SDF-1/CXCR4 signaling axis. PGE 2 suppresses the expression of MMP-2 in Tie2Cre Dgke fl/fl mouse kidneys. DGKE nephropathy is invariably associated with proteinuria (3,25,58). Proteinuria was also present in 100% of endothelial specific Tie2-Cre Dgke fl/fl knockout mice at a later time compared with the appearance of the hematologic signs, which suggests that the filtration barrier is secondarily affected by primary endothelial abnormalities. To gain insights into the pathogenesis of the proteinuria in the Tie2-Cre Dgke fl/fl knockout mice, we performed transmission electron microscopy (TEM) of renal cortexes of these mice at 3 months of age, when albuminuria is detectable.

Tie2-Cre
Dgke fl/fl knockout mouse glomeruli showed moderate podocyte foot process effacement, subendothelial widening, and, interestingly, segmental thickening of the glomerular basement membrane (GBM) (Figure 8,  A and B), but all these abnormalities were not detectable in dmPGE 2 -treated Tie2-Cre Dgke fl/fl mice, in concordance with the normalized albuminuria (Figure 8, C and D, and Figure 5F). For this reason, we investigated if the proteinuria in Tie2-Cre Dgke fl/fl mice could result from abnormalities of the GBM induced by the activated endothelium (59). In the bone marrow niche, SDF-1 and CXCR4 are essential regulators of the homing of hematopoietic progenitor cells (HPCs) (60)(61)(62)(63), and inhibition of CXCR4 is used to force HPCs to egress from their niche into the bloodstream, when large numbers of HPCs need to be harvested for bone marrow transplantation (64). The mobilizing effect of CXCR4 inhibitors and other hematopoietic cell mobilizers is mediated by the induction of several proteinases, including MMPs (65). Considering that the expression of CXCR4 in endothelial cells can be induced by PGE 2 (55-57), we tested by Q-PCR if impaired endothelial induction of PGE 2 in the absence of Dgke would be indirectly associated with increased expression of MMP-2 and MMP-9, the MMPs most abundantly expressed in the glomeruli (66), compared with controls. We found that the expression of MMP-2 was increased in DGKE-knockdown HUVECs and HMECs and normalized by supplementing the culture medium with PGE 2 ( Figure 8E and Supplemental Figure 2D). MMP-2, but not MMP-9, expression was also higher in cortex extracts of Tie2-Cre Dgke fl/fl mice in vivo, but not in kidneys of mice 28 days after they were injured by folic acid injection (67) (Supplemental Figure 7). We further confirmed by gel zymography analysis of cortex lysates that the total MMP-2 protein levels and its proteolytic activity were increased in Tie2-Cre Dgke fl/fl mice but not in Tie2-Cre Dgke fl/fl mice treated with dmPGE 2 ( Figure 8F). These results indicate that MMP-2 activity is high in endothelial specific Tie2-Cre Dgke fl/fl mouse kidneys and suggest that chronic impairment of the VEGF signaling in these mice may favor the remodeling of the GBM, maybe by controlling PGE 2 and CXCR4-dependent MMP-2 expression. Further studies that are beyond the scope of this work will be needed to further explore this possibility.

Discussion
In this work we have investigated the molecular mechanisms by which biallelic loss-of-function mutations in DGKE cause a form of TMA and aHUS that it is not associated with defects of the alternative complement pathway (2, 3). By generating a potentially novel conditional knockout mouse for the gene Dgke, we demonstrated that endothelial specific deletion of Dgke is sufficient to phenocopy in full the human disease, which is remarkably similar to the disease reported in subjects treated with bevacizumab (2), highlighting the centrality of the endothelium in the DGKE disorder and suggesting that podocytes, which also express DGKE, are only secondarily affected. Mechanistically, we found that the shortage of PIP2, the intracellular substrate of PI3K, is the primary biochemical defect that causes the DGKE disease resulting in defective PIP3-dependent Akt activation in endothelial cells downstream of VEGFR2, as demonstrated in vitro and in vivo by the experiments in which a PTEN inhibitor was administered to Tie2-Cre Dgke fl/fl mice. Thus, our data point at a central role of a defect of the Akt signaling in the endothelium of Tie2-Cre Dgke fl/fl mice and speak against a potential role of increased PKC activity in determining the DGKE phenotype, although this possibility cannot be completely excluded. Consistent with our in vivo data is also the conclusion, recently reported in an in vitro study, that by causing shortage of PIP2 (the common cellular substrate of both PI3K and PLC) loss of Dgke does not affect DAG signaling (8).
A somewhat unexpected connection that emerges from our studies is the relation between endothelial VEGF and Cox2/PGE 2 signaling in endothelial cells. Although we previously reported the defect of Cox2 expression and PGE 2 production in the constitutive Dgke-knockout mice, it was not clear how these abnormalities would be connected with the observed phenotype and if their correction would have been able to rescue the disease in Dgke-defective mice. The in vitro and in vivo experiments that we have reported here show that Dgke is required in endothelial cells to induce the expression of Cox2 and the production of PGE 2 downstream of VEGFR2 and to promote the maintenance of the kidney microvasculature. This conclusion is supported by the ability of the endothelial overexpression of Cox2 and of the subcutaneous infusion of dmPGE 2 to rescue the hematologic and renal manifestations observed in Tie2-Cre Dgke fl/fl mice. To this point, it is worthwhile to notice that treatment with dmPGE 2 both rescued capillary occlusion and renal function, which could be explained by a vasodilator effect of PGE 2 on the glomerular microvasculature, Finally, the studies that we have presented here imply an unanticipated interaction between VEGF and SDF-1/CXCR4 signaling in the kidney. A hypothetical general model that could be drawn from our observations would suggest the existence of a balance between proangiogenic VEGF-PGE 2 -mediated signaling that would cause, among other responses, the GBM remodeling by activating MMPs and negative feedback sustained by the counteracting SDF-1/CXCR4 signaling triggered by PGE 2 . According to such a model, the lack of Dgke and PGE 2 would impair CXCR4-dependent suppression of MMP-2 in endothelial cells, and it would eventually result in glomerular filtration barrier damage (Graphical abstract). Although further experimental evidence has to be produced to better define the interplay between PGE 2 and CXCR4 expression in vivo, our results reveal the existence of complex autocrine signaling events downstream of VEGFR2 that could conceivably be manipulated to modify the clinical course of aHUS and of other diseases characterized by endothelial activation or abnormal angiogenesis.

Methods
Animal studies. All mice were on a C57BL/6J background. The Cox2-COE mice were provided by Harvey R. Herschman, University of California, Los Angeles (Los Angeles, California, USA), and were previously described (38).
Generation of Dgke conditional knockout mice. These mice were generated by injecting ES cells with a knockout first construct, from the KOMP (https://www.komp.org) (30). The knockout first allele is initially a nonexpressive form, but it can be converted to a conditional allele, which carries 2 LoxP sites flanking exon 2 of Dgke, via Flp recombination. We confirmed by genomic sequencing that this exon was deleted after Cre recombination. Dgke-floxed mice are viable and fertile, and they produce progeny at the expected Mendelian ratio. Tie2-Cre mice were purchased from The Jackson Laboratory (stock 008863) and were previously described (68). CMV-Cre mice were purchased from The Jackson Laboratory (stock 006054). WT mice bred from the colony were used as controls, unless otherwise specified.
Endothelial cell culture. HUVECs and human dermal microvascular endothelial cells (HMEC-1) were obtained from the American Type Culture Collection. Monolayers were cultured in medium 199 (Gibco, Thermo Fisher Scientific) supplemented with 20% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), Mouse platelet isolation and in vitro platelet aggregation studies. Mouse platelets were prepared as previously described (71,72). Blood from anesthetized mice was drawn from the retro-orbital plexus and collected in 1.5 mL polypropylene tubes containing 300 μL of enoxaparin (0.3 mg/mL; Sanofi-Aventis). The blood was centrifuged at 100g for 5 minutes, and the platelet-rich plasma (PRP) was collected in a fresh tube. PRP (platelet count; 2 × 10 8 /mL) from WT and endothelial specific knockout DGKE was stirred (178 RCF) at 37°C for 2 minutes in a whole blood/optical lumi-aggregometer (Chrono-log, model 700-2) before the addition of agonists (collagen or ADP). Aggregation was measured as percentage change in light transmission, where 100% refers to transmittance through the blank sample (platelet-poor plasma). PGE 2 infusion with subcutaneous osmotic minipumps. Mice were treated with dmPGE 2 (Cayman Chemical) and vehicle control through a subcutaneously implanted ALZET 1004 mini-osmotic pump (DURECT). Minipumps were loaded with 100 μL of dmPGE 2 solution in sterile PBS. A release rate of 0.11 μL/h administered a total amount of 30 μg/kg dmPGE 2 daily, a dose that resulted in elevated serum PGE 2 without obvious adverse side effects, during the 28 days of the experimental setup.
Flow cytometry. Fresh mouse kidney cortexes were digested in 2 mL of protease solution -5 mM CaCl 2 , 10 mg/mL Bacillus licheniformis protease (Creative Enzymes, NATE0633), and 125 U/mL DNase I (Roche) in Dulbecco's PBS -on ice for 30 minutes. The lysates were filtered through 70 μm cell strainers. Single-cell suspension was collected by rinsing with FACS buffer. Suspensions were lysed with red blood cell lysis buffer (Quality Biological) to remove red blood cells. After washing with 1× PBS, 1 × 10 6 cells were resuspended in 100 μL of FACS buffer and preincubated with anti-mouse CD16/32 antibody (Bio-Legend, 101302) for blocking prior to labeling with CD144-APC (BioLegend, 138012) and CD184-BV421 (BD Biosciences, 562738) antibodies for 30 minutes at room temperature. Cells were then washed with FACS buffer and analyzed on BD LSR II Violet at the University of Iowa Flow Cytometry Facility. All flow cytometry results were analyzed using FlowJo software (Version 9).
Zymography. Kidney cortical tissues were lysed in lysis buffer (20 mM Tris-HCl, 125 mM NaCl, and 1% Triton X-100, pH 8.5) with protease inhibitor cocktail (Roche). Protein concentration was calculated by NanoDrop (Thermo Fisher Scientific). Samples were separated on Tris-glycine gels with 0.1% gelatin (Invitrogen, Thermo Fisher Scientific). After electrophoresis, the gels were incubated in 1× renaturing buffer (Thermo Fisher Scientific) for 2 hours at room temperature, then in 1× developing buffer (Thermo Fisher Scientific) for 48 hours. Gels were stained with Coomassie Blue R-250 (Bio-Rad) and destained in 20% ethanol and 10% acetic acid. MMP-2 activity was detected as clear bands on the blue background.
Statistics. All quantitative data are presented as mean ± SD. Statistical significance (P ≤ 0.05 was considered significant) was calculated using the 2-tailed unpaired Student's t test for 2 groups and 1-way ANOVA for multiple-group comparison by Prism 7 (GraphPad Software).
Study approval. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Iowa (protocol 7021976).