Adiponectin Reduces Glomerular Endothelial Glycocalyx Disruption and Restores Glomerular Barrier Function in a Mouse Model of Type 2 Diabetes.

Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the tumor necrosis factor-α (TNF-α)-mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps'alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps'alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes.

Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

Mineralocorticoid receptor antagonism in diabetes reduces albuminuria by preserving the glomerular endothelial glycocalyx.

The glomerular endothelial glycocalyx (GEnGlx) forms the first part of the glomerular filtration barrier. Previously, we showed that mineralocorticoid receptor (MR) activation caused GEnGlx damage and albuminuria. In this study, we investigated whether MR antagonism could limit albuminuria in diabetes and studied the site of action. Streptozotocin-induced diabetic Wistar rats developed albuminuria, increased glomerular albumin permeability (Ps'alb), and increased glomerular matrix metalloproteinase (MMP) activity with corresponding GEnGlx loss. MR antagonism prevented albuminuria progression, restored Ps'alb, preserved GEnGlx, and reduced MMP activity. Enzymatic degradation of the GEnGlx negated the benefits of MR antagonism, confirming their dependence on GEnGlx integrity. Exposing human glomerular endothelial cells (GEnC) to diabetic conditions in vitro increased MMPs and caused glycocalyx damage. Amelioration of these effects confirmed a direct effect of MR antagonism on GEnC. To confirm relevance to human disease, we used a potentially novel confocal imaging method to show loss of GEnGlx in renal biopsy specimens from patients with diabetic nephropathy (DN). In addition, patients with DN randomized to receive an MR antagonist had reduced urinary MMP2 activity and albuminuria compared with placebo and baseline levels. Taken together, our work suggests that MR antagonists reduce MMP activity and thereby preserve GEnGlx, resulting in reduced glomerular permeability and albuminuria in diabetes.

Minimal Change Disease Is Associated With Endothelial Glycocalyx Degradation and Endothelial Activation.

Introduction: Minimal change disease (MCD) is considered a podocyte disorder triggered by unknown circulating factors. Here, we hypothesized that the endothelial cell (EC) is also involved in MCD. Methods: We studied 45 children with idiopathic nephrotic syndrome (44 had steroid sensitive nephrotic syndrome [SSNS], and 12 had biopsy-proven MCD), 21 adults with MCD, and 38 healthy controls (30 children, 8 adults). In circulation, we measured products of endothelial glycocalyx (EG) degradation (syndecan-1, heparan sulfate [HS] fragments), HS proteoglycan cleaving enzymes (matrix metalloprotease-2 [MMP-2], heparanase activity), and markers of endothelial activation (von Willebrand factor [vWF], thrombomodulin) by enzyme-linked immunosorbent assay (ELISA) and mass spectrometry. In human kidney tissue, we assessed glomerular EC (GEnC) activation by immunofluorescence of caveolin-1 (n = 11 MCD, n = 5 controls). In vitro, we cultured immortalized human GEnC with sera from control subjects and patients with MCD/SSNS sera in relapse (n = 5 per group) and performed Western blotting of thrombomodulin of cell lysates as surrogate marker of endothelial activation. Results: In circulation, median concentrations of all endothelial markers were higher in patients with active disease compared with controls and remained high in some patients during remission. In the MCD glomerulus, caveolin-1 expression was higher, in an endothelial-specific pattern, compared with controls. In cultured human GEnC, sera from children with MCD/SSNS in relapse increased thrombomodulin expression compared with control sera. Conclusion: Our data show that alterations involving the systemic and glomerular endothelium are nearly universal in patients with MCD and SSNS, and that GEnC can be directly activated by circulating factors present in the MCD/SSNS sera during relapse.

Circulating hyaluronan as a marker of endothelial glycocalyx damage in dogs with myxomatous mitral valve disease and dogs in a hypercoagulable state.

The endothelial glycocalyx (eGlx) lines the luminal surface of endothelial cells, maintaining vascular health. Glycocalyx damage is pathophysiologically important in many diseases across species however few studies have investigated its breakdown in naturally occurring disease in dogs. The aims of the study were to investigate eGlx damage in dogs with myxomatous mitral valve disease (MMVD) diagnosed on echocardiography, and dogs in a hypercoagulable state diagnosed using thromboelastography (TEG), by measuring serum hyaluronan concentrations. Serum hyaluronan was quantified in dogs with MMVD (n = 27), hypercoagulability (n = 21), and in healthy controls dogs (n = 18). Serum hyaluronan concentrations were measured using a commercially-available ELISA validated for use in dogs. Hyaluronan concentrations were compared among groups using Kruskal-Wallis tests, and post-hoc with Dunn's tests. Serum hyaluronan concentrations (median [range]) were significantly increased in dogs with MMVD (62.4 [22.8-201] ng/mL; P = 0.031) and hypercoagulability (92.40 [16.9-247.6] ng/mL; P < 0.001) compared to controls (45.7 [8.7-80.2] ng/mL). Measurement of serum hyaluronan concentration offers a clinically applicable marker of eGlx health and suggests the presence of eGlx damage in dogs with MMVD and dogs in a hypercoagulable state.

CRISPR/Cas9-Engineered HLA-Deleted Glomerular Endothelial Cells as a Tool to Predict Pathogenic Non-HLA Antibodies in Kidney Transplant Recipients.

BACKGROUND: After kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs). METHODS: W e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II-deficient glomerular endothelial cells (CiGEnCΔHLA) that had been previously generated through CRISPR/Cas9-induced B2M and CIITA gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens. RESULTS: W e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnCΔHLA clone. CiGEnCΔHLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result (P<0.001). Stratification of 3-month allograft biopsy specimens (n=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (P=0.002), microvascular inflammation (P=0.003), and ABMRh (P=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (P=0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh. CONCLUSION: The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.

Cystatin C and α-1-Microglobulin Predict Severe Acute Kidney Injury in Patients with Hemorrhagic Fever with Renal Syndrome.

Puumala orthohantavirus causes hemorrhagic fever with renal syndrome (HFRS) characterized by acute kidney injury (AKI), an abrupt decrease in renal function. Creatinine is routinely used to detect and quantify AKI; however, early AKI may not be reflected in increased creatinine levels. Therefore, kidney injury markers that can predict AKI are needed. The potential of the kidney injury markers urea, cystatin C, α1-microglobulin (A1M) and neutrophil gelatinase-associated lipocalin (NGAL) to detect early AKI during HFRS was studied by quantifying the levels of these markers in consecutively obtained plasma (P) and urine samples (U) for 44 HFRS patients. P-cystatin C and U-A1M levels were significantly increased during early HFRS compared to follow-up. In a receiver operating characteristic (ROC) curve analysis, P-cystatin C, U-A1M and P-urea predicted severe AKI with area under the curve 0.72, 0.73 and 0.71, respectively, whereas the traditional kidney injury biomarkers creatinine and U-albumin did not predict AKI. Nearly half of the HFRS patients (41%) fulfilled the criteria for shrunken pore syndrome, which was associated with the level of inflammation as measured by P-CRP. P-cystatin C and U-A1M are more sensitive and earlier markers compared to creatinine in predicting kidney injury during HFRS.

Metabolomic analysis of Shiga toxin 2a-induced injury in conditionally immortalized glomerular endothelial cells.

INTRODUCTION: Shiga toxin 2a (Stx2a) induces hemolytic uremic syndrome (STEC HUS) by targeting glomerular endothelial cells (GEC). OBJECTIVES: We investigated in a metabolomic analysis the response of a conditionally immortalized, stable glomerular endothelial cell line (ciGEnC) to Stx2a stimulation as a cell culture model for STEC HUS. METHODS: CiGEnC were treated with tumor necrosis factor-(TNF)α, Stx2a or sequentially with TNFα and Stx2a. We performed a metabolomic high-throughput screening by lipid- or gas chromatography and subsequent mass spectrometry. Metabolite fold changes in stimulated ciGEnC compared to untreated cells were calculated. RESULTS: 320 metabolites were identified and investigated. In response to TNFα + Stx2a, there was a predominant increase in intracellular free fatty acids and amino acids. Furthermore, lipid- and protein derived pro-inflammatory mediators, oxidative stress and an augmented intracellular energy turnover were increased in ciGEnC. Levels of most biochemicals related to carbohydrate metabolism remained unchanged. CONCLUSION: Stimulation of ciGEnC with TNFα + Stx2a is associated with profound metabolic changes indicative of increased inflammation, oxidative stress and energy turnover.

Early Acute Microvascular Kidney Transplant Rejection in the Absence of Anti-HLA Antibodies Is Associated with Preformed IgG Antibodies against Diverse Glomerular Endothelial Cell Antigens.

BACKGROUND: Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed. METHODS: We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs. RESULTS: We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals. CONCLUSIONS: Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that in vitro cell-based assays are needed to improve risk assessments before transplant.

Poly(ethylene glycol)-crosslinked gelatin hydrogel substrates with conjugated bioactive peptides influence endothelial cell behavior.

The basement membrane is a specialized extracellular matrix substrate responsible for support and maintenance of epithelial and endothelial structures. Engineered basement membrane-like hydrogel systems have the potential to advance understanding of cell-cell and cell-matrix interactions by allowing precise tuning of the substrate or matrix biochemical and biophysical properties. In this investigation, we developed tunable hydrogel substrates with conjugated bioactive peptides to modulate cell binding and growth factor signaling by endothelial cells. Hydrogels were formed by employing a poly(ethylene glycol) crosslinker to covalently crosslink gelatin polymers and simultaneously conjugate laminin-derived YIGSR peptides or vascular endothelial growth factor (VEGF)-mimetic QK peptides to the gelatin. Rheological characterization revealed rapid formation of hydrogels with similar stiffnesses across tested formulations, and swelling analysis demonstrated dependency on peptide and crosslinker concentrations in hydrogels. Levels of phosphorylated VEGF Receptor 2 in cells cultured on hydrogel substrates revealed that while human umbilical vein endothelial cells (HUVECs) responded to both soluble and conjugated forms of the QK peptide, conditionally-immortalized human glomerular endothelial cells (GEnCs) only responded to the conjugated presentation of the peptide. Furthermore, whereas HUVECs exhibited greatest upregulation in gene expression when cultured on YIGSR- and QK-conjugated hydrogel substrates after 5 days, GEnCs exhibited greatest upregulation when cultured on Matrigel control substrates at the same time point. These results indicate that conjugation of bioactive peptides to these hydrogel substrates significantly influenced endothelial cell behavior in cultures but with differential responses between HUVECs and GEnCs.

MicroRNA-532-5p Regulates Pericyte Function by Targeting the Transcription Regulator BACH1 and Angiopoietin-1.

MicroRNAs regulate endothelial function and angiogenesis, but their implication in pericyte biology remains undetermined. A PCR array, covering a panel of 379 human microRNAs, showed microRNA-532-5p to be one of the most differentially modulated by hypoxia, which was confirmed by qPCR in both skeletal muscle and adventitial pericytes. Furthermore, microRNA-532-5p was upregulated in murine muscular pericytes early after experimentally induced ischemia, decreasing below baseline after reperfusion. Transfection of human pericytes with anti-microRNA, microRNA-mimic, or controls indicates microRNA-532-5p modulates pro-angiogenic activity via transcriptional regulation of angiopoietin-1. Tie-2 blockade abrogated the ability of microRNA-532-5p-overexpressing pericytes to promote endothelial network formation in vitro. However, angiopoietin-1 is not a direct target of microRNA-532-5p. In silico analysis of microRNA-532-5p inhibitory targets associated with angiopoietin-1 transcription indicated three potential candidates, BACH1, HIF1AN, and EGLN1. Binding of microRNA-532-5p to the BACH1 3' UTR was confirmed by luciferase assay. MicroRNA-532-5p silencing increased BACH1, while a microRNA-532-5p mimic decreased expression. Silencing of BACH1 modulated angiopoietin-1 gene and protein expression. ChIP confirmed BACH1 transcriptional regulation of angiopoietin-1 promoter. Finally, microRNA-532-5p overexpression increased pericyte coverage in an in vivo Matrigel assay, suggesting its role in vascular maturation. This study provides a new mechanistic understanding of the transcriptional program orchestrating angiopoietin-1/Tie-2 signaling in human pericytes.

Association of Elevated Urinary miR-126, miR-155, and miR-29b with Diabetic Kidney Disease.

Effective diabetic kidney disease (DKD) biomarkers remain elusive, and urinary miRNAs represent a potential source of novel noninvasive disease sentinels. We profiled 754 miRNAs in pooled urine samples from DKD patients (n = 20), detecting significantly increased miR-126, miR-155, and miR-29b compared with controls (n = 20). These results were confirmed in an independent cohort of 89 DKD patients, 62 diabetic patients without DKD, and 41 controls: miR-126 (2.8-fold increase; P < 0.0001), miR-155 (1.8-fold increase; P < 0.001), and miR-29b (4.6-fold increase; P = 0.024). Combined receiver operating characteristic curve analysis resulted in an area under the curve of 0.8. A relative quantification threshold equivalent to 80% sensitivity for each miRNA gave a positive signal for 48% of DKD patients compared with 3.6% of diabetic patients without DKD. Laser-capture microdissection of renal biopsy specimens, followed by quantitative RT-PCR, detected miR-155 in glomeruli and proximal and distal tubules, whereas miR-126 and miR-29b were most abundant in glomerular extracts. Subsequent experiments showed miR-126 and miR-29b enrichment in glomerular endothelial cells (GEnCs) compared with podocytes, proximal tubular epithelial cells, and fibroblasts. Significantly increased miR-126 and miR-29b were detected in GEnC conditioned medium in response to tumor necrosis factor-α and transforming growth factor-ß1, respectively. Our data reveal an altered urinary miRNA profile associated with DKD and link these variations to miRNA release from GEnCs.

Heme oxygenase-1 is a potent inhibitor of placental ischemia-mediated endothelin-1 production in cultured human glomerular endothelial cells.

Preeclampsia is a pregnancy-specific disorder of maternal hypertension and reduced renal hemodynamics linked to reduced endothelial function. Placental ischemia is thought to be the culprit of this disease, as it causes the release of factors like tumor necrosis factor (TNF)-α that induce vascular endothelin-1 (ET-1) production. Interestingly, placental ischemia-induced hypertension in rats [reduced uterine perfusion pressure (RUPP) model] is abolished by ETA receptor blockade, suggesting a critical role for ET-1. Although it has been found that systemic induction of heme oxygenase (HO)-1 is associated with reduced ET-1 production and attenuated hypertension, it is unclear whether HO-1 directly modulates the increased ET-1 response to placental factors. We tested the hypothesis that HO-1 or its metabolites inhibit ET-1 production in human glomerular endothelial cells induced by serum of RUPP rats or TNF-α. Serum (5%) from RUPP hypertensive (mean arterial blood pressure 119 ± 9 mmHg) vs. normotensive pregnant (NP, 101 ± 6 mmHg, P < 0.001) rats increased ET-1 production (RUPP 168.8 ± 18.1 pg/ml, NP 80.3 ± 22.7 pg/ml, P < 0.001, n = 12/group). HO-1 induction [25 µM cobalt photoporphyrin (CoPP)] abolished RUPP serum-induced ET-1 production (1.6 ± 0.8 pg/ml, P < 0.001), whereas bilirubin (10 µM) significantly attenuated ET-1 release (125.3 ± 5.2 pg/ml, P = 0.005). Furthermore, TNF-α-induced ET-1 production (TNF-α 31.0 ± 8.4 vs. untreated 7.5 ± 0.4 pg/ml, P < 0.001) was reduced by CoPP (1.5 ± 0.8 pg/ml, P < 0.001) and bilirubin (10.5 ± 4.3 pg/ml, P < 0.001). These results suggest that circulating factors released during placental ischemia target the maternal glomerular endothelium to increase ET-1, and that pharmacological induction of HO-1 or bilirubin could be a treatment strategy to block this prohypertensive pathway in preeclampsia.

Heme Drives Susceptibility of Glomerular Endothelium to Complement Overactivation Due to Inefficient Upregulation of Heme Oxygenase-1.

Atypical hemolytic uremic syndrome (aHUS) is a severe disease characterized by microvascular endothelial cell (EC) lesions leading to thrombi formation, mechanical hemolysis and organ failure, predominantly renal. Complement system overactivation is a hallmark of aHUS. To investigate this selective susceptibility of the microvascular renal endothelium to complement attack and thrombotic microangiopathic lesions, we compared complement and cyto-protection markers on EC, from different vascular beds, in in vitro and in vivo models as well as in patients. No difference was observed for complement deposits or expression of complement and coagulation regulators between macrovascular and microvascular EC, either at resting state or after inflammatory challenge. After prolonged exposure to hemolysis-derived heme, higher C3 deposits were found on glomerular EC, in vitro and in vivo, compared with other EC in culture and in mice organs (liver, skin, brain, lungs and heart). This could be explained by a reduced complement regulation capacity due to weaker binding of Factor H and inefficient upregulation of thrombomodulin (TM). Microvascular EC also failed to upregulate the cytoprotective heme-degrading enzyme heme-oxygenase 1 (HO-1), normally induced by hemolysis products. Only HUVEC (Human Umbilical Vein EC) developed adaptation to heme, which was lost after inhibition of HO-1 activity. Interestingly, the expression of KLF2 and KLF4-known transcription factors of TM, also described as possible transcription modulators of HO-1- was weaker in micro than macrovascular EC under hemolytic conditions. Our results show that the microvascular EC, and especially glomerular EC, fail to adapt to the stress imposed by hemolysis and acquire a pro-coagulant and complement-activating phenotype. Together, these findings indicate that the vulnerability of glomerular EC to hemolysis is a key factor in aHUS, amplifying complement overactivation and thrombotic microangiopathic lesions.

The adipokine leptin modulates adventitial pericyte functions by autocrine and paracrine signalling.

Transplantation of adventitial pericytes (APCs) improves recovery from tissue ischemia in preclinical animal models by still unknown mechanisms. This study investigates the role of the adipokine leptin (LEP) in the regulation of human APC biological functions. Transcriptomic analysis of APCs showed components of the LEP signalling pathway are modulated by hypoxia. Kinetic studies indicate cultured APCs release high amounts of immunoreactive LEP following exposure to hypoxia, continuing upon return to normoxia. Secreted LEP activates an autocrine/paracrine loop through binding to the LEP receptor (LEPR) and induction of STAT3 phosphorylation. Titration studies using recombinant LEP and siRNA knockdown of LEP or LEPR demonstrate the adipokine exerts important regulatory roles in APC growth, survival, migration and promotion of endothelial network formation. Heterogeneity in LEP expression and secretion may influence the reparative proficiency of APC therapy. Accordingly, the levels of LEP secretion predict the microvascular outcome of APCs transplantation in a mouse limb ischemia model. Moreover, we found that the expression of the Lepr gene is upregulated on resident vascular cells from murine ischemic muscles, thus providing a permissive milieu to transplanted LEP-expressing APCs. Results highlight a new mechanism responsible for APC adaptation to hypoxia and instrumental to vascular repair.

VEGF regulates local inhibitory complement proteins in the eye and kidney.

Outer retinal and renal glomerular functions rely on specialized vasculature maintained by VEGF that is produced by neighboring epithelial cells, the retinal pigment epithelium (RPE) and podocytes, respectively. Dysregulation of RPE- and podocyte-derived VEGF is associated with neovascularization in wet age-related macular degeneration (ARMD), choriocapillaris degeneration, and glomerular thrombotic microangiopathy (TMA). Since complement activation and genetic variants in inhibitory complement factor H (CFH) are also features of both ARMD and TMA, we hypothesized that VEGF and CFH interact. Here, we demonstrated that VEGF inhibition decreases local CFH and other complement regulators in the eye and kidney through reduced VEGFR2/PKC-α/CREB signaling. Patient podocytes and RPE cells carrying disease-associated CFH genetic variants had more alternative complement pathway deposits than controls. These deposits were increased by VEGF antagonism, a common wet ARMD treatment, suggesting that VEGF inhibition could reduce cellular complement regulatory capacity. VEGF antagonism also increased markers of endothelial cell activation, which was partially reduced by genetic complement inhibition. Together, these results suggest that VEGF protects the retinal and glomerular microvasculature, not only through VEGFR2-mediated vasculotrophism, but also through modulation of local complement proteins that could protect against complement-mediated damage. Though further study is warranted, these findings could be relevant for patients receiving VEGF antagonists.

Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.

Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines.

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor α: a contributor to endothelial cell glycocalyx dysfunction.

The endothelial surface glycocalyx is a hydrated mesh in which proteoglycans are prominent. It is damaged in diseases associated with elevated levels of tumor necrosis factor α (TNF-α). We investigated the mechanism of TNF-α-induced disruption of the glomerular endothelial glycocalyx. We used conditionally immortalized human glomerular endothelial cells (GEnCs), quantitative PCR arrays, Western blotting, immunoprecipitation, immunofluorescence, and dot blots to examine the effects of TNF-α. TNF-α induced syndecan 4 (SDC4) mRNA up-regulation by 2.5-fold, whereas cell surface SDC4 and heparan sulfate (HS) were reduced by 36 and 30%, respectively, and SDC4 and sulfated glycosaminoglycan in the culture medium were increased by 52 and 65%, respectively, indicating TNF-α-induced shedding. Small interfering (siRNA) knockdown of SDC4 (by 52%) caused a corresponding loss of cell surface HS of similar magnitude (38%), and immunoprecipitation demonstrated that SDC4 and HS are shed as intact proteoglycan ectodomains. All of the effects of TNF-α on SDC4 and HS were abrogated by the metalloproteinase (MMP) inhibitor batimastat. Also abrogated was the associated 37% increase in albumin passage across GEnC monolayers. Specific MMP9 knockdown by siRNA similarly blocked TNF-α effects. SDC4 is the predominant HS proteoglycan in the GEnC glycocalyx. TNF-α-induced MMP9-mediated shedding of SDC4 is likely to contribute to the endothelial glycocalyx disruption observed in diabetes and inflammatory states.

Podocytes regulate neutrophil recruitment by glomerular endothelial cells via IL-6-mediated crosstalk.

Stromal cells actively modulate the inflammatory process, in part by influencing the ability of neighboring endothelial cells to support the recruitment of circulating leukocytes. We hypothesized that podocytes influence the ability of glomerular endothelial cells (GEnCs) to recruit neutrophils during inflammation. To address this, human podocytes and human GEnCs were cultured on opposite sides of porous inserts and then treated with or without increasing concentrations of TNF-α prior to addition of neutrophils. The presence of podocytes significantly reduced neutrophil recruitment to GEnCs by up to 50% when cultures were treated with high-dose TNF-α (100 U/ml), when compared with GEnC monocultures. Importantly, this phenomenon was dependent on paracrine actions of soluble IL-6, predominantly released by podocytes. A similar response was absent when HUVECs were cocultured with podocytes, indicating a tissue-specific phenomenon. Suppressor of cytokine signaling 3 elicited the immunosuppressive actions of IL-6 in a process that disrupted the presentation of chemokines on GEnCs by altering the expression of the duffy Ag receptor for chemokines. Interestingly, suppressor of cytokine signaling 3 knockdown in GEnCs upregulated duffy Ag receptor for chemokines and CXCL5 expression, thereby restoring the neutrophil recruitment. In summary, these studies reveal that podocytes can negatively regulate neutrophil recruitment to inflamed GEnCs by modulating IL-6 signaling, identifying a potential novel anti-inflammatory role of IL-6 in renal glomeruli.