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TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis

2013; Elsevier BV; Volume: 85; Issue: 1 Linguagem: Inglês

10.1038/ki.2013.286

1523-1755

Chang Xu, Anthony Chang, Bradley K. Hack, Michael T. Eadon, Seth L. Alper, Patrick N. Cunningham,

Sepsis Diagnosis and Treatment

Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration permselectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower, whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-α activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse. Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration permselectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower, whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-α activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse. Acute kidney injury (AKI) is a frequent and serious complication of sepsis. The incidence of AKI is about 40% in patients with severe sepsis and septic shock. Moreover, there is strong evidence that AKI in patients with severe sepsis is associated with a higher mortality rate.1.Oppert M. Engel C. 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Properties of the glomerular barrier and mechanisms of proteinuria.Physiol Rev. 2008; 88: 451-487Crossref PubMed Scopus (608) Google Scholar In this study, we investigated the changes of glomerular endothelial fenestrae and ESL during severe experimental endotoxemia and TNF-induced AKI, and test the hypothesis that such changes may be related to signaling through TNFR1. We measured plasma urea levels as an indicator of glomerular filtration rate (GFR), and urine albumin-to-creatinine ratio to assess injury to the GFB. In wild-type (WT) mice, plasma urea levels increased from 28.8±2.8 to 112.5±9.5mg/dl (P<0.01) 24h after injection of LPS (10mg/kg; Figure 1a). LPS also induced significant weight loss (12.5±1.1%, P<0.01) compared with mice treated with normal saline (control; 2.6±0.6%; Figure 1c). The urinary albumin-to-creatinine ratio increased about 10-fold, from an initial value of 0.03±0.01 to a 24-h value of 0.30±0.06 (P<0.05; Figure 1b), despite the rapid decline in GFR. TNF-α release into the circulation followed LPS administration, and Tnfr1−/− mice were resistant to LPS-induced AKI.7.Cunningham P.N. Dyanov H.M. Park P. et al.Acute renal failure in endotoxemia is caused by TNF acting directly on TNF receptor-1 in kidney.J Immunol. 2002; 168: 5817-5823Crossref PubMed Scopus (313) Google Scholar We confirmed this finding and showed that plasma urea level was not elevated in Tnfr1−/− mice 24h after LPS injection, despite similar LPS-induced weight loss in Tnfr1−/− and WT mice (Figure 1a and c). In addition to protection from a fall in GFR, Tnfr1−/− mice had reduced albuminuria in response to LPS. Tnfr1−/− mice had a urine albumin-to-creatinine ratio of only 0.03±0.01 after LPS, significantly less than WT mice after LPS (0.30±0.6, P<0.05), and no different from WT control mice (Figure 1b). We did not compare Tnfr1−/− mice treated with normal saline with WT control mice, as previous data demonstrate similar baseline values of urinary albumin excretion and GFR in vehicle-treated WT and Tnfr1−/− mice.7.Cunningham P.N. Dyanov H.M. Park P. et al.Acute renal failure in endotoxemia is caused by TNF acting directly on TNF receptor-1 in kidney.J Immunol. 2002; 168: 5817-5823Crossref PubMed Scopus (313) Google Scholar,36.Chen C.C. Pedraza P.L. Hao S. et al.TNFR1-deficient mice display altered blood pressure and renal responses to ANG II infusion.Am J Physiol Renal Physiol. 2010; 299: F1141-F1150Crossref PubMed Scopus (37) Google Scholar Our results support the idea that TNF, acting through TNFR1, is a key mediator of LPS-induced AKI and albuminuria. As transport of water across the glomerular capillary wall occurs predominantly through the endothelial fenestrae, a reduction in the diameter and/or density of endothelial fenestrae can reduce endothelial filtration area and glomerular ultrafiltration coefficient (Kf). To explore whether sepsis-induced acute renal failure is accompanied by morphological changes in glomerular fenestrae, and whether such changes require TNFR1, we compared the ultrastructural morphology of the glomerular endothelium in LPS-untreated and LPS-treated WT mice with that of LPS-treated Tnfr1−/− mice. The glomerular capillary wall in control mice, as imaged by TEM, is shown lined with fenestrated endothelium, with fenestrae appearing circular when viewed en face in electron microscopic images (Figure 2a and d). However, LPS-treated WT mice show extensive detachment of glomerular ECs from their glomerular basement membranes (arrowheads, Figure 2b). The majority of glomerular ECs were often swollen, devoid of fenestrae, and detached from their glomerular basement membranes (although intact fenestrae are evident at the bottom right of Figure 2b). The glomerular basement membrane itself and adjacent podocytes were normal without podocyte detachment or effacement (Figure 2b). However, in LPS-treated Tnfr1−/− mice, glomerular ECs appear normal, with minimal detachment from the glomerular basement membranes (Figure 2c). Fenestral density per μm capillary length as measured in electron micrographs was 3.6±0.5 in the WT control mice, which was significantly higher than that in the WT mice 24h after the LPS injection (0.6±0.2). In contrast, fenestral density in the Tnfr1−/− mice 24h post LPS injection (3.2±0.3) was indistinguishable from that of WT control (Figure 1d). In en face electron microscopic images, the fenestral diameters were much larger in the LPS-treated mice (195±16.4nm) than in saline-injected WT controls (64.2±2.4nm; Figure 2e). The average diameter of the endothelial fenestrae in LPS-treated Tnfr1−/− mice was 75.5±2.5nm, which was significantly smaller than that in LPS-treated WT mice (Figure 1e). In conclusion, LPS treatment significantly increased the size of glomerular EC fenestrae but decreased fenestral density, and both effects were completely prevented by the absence of TNFR1. Although LPS increased fenestral diameter, the fenestrated fraction along the glomerular capillary loop (average fenestral density/μm × average fenestral diameter in μm) was around 12%, which was much smaller than the 23% value in untreated WT mice. To confirm the importance of circulating TNF acting alone, we injected recombinant TNF intravenously into mice. Injected TNF (2.5μg) indeed not only decreased GFR but also produced moderate tubular injury resembling that associated with LPS injection (Figure 3). This TNF-induced AKI corresponds to a serum level of TNF of 6.7±1.3ng/ml measured 2h after TNF injection, which falls in the same range as that 2h after LPS challenge (3–10ng/ml).37.Marino M.W. Dunn A. Grail D. et al.Characterization of tumor necrosis factor-deficient mice.Proc Natl Acad Sci U S A. 1997; 94: 8093-8098Crossref PubMed Scopus (693) Google Scholar,38.Alexander J.J. Jacob A. Cunningham P. et al.TNF is a key mediator of septic encephalopathy acting through its receptor, TNF receptor-1.Neurochem Int. 2008; 52: 447-456Crossref PubMed Scopus (179) Google Scholar In contrast, AKI was not induced by low-dose TNF (0.5μg), yielding a serum TNF level of 0.6±0.3ng/ml (Figure 3a). To explore whether TNF alone induces morphological changes in glomerular fenestrae similar to those of LPS-induced AKI, we compared the ultrastructural morphology of the glomerular endothelium in TNF-treated and matched control mice. The glomerular capillary wall in control mice, as imaged by TEM, was lined with fenestrated endothelium. Fenestrae viewed en face in electron microscopic images appeared circular (Figure 4a and c). In contrast, TNF-treated mice showed extensive loss of fenestrae (Figure 4b). En face electron microscopic images revealed fenestral diameters much larger in TNF-treated mice (141.5±10.7nm) than in saline-injected controls (77.1±2.7nm; Figure 4c and d). In conclusion, treatment with TNF alone had a similar effect as LPS on glomerular EC fenestrae; both significantly increased the size of glomerular EC fenestrae but decreased fenestral density.Figure 4Transmission electron microscopic ultrastructure of glomerular endothelial cells in glomeruli of wild-type mice 24h after intravenous administration of saline (control, a, c) or 2.5μg of tumor necrosis factor-α (TNF-α; b, d). (a, b) Loss of endothelial fenestrae in the glomerular capillaries in TNF-treated mice compared with controls. (c, d) High-magnification electron micrographs, with en face view of the fenestrae, showing enlarged glomerular endothelial fenestrae in TNF-treated mice. Scale bar, 1μm. Arrows indicate endothelial fenestrae.View Large Image Figure ViewerDownload (PPT) Vascular endothelial growth factor (VEGF) is an important molecule known to induce fenestrae in vivo. It has been reported that kidney but not plasma VEGF protein levels significantly decreased 24h after LPS injection, which was associated with increased circulation of soluble Flt-1.39.Yano K. Liaw P.C. Mullington J.M. et al.Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality.J Exp Med. 2006; 203: 1447-1458Crossref PubMed Scopus (228) Google Scholar We examined the effect of LPS on the expression of VEGF in mouse kidneys. LPS treatment significantly decreased kidney VEGF mRNA levels measured by reverse transcriptase-PCR at 6 and 24h after injection (Figure 5a). Similarly, kidney VEGF protein levels were significantly decreased to 55.6±3.8% of control levels (100.0±7.7, P<0.01) 24h after LPS treatment (Figure 5b). We also investigated whether LPS affects the expression of the main VEGF receptor, VEGFR2, in glomerular ECs. In control kidneys, VEGFR2 was highly expressed in glomeruli as detected by immunofluorescence, but levels of neither VEGFR2 protein (Figure 6a and b) nor mRNA (Figure 6c) were significantly changed 24h after LPS treatment (Figure 6c).Figure 6Effect of lipopolysaccharide (LPS) on vascular endothelial growth factor receptor 2 (VEGFR2) expression in the kidney. (a, b) Indirect immunofluorescence photomicrographs of frozen kidney cortex sections from wild-type (WT) control mice (a) and WT mice treated 24h with 10/ mg/kg LPS (b), incubated with anti-VEGFR2 antibody. LPS did not change VEGFR2 expression in glomeruli. (c) Time-dependent effect of LPS (10mg/kg, 6, 24, and 48h) on renal VEGFR2 mRNA expression, as normalized to 18S mRNA expression. Values are means±s.e.m. for 4–6 animals. Analysis of variance shows no significant difference between the LPS and control groups. Scale bar 20μm.View Large Image Figure ViewerDownload (PPT) To examine whether LPS-induced AKI is associated with damage of the glomerular ESL, kidney cryostat sections taken from mice 24h after LPS or control injections were stained with wheat germ agglutinin (WGA), a lectin that binds to negatively charged sugar residues of glycoproteins, such as sialic acid.40.Bhavanandan V.P. Katlic A.W. The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid.J Biol Chem. 1979; 254: 4000-4008Abstract Full Text PDF PubMed Google Scholar WGA-labeled glomerular ECs in both control and LPS-treated mice, as shown by co-staining with endothelial markers vascular endothelial (VE)-cadherin and CD31. LPS treatment decreased WGA staining of glomerular ECs (Figure 7a–f) by 33% relative to control glomeruli (P<0.01; Figure 7o). We further confirmed that LPS injection disrupted the endothelial ESL by studying its effect on the most abundant PGs of the ESL, those containing HS glycosaminoglycan chains. Some of these PGs are secreted and others are membrane bound.41.Sörensson J. Björnson A. Ohlson M. et al.Synthesis of sulfated proteoglycans by bovine glomerular endothelial cells in culture.Am J Physiol Renal Physiol. 2003; 284: F373-F380Crossref PubMed Scopus (41) Google Scholar,42.Singh A. Satchell S.C. Neal C.R. et al.Glomerular endothelial glycocalyx constitutes a barrier to protein permeability.J Am Soc Nephrol. 2007; 18: 2885-2893Crossref PubMed Scopus (207) Google Scholar Immunostaining with anti-HS antibody mostly colocalized with VE-cadherin (data not shown), and again revealed substantial reduction in WT mice after LPS exposure (Figure 7m and n). TNF injection itself also reduced in WGA staining in glomerular ECs (Figure 7j–l). To identify the changes to heparanase expression that might be responsible for LPS-induced ESL damage, heparanase localization and levels were examined by confocal microscopy and immunoblotting. Heparanase was highly expressed in glomeruli, as shown by co-staining with nephrin (Figure 8). LPS treatment of mice markedly increased glomerular loop staining of heparanase (Figure 8a–f). Immunoblotting also revealed increased heparanase polypeptide levels in LPS-treated kidneys (279.6±31.9%) compared with the control group (100.0±13.8%, P<0.01; Figure 8g). TNF treatment similarly increased glomerular heparanase expression (data not shown). Neither glomerular heparanase staining nor glomerular WGA staining changed significantly in LPS-treated Tnfr1−/− mice compared with control untreated mice, as shown in Supplementary Figure S1 online. Immunoblotting also confirmed unchanged heparanase protein levels in LPS-treated Tnfr1−/− kidneys as compared with the control group (data not shown). Download .jpg (.48 MB) Help with files Supplementary figure To investigate whether the glomerular endothelial cell TJs were disrupted in LPS and TNF-induced endotoxemia, we examined localization and abundance of VE-cadherin, an endothelium-specific member of the cadherin family, and of platelet endothelial cell adhesion molecule-1 (CD31), an immunoglobulin-like cell adhesion molecule concentrated at sites of endothelial cell–cell contact.43.Albelda S.M. Muller W.A. Buck C.A. et al.Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.J Cell Biol. 1991; 114: 1059-1068Crossref PubMed Scopus (611) Google Scholar Confocal immunofluorescence studies on frozen kidney sections showed that levels of VE-cadherin and CD31 in glomerular ECs were not decreased in mice 24h after treatment with either LPS or TNF (Figure 7). Our results show that LPS and intravenous TNF itself induce similar forms of renal damage, including ultrastructural alterations of glomerular endothelial fenestrae and diffuse alteration of glomerular ESL components, together contributing to increased albumin permeability and decreased GFR. The absence of these changes in glomerular endothelial morphology in LPS-treated Tnfr1−/− mice, in parallel with GFR preservation, demonstrates a key role for TNF-mediated glomerular endothelial injury in LPS-induced AKI, and strongly suggests a key role in the syndrome of sepsis-induced AKI. In this study, we demonstrate by TEM that LPS causes glomerular EC swelling and loss of fenestrae, without overt podocyte injury. Similar renal pathology has been noted in patients with pre-eclampsia.44.Karumanchi S.A. Maynard S.E. Stillman I.E. et al.Preeclampsia: a renal perspective.Kidney Int. 2005; 67: 2101-2113Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar In patients with type 2 diabetes, loss of glomerular EC fenestration correlated with albuminuria and GFR reduction,45.Weil E.J. Lemley K.V. Mason C.C. et al.Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy.Kidney Int. 2012; 82: 1010-1017Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar although significant podocyte detachment was also observed in this report. Reduced numbers and increased diameters of glomerular EC fenestrae are quantifiable structural features of nephropathy in LPS-induced sepsis. Ours is the first study to demonstrate an association between loss of normal glomerular EC fenestration and declining GFR in an established endotoxin model of sepsis. A reduction in density of endothelial fenestrations with consequently reduced glomerular hydraulic permeability could be responsible for the decline in GFR. This is also the first study to demonstrate a similar loss of fenestrae in AKI induced by intravenous administration of TNF. The underlying mechanisms for the changes of glomerular endothelial fenestrae in sepsis were investigated. Knockout of TNFR1, which in kidney is predominantly expressed in the glomerular endothelium,8.Al-Lamki R.S. Wang J. Skepper J.N. et al.Expression of tumor necrosis factor receptors in normal kidney and rejecting renal transplants.Lab Invest. 2001; 81: 1503-1515Crossref PubMed Scopus (109) Google Scholar prevented LPS-induced loss of endothelial fenestrae. TNF-α alone induced a