Endothelial dysfunction and renal fibrosis in endotoxemia-induced oliguric kidney injury: possible role of LPS-binding protein - PubMed (original) (raw)
doi: 10.1186/s13054-014-0520-2.
Alessandra Stasi, Angelica Intini, Margherita Gigante, Anna Maria Di Palma, Chiara Divella, Giuseppe Stefano Netti, Clelia Prattichizzo, Paola Pontrelli, Antonio Crovace, Francesco Staffieri, Enrico Fiaccadori, Nicola Brienza, Giuseppe Grandaliano, Giovanni Pertosa, Loreto Gesualdo
- PMID: 25261195
- PMCID: PMC4205288
- DOI: 10.1186/s13054-014-0520-2
Endothelial dysfunction and renal fibrosis in endotoxemia-induced oliguric kidney injury: possible role of LPS-binding protein
Giuseppe Castellano et al. Crit Care. 2014.
Abstract
Introduction: The pathophysiology of endotoxemia-induced acute kidney injury (AKI) is characterized by an intense activation of the host immune system and renal resident cells by lipopolysaccharide (LPS) and derived proinflammatory products. However, the occurrence of renal fibrosis in this setting has been poorly investigated. The aim of the present study was to investigate the possible association between endothelial dysfunction and acute development of tissue fibrosis in a swine model of LPS-induced AKI. Moreover, we studied the possible effects of coupled plasma filtration adsorption (CPFA) in this setting.
Methods: After 9 hours from LPS infusion and 6 hours of CPFA treatment, histologic and biochemical changes were analyzed in pigs. Apoptosis and endothelial dysfunction were assessed on renal biopsies. The levels of LPS-binding protein (LBP) were quantified with enzyme-linked immunosorbent assay (ELISA). Endothelial cells (ECs) were stimulated in vitro with LPS and cultured in the presence of swine sera and were analyzed with FACS and real-time RT-PCR.
Results: In a swine model of LPS-induced AKI, we observed that acute tubulointerstitial fibrosis occurred within 9 hours from LPS injection. Acute fibrosis was associated with dysfunctional alpha-smooth muscle actin (α-SMA)+ ECs characterized by active proliferation (Ki-67+) without apoptosis (caspase-3-). LPS led to EC dysfunction in vitro with significant vimentin and N-cadherin expression and increased collagen I mRNA synthesis. Therapeutic intervention by citrate-based CPFA significantly prevented acute fibrosis in endotoxemic animals, by preserving the EC phenotype in both peritubular capillaries and renal arteries. We found that the removal of LBP from plasma was crucial to eliminate the effects of LPS on EC dysfunction, by blocking LPS-induced collagen I production.
Conclusions: Our data indicate that EC dysfunction might be pivotal in the acute development of tubulointerstitial fibrosis in LPS-induced AKI. Selective removal of the LPS adaptor protein LBP might represent a future therapeutic option to prevent EC dysfunction and tissue fibrosis in endotoxemia-induced AKI.
Figures
Figure 1
LPS-induced AKI is associated with early development of tissue fibrosis. In a swine model of LPS-induced AKI, Masson trichrome staining (A) revealed extensive collagen deposition at the interstitial level (thin black arrow), along capillaries (zoomed image), and diffuse glomerular thrombi (thick black arrow) after 9 hours from LPS infusion compared with T0 and T9 of the control group. The pictures are displayed at 4× magnification (A, first line) and 10× magnification (A, second line). (C) In basal condition, no α-SMA+ cells (green) were localized in the interstitium and within glomeruli (white arrows). A dramatic increase in α-SMA expression was observed at tubulointerstitial level and within glomeruli and the Bowman capsule (white arrow). An early tubulointerstitial expression of α-SMA was found already at 1 hour with LPS injection. Magnification 630×. To-pro 3 was used to counterstain nuclei (blue). The quantitative analyses of Masson trichrome (B) and α-SMA staining (D) were obtained as described in the Methods section and expressed as median ± IQR of at least five independent pigs for each group. The ELISA tests (E, F) showed a significant increase of KIM-1 and cystatin C in swine sera at 6 and 9 hours from LPS administration compared with T0.
Figure 2
Recovery of hypotension, oligoanuria, and renal function in endotoxemic animals. (A) Endotoxemic pigs developed hypotension after 3 hours of LPS infusion. CPFA treatment restored blood pressure to basal condition. (B) Presence of oligoanuria was a clear sign of LPS-induced AKI. Urinary output was significantly reversed after 6 hours of CPFA treatment. CPFA treatment significantly reduced serum KIM-1 (C) and cystatin C (D) at 6 and 9 hours from LPS infusion.
Figure 3
Characterization of renal EC dysfunction in vivo . ECs were double-stained for CD31 (red) and α-SMA marker (green) further to demonstrate the occurrence of endothelial dysfunction. In renal vessels of endotoxemic tissue (C, dotted white arrow), CD31+EC expressed α-SMA marker and migrated from the intima to the media of the vessel wall (E, zoomed image), in otherwise basal condition (A, dotted white arrow). In the interstitium of T9 control pig (A, B) CD31+/α-SMA+ cells were rarely detectable. After 9 hours from LPS infusion, the number of these cells increased dramatically within glomeruli (C, white arrows) and interstitium (D, white arrows). Zoomed image of EC (F) co-expressing CD31 and α-SMA in endotoxemic tissue. Results are expressed as median ± IQR of the numbers of CD31+/α-SMA+ cells/HPF of at least five independent pigs for each group (G). Magnification, 630×. To-pro 3 was used to counterstain nuclei (blue).
Figure 4
Characterization of dysfunctional ECs in endotoxemia. Immunohistochemical analysis for caspase-3 showed rare apoptotic EC in endotoxemic pigs within glomeruli (D), at vascular (E) and peritubular levels (F, zoomed image, thin black arrow). Moreover, an increased number of tubular cells underwent apoptosis (F, zoomed image, thick black arrow). Caspase-3 nuclear expression in tubular cells (G) was analyzed as reported in the Methods section and expressed as median ± IQR of at least five independent pigs for each group. Magnification 10× (A, B, D, E) and 20× (C, F). Double immunofluorescence analysis (CD31 red; Ki-67 green) revealed Ki-67+/CD31+ proliferating ECs in endotoxemic pigs (I) compared with the T9 control group (H). Single-color images (J, K) underlined the co-localization of C31 (red J) and Ki-67 (green K) markers on ECs. Results are expressed as median ± IQR of the numbers of Ki-67+/CD31+ cells/HPF of at least five independent pigs for each group (L). Magnification, 630×. To-pro 3 was used to counterstain nuclei (blue).
Figure 5
LPS induced EC dysfunction and collagen I mRNA synthesis. Cultured ECs were incubated with LPS, 2 μg/ml, or LPS, 4 μg/ml, for 24 hours. (A) EC viability was evaluated with FACS analysis (AnnV/PI). Only a small percentage of ECs underwent apoptosis after LPS, 2 μg/ml (8.9% ±1.6 versus basal 5. 9% ±1.2) and LPS, 4 μg/ml (7.8% ±1.6) stimulation compared with ECs treated with H2O2 100 μ_M_ for 24 hours (positive control). (B) MTT cell-viability assay highlighted a significant proliferation of ECs after LPS, 2 μg/ml (0.720 ± 0.03 versus basal 24-hour 0.469 ± 0.004; P = 0.03) and LPS 4 μg/ml (0.597 ± 0.03 versus basal 24 hour 0.469 ± 0.004, P = 0.04) stimulation. (C, D) FACS analysis of EC showed the phenotypic changes induced by 24 hours of LPS stimulations. Results are expressed as mean ± SD and are representative of three independent experiments. (E) Real-time RT-PCR revealed the mRNA expression levels of collagen I. The gene relative expression was normalized to the expression of GAPDH. The histograms represent the mean ± SEM and are representative of three independent experiments.
Figure 6
Prevention of LPS-induced renal fibrosis by CPFA treatment. Renal biopsies after CPFA treatment showed a significant reduction in collagen deposits (A, thin arrow), in particular along capillaries (zoomed image), and glomerular thrombi (thick arrow) compared with endotoxemic condition. Magnification 6×. (C) Immunofluorescence analysis showed a strong expression of interstitial α-SMA (green) in endotoxemic pigs that was dramatically decreased by CPFA treatment (white arrow). Magnification, 630×. To-pro 3 was used to counterstain nuclei (blue). (E) Caspase-3 staining highlighted the efficacy of treatment (black arrows) in reversing LPS-induced tubular apoptosis. Magnification, 14×. The quantitative analyses of Masson trichrome (B), α-SMA (D), and caspase-3 (F) staining were obtained as described in the Methods section and expressed as median ± IQR of at least five independent pigs for each group.
Figure 7
Inhibition of EC dysfunction by CPFA treatment. (A) In endotoxemic animals, the reduced CD31 (red) expression at vascular level (dotted white arrow) was reversed after 6 hours of CPFA treatment (dotted white arrow). The increased number of CD31+/α-SMA+ cells induced by LPS infusion (white arrows) was significantly hampered in treated pigs (white arrows). (B) Results are expressed as median ± IQR of the numbers of CD31+/α-SMA+ cells/HPF of at least five independent animals for each group. (C) Comparison of zoomed peritubular capillaries of endotoxemic (white arrow) and treated endotoxemic pigs (white arrow). Magnification, 630×. To-pro 3 was used to counterstain nuclei (blue).
Figure 8
LBP is pivotal in LPS-mediated endothelial dysfunction. (A) ELISA revealed the increased level of LBP in sera of endotoxemic pigs comparing with the control group at T0 and T9. A considerable reduction in serum LBP levels was found after 6 hours of CPFA treatment. (B) Plasma samples drawn from the CPFA circuit were also analyzed. A significant decrease of LBP was found in the plasma filtrate effluent from the sorbent cartridge 1 hour after circuit installation (T4 plasma postcartridge). After 6 hours from the start of treatment, LBP levels in plasma postcartridge (T9 plasma postcartridge) were lower than in plasma precartridge. The histograms represent the median ± IQR of at least five independent animals for each group. (C-E) Cultured ECs were treated with sera of healthy (CTR), endotoxemic (LPS), and CPFA-treated endotoxemic (LPS CPFA) pigs. (C, D) FACS analysis of ECs showed phenotypic changes after 12 hours of LPS sera incubation. In the presence of LPS CPFA sera, EC preserved their phenotypes. After LBP addition in LPS CPFA sera, ECs showed phenotypic changes (LBP + LPS CPFA). (D) Results are representative of three independent experiments. (E) The expression of collagen I was detected by real-time RT-PCR. The gene relative expression was normalized to the expression of GAPDH. The histograms represent the mean ± SEM and are representative of three independent experiments.
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