Effects of acute aerobic exercise on leukocyte inflammatory gene expression in systemic lupus erythematosus.

UNLABELLED: Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC). METHODS: All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge. RESULTS: In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end of exercise (e.g., TLR3, IFNG, GATA3, FOXP3, STAT4) with a subsequent up-regulation occurring upon recovery. Exercise regulated the expression of inflammatory genes in the blood leukocytes of the SLE patients and HC, although the SLE groups exhibited fewer modulated genes and less densely connected networks (number of nodes: 29, 40 and 58; number of edges: 29, 60 and 195; network density: 0.07, 0.08 and 0.12, for SLEACTIVE, SLEINACTIVE and HC, respectively). CONCLUSION: The leukocytes from the SLE patients, irrespective of disease activity, showed a down-regulated inflammatory geneexpression immediately after acute aerobic exercise, followed by an up-regulation at recovery. Furthermore, less organized gene networks were observed in the SLE patients, suggesting that they may be deficient in triggering a normal exercised-induced immune transcriptional response.

Lung inflammation is induced by renal ischemia and reperfusion injury as part of the systemic inflammatory syndrome.

INTRODUCTION: Ischemia and reperfusion injury (IRI) are mainly caused by leukocyte activation, endothelial dysfunction and production of reactive oxygen species. Moreover, IRI can lead to a systemic response affecting distant organs, such as the lungs. AIM: The objective was to study the pulmonary inflammatory systemic response after renal IRI. METHODS: Male C57Bl/6 mice were subjected to 45 min of bilateral renal ischemia, followed by 4, 6, 12, 24 and 48 h of reperfusion. Blood was collected to measure serum creatinine and cytokine concentrations. Bronchoalveolar lavage fluid (BALF) was collected to determine the number of cells and PGE(2) concentration. Expressions of iNOS and COX-2 in lung were determined by Western blot. Gene analyses were quantified by real time PCR. RESULTS: Serum creatinine increased in the IRI group compared to sham mainly at 24 h after IRI (2.57 +/- 0.16 vs. 0.43 +/- 0.07, p < 0.01). The total number of cells in BAL fluid was higher in the IRI group in comparison with sham, 12 h (100 x 10(4) +/- 15.63 vs. 18.1 x 10(4) +/- 10.5, p < 0.05) 24 h (124 x 10(4) +/- 8.94 vs. 23.2 x 10(4) +/- 3.5, p < 0.05) and 48 h (79 x 10(4) +/- 15.72 vs. 22.2 x 10(4) +/- 4.2, p < 0.05), mainly by mononuclear cells and neutrophils. Pulmonary COX-2 and iNOS were up-regulated in the IRI group. TNF-alpha, IL-1beta, MCP-1, KC and IL-6 mRNA expression were up-regulated in kidney and lungs 24 h after renal IRI. ICAM-1 mRNA was up-regulated in lungs 24 h after renal IRI. Serum TNF-alpha, IL-1beta and MCP-1 and BALF PGE(2) concentrations were increased 24 h after renal IRI. CONCLUSION: Renal IRI induces an increase of cellular infiltration, up-regulation of COX-2, iNOS and ICAM-1, enhanced chemokine expression and a Th1 cytokine profile in lung demonstrating that the inflammatory response is indeed systemic, possibly leading to an amplification of renal injury.

Improved renal function after kidney transplantation is associated with heme oxygenase-1 polymorphism.

Heme oxygenase-1 (HO-1) has a microsatellite polymorphism based on the number of guanosine-thymidine nucleotide repeats (GT) repeats that regulates expression levels and could have an impact on organ survival post-injury. We correlated HO-1 polymorphism with renal graft function. The HO-1 gene was sequenced (N = 181), and the allelic repeats were divided into subclasses: short repeats (S) (<27 repeats) and long repeats (L) (>/=27 repeats). A total of 47.5% of the donors carried the S allele. The allograft function was statistically improved six months, two and three yr after transplantation in patients receiving kidneys from donors with an S allele. For the recipients carrying the S allele (50.3%), the allograft function was also better throughout the follow-up, but reached statistical significance only three yr after transplantation (p = 0.04). Considering only those patients who had chronic allograft nephropathy (CAN; 74 of 181), allograft function was also better in donors and in recipients carrying the S allele, two and three yr after transplantation (p = 0.03). Recipients of kidney transplantation from donors carrying the S allele presented better function even in the presence of CAN.

High serum levels of soluble Fas (sFas) in CKD patients: effects of renal clearance, reabsorption and synthesis.

PURPOSE: Increased serum concentrations of soluble Fas (sFas) have been reported in patients with chronic kidney disease (CKD). However, little is known about the renal clearance of sFas, whether sFas is reabsorbed in the renal tubules, or the behavior of sFas synthesis in CKD. MATERIALS AND METHODS: We studied 69 patients with CKD (60+/-15 years old, creatinine clearance 37+19 ml/min/1.73 m2) and 14 healthy subjects (61+/-17 years, creatinine clearance 79+/-24 ml/min/1.73 m2). ELISA was used to measure the levels of sFas (pg/mL) and retinol binding protein (RBP - mg/L). RT-PCR was used to quantify sFasmRNA of leukocytes. RESULTS: Serum sFas levels were significantly higher in patients with CKD (2781+/-1214 vs. 2196+/-773, p=0.02). The concentrations of sFas in 24-hour urine samples (23+/-27 vs. 40+/-17, p=0.006) and sFas Clearance (0.019+/-0.022 vs. 0.036+/-0.020, p=0.01) were significantly lower in patients with CKD. sFas clearance correlated with creatinine clearance (r=0.25, p=0.02). Urine concentrations of RBP correlated with sFas concentrations in the urine (r=0.80, p<0.001). sFasmRNA were higher in patients with CKD (3.9+/-1.8 vs. 2.5+/-0.9, p<0.001). CONCLUSIONS: In CKD patients, the decrease in renal function is followed by a decrease in sFas clearance and an increase in serum sFas. In patients with proximal tubule dysfunction (high urinary RBP concentrations), urinary sFas is also increased, suggesting that sFas is reabsorbed by the proximal tubule. It is possible that an increase in sFas synthesis also contributes to the increase of serum sFas concentrations in uremia.

The role of toll-like receptor 4 in cisplatin-induced renal injury.

BACKGROUND: Toll-like receptors recognize pattern-associated molecules found in pathogens as well as in endogen cells and in matrix degradation products. Despite the effectiveness of cisplatin against various solid tumors the administered dose is limited by its nephrotoxicity, namely, induction of tubular cell apoptosis. Herein, we investigated whether the cell toxicity of cisplatin was mediated by toll-like receptor 4 signaling. METHODS: C3H/He J (Toll-like receptor 4 deficient) and C3H/HePas (control) were treated with cisplatin (20 mg/kg). We evaluated renal function as well as expression of (HO-1) heme oxygenase 1 and MCP-1 mRNAs. RESULTS: Animals deficient in Toll-like receptor 4 showed less renal dysfunction after cisplatin therapy, which was more evident at later time points. Moreover, MCP-1 mRNA expression in kidneys from these animals were lower than controls, mainly at 96 hours after treatment. No differences were seen in HO-1 mRNA expression. CONCLUSIONS: These results suggested that cisplatin-induced renal toxicity is mediated in part though toll-like receptor 4.

Mesenchymal stem cells ameliorate tissue damages triggered by renal ischemia and reperfusion injury.

BACKGROUND: Ischemia and reperfusion injury (I/R) is the major cause of acute renal failure (ARF) with high mortality rates. Because alternative therapies are needed, we investigated the use of stem cell therapy to modulate inflammation in a renal I/R model. METHODS: To study kidney I/R injury, we clamped bilateral pedicles for 60 minutes. Mesenchymal stem cells (MSC), which had been isolated and cultivated in plastic flasks, were administered to mice 6 hours after injury. Real-time polymerase chain reaction was used to quantify interleukin (IL)-4 and IL-1beta mRNAs. Proliferative nuclear cell antigen (PCNA) was used to calculate tubular regeneration. RESULTS: Administration of MSC attenuated renal injury; serum creatinine and plasma urea levels were significantly reduced 24 hours after reperfusion. PCNA immunohistochemistry showed that regeneration occurred faster in renal tissues of animals that received MSC than in tissues of control animals. Analyses of cytokine expression in renal tissue demonstrated a greater level of anti-inflammatory cytokines in MSC-treated animals. CONCLUSION: These results showed an antiinflammatory pattern in MSC-treated animals, demonstrating the potential of MSC to modulate I/R, leading to earlier regeneration of damaged renal tissue.

The role of immunosuppressive drugs in aggravating renal ischemia and reperfusion injury.

UNLABELLED: Renal ischemia followed by reperfusion leads to acute renal failure in both native kidneys and renal allografts. Cyclosporine has known nephrotoxic effects. Thus, cyclosporine therapy subsequent to ischemia/reperfusion (I/R) injury may further exacerbate graft dysfunction. Rapamycin is a newer agent that suppresses the immune system by a different mechanism. In the present study, the effects of Cyclosporine and rapamycin at low and higher concentrations were investigated in an I/R-induced injury model. METHODS: Cyclosporine (100 mg/kg or 50 mg/kg), rapamycin (3 mg/kg per day or 1.5 mg/kg), or both were administered to mice before being subjected to 45 minutes of ischemia. Blood and kidney samples were collected at 24, 48, and 120 hours after surgery. We quantified acute tubular necrosis and tubular regeneration. RESULTS: Animals subjected to I/R showed impaired renal function that peaked at 24 hours (2.05 +/- 0.23 mg/dL), decreasing thereafter. Treatment with higher concentrations of cyclosporine or rapamycin caused even more renal dysfunction at 48 hours, which was sustained up to 120 hours after reperfusion (1.53 +/- 0.6 mg/dL), when compared to the low concentrations of cyclosporine or rapamycin (1.08 +/- 0.19 mg/dL; 0.99 +/- 0.14 mg/dL, P < .05, respectively). Cyclosporine delayed tubular regeneration, which was higher in controls at day 5 (67.0% vs 37.6%, P < .05). CONCLUSIONS: These results demonstrated that cyclosporine or rapamycin might further aggravate ischemically injured organs, negatively affecting posttransplantation recovery in a concentration-dependent fashion.

A role for HO-1 in renal function impairment in animals subjected to ischemic and reperfusion injury and treated with immunosuppressive drugs.

INTRODUCTION: Ischemia/reperfusion injury (IRI) represents the single major antigen-independent factor implicated in pathogenesis of chronic graft dysfunction. Tacrolimus is a calcineurin inhibitor, which has been suggested to be helpful in cyclosporine-related chronic toxicity. Rapamycin has antiproliferative properties that may impair renal regeneration after IRI. Therefore, immunosuppressive drugs might impair renal graft outcome in those organs suffering IRI. MATERIAL AND METHODS: C57B1/6 male mice subjected to 45 minutes of renal pedicle ligation were reperfused for 24 hours. Mice were treated with rapamycin, cyclosporine, or tacrolimus. Blood and renal tissue samples were collected at 24 hours after IRI. Urea levels were measured. Heme Oxygenase 1 (HO-1) gene transcript was amplified by a real-time polymerase chain reaction technique. RESULTS: Animals treated with cyclosporine and subjected to IRI showed impaired renal function that peaked at 24 hours. Additional pretreatment with rapamycin produced even more impairment of renal function, when compared with controls. However, tacrolimus pretreatment was associated with a better renal outcome. HO-1 expression was upregulated after IRI by 2.6 arbitrary units at 24 hours. Rapamycin showed worse impairment of renal function. CONCLUSION: Tacrolimus was not associated with worsening renal function when compared with animals just subjected to IRI. Upregulation of HO-1 may be an attractive approach to limit graft injury.

The effects of rapamycin in the progression of renal fibrosis.

UNLABELLED: Renal fibrosis is a hallmark of end-stage renal diseases and of chronic allograft nephropathy (CAN). Rapamycin, besides its action through blockade of lymphocyte proliferation, also has antiproliferative, antiviral, and antitumor actions. Its use in clinical in patients with CAN has recently been advocated. OBJECTIVES: Our goal was to evaluate the effect of rapamycin in an established model of renal fibrosis, unilateral ureteral obstruction. MATERIALS AND METHODS: C57BL/6 mice were divided into two groups, treated or not with daily doses of rapamycin (0.2 mg/kg) beginning on day-1. The obstruction was performed as day 0. Blood and kidney tissues were collected at 1, 4, 7, and 14 days after the surgery to quantify bone morphogenic protein (BMP)-7 and transforming growth factor (TGF)-beta mRNA by real time PCR. RESULTS: Daily treatment with rapamycin caused a significant reduction in serum creatinine at day 1 (0.57 +/- 0.03 vs 0.95 +/- 0.15 mg/dL, P = .002) and at day 14 (0.56 +/- 0.04 vs 0.73 +/- 0.07 mg/dL, P = .040). This profile was corroborated by histological morphometric analyses showing less fibrosis at day 14. However, rapamycin surprisingly induced an upregulation of TGF-beta at day 4 (3.05 +/- 0.46 vs 1.85 +/- 0.41, P = .006) and at day 7 (6.33 +/- 0.55 vs 4.97 +/- 0.38, P = .024) with a reduced expression by day 14 (4.03 +/- 1.07 vs 7.89 +/- 0.83, P < .001). Surprisingly, rapamycin also promoted an increment in BMP-7, completely reversing the ratio of TGF-beta to BMP-7, allowing a more protective phenotype. CONCLUSION: Rapamycin slightly ameliorated the renal dysfunction and, at later time points, induced less fibrosis and less decrease in the TGF-beta to BMP-7 ratio.

The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells.

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 +/- 5.85% (from 162 +/- 10 to 73 +/- 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 +/- 2.7 vs 91.2 +/- 0.9%, P < 0.02; 35.96 +/- 11.64 vs 22.19 +/- 9.65%, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 microM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.

FOXP3-positive regulatory cells inside the allograft and the correlation with rejection.

BACKGROUND: Foxp3 is a transcription factor associated with regulatory T cells. Little is known about the role of Foxp3+ regulatory T cells in relation to graft rejection in humans. METHODS: By using a quantitative polymerase chain reaction assay, we measured the levels of messenger RNA (mRNA) for Foxp3 in 27 samples obtained at allograft nephrectomy for acute nonvascular rejection (ANVR; n = 7), or acute vascular rejection (AVR; n = 15), or loss due to a nonimmune cause (LNIC; n = 5, as control). Granzyme B was also analyzed as a positive control for the host-driven immune response. RESULTS: Median Foxp3 mRNA levels correlated with the severity of rejection: LNIC 1.000, ANVR 1.429, and AVR 3.904 (P = .022 for LNIC and AVR by the Kruskal-Wallis test). The receiver operating characteristic curve for AVR demonstrated an area under the curve of 0.733 (P = .04; 95% CI, 0.528-0.939). The levels of granzyme B mRNA also showed the same profile but did not reach statistical significance. CONCLUSIONS: The presence of mRNA for Foxp3 inside the graft suggested specific homing during severe episodes of acute rejection. Its presence may indicate the development of host immunoregulatory responses during the ongoing cytolytic activity. In addition, assessment of Foxp3 mRNA inside the graft may distinguish vascular from nonvascular rejection.

Dual response of animals with chronic cyclosporine nephrotoxicity to an acute ischemic injury.

Normal kidneys regenerate after acute injury with little development of chronic fibrosis. However, the long-term effects of an acute injury in kidneys with established chronic toxicity induced by cyclosporine (CsA) are not entirely clear. To study the consequences of an ischemia and reperfusion (IR) injury in long-term CsA-treated rats, male Wistar rats (250-300 g) were treated daily with CsA (10 mg/kg) or vehicle (olive oil 1 mL/kg) for 28 days. On day 21, ischemia was performed by clamping the renal vessel for 1 hour. Blood samples were collected on days 0 and 21 (before IR) as well as days 22 and 28. On day 28, the kidneys were collected to examine the mRNA expression of MCP-1 by real-time PCR. For renal function, serum creatinine levels were measured. Twenty-four hours after reperfusion, long-term CsA-treated animals showed better renal function compared with the control group, as demonstrated by serum creatinine levels: 2.2 +/- 0.13 mg/dL vs 2.9 +/- 0.18 mg/dL, respectively (P < .05). However, 1 week after IR, the renal function was worse among the long-term CsA-treated group than the controls: 1.16 +/- 0.08 mg/dL vs 0.8 +/- 0.09 mg/dL, respectively (P < .05). Interestingly, CsA treatment was associated with lower MCP-1 mRNA expression than that in the control group: mean MCP-1 mRNA expression 0.58 +/- 0.13 vs 1.02 +/- 0.12, respectively (P < .05). In conclusion, animals with chronic CsA nephrotoxicity were protected from an acute renal injury, possibly through decreased chemokine production, although at later time points, renal function was clearly impaired, probably by the acceleration of vasculopathy caused by nephrotoxicity.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure.

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

High expression of Tim-3 mRNA in urinary cells from kidney transplant recipients with acute rejection.

TIM-3 is a recently described molecule specifically expressed on Th1 differentiated T cells. We explored the usefulness of urinary mRNA profiles in the diagnosis of renal acute rejection (AR). Sixty urinary samples from renal transplant recipients simultaneously collected to allograft biopsy, (AR = 30 and No-AR =30), and 12 urinary samples from stable renal transplants were analyzed. Urinary mRNA encoding for TIM-3 and IFN-gamma were quantified using real time RT-PCR. TIM-3 mRNA was highly expressed in AR (559.19 +/- 644.41) compared to No-AR (3.78 +/- 7.20), and stable transplants (0.54 +/- 0.76), p < 0.001. To a lesser degree, IFN-gamma mRNA transcripts were also increased in AR (50.40 +/- 38.71), compared with No-AR (4.69 +/- 12.62), and stable transplants (0.38 +/- 0.44) p < 0.001. The highest expression of TIM-3 in AR makes it a promising noninvasive test for its diagnosis.

Oligozoospermia and heat-shock protein expression in ejaculated spermatozoa.

BACKGROUND: Heat-shock protein A2 (HspA2) is correlated with sperm maturity, function and fertility, and a dysfunctional expression of such a gene results in abnormal spermatogenesis. The purpose of this study was to compare HspA2 gene expression in spermatozoa from oligozoospermic men and normozoospermic controls. METHODS: Semen was obtained and analysed according to World Health Organization (World Health Organization, 1999) guidelines, morphology by Kruger's strict criteria. Seventeen patients with oligozoospermia and 21 fertile controls were studied. Total RNA was extracted from ejaculated and Percoll density-gradient-separated spermatozoa followed by semiquantitative RT-PCR analysis. The relative expression level of HspA2 was analysed according to the expression level of the housekeeping beta-actin gene. Serum hormonal profiles (FSH, LH and testosterone) and a peripheral karyotype were also performed. RESULTS: All patients possessed normal karyotype, and no significant hormonal differences were found between the two groups. The study group had significantly lower sperm concentration and normal morphology than the controls. Semiquantitative RT-PCR analysis of HspA2 showed significantly lower expression levels in the oligoteratozoospermic men when compared to controls (P=0.0021). CONCLUSIONS: The HspA2 gene was down-regulated in sperm from infertile men with idiopathic oligoteratozoospermia, suggesting that such anomalies of gene expression might be associated with pathogenesis in some subtypes of male infertility.

The role of heme oxygenase 1 in rapamycin-induced renal dysfunction after ischemia and reperfusion injury.

Ischemia and reperfusion injury (IRI) is the main etiology of acute renal failure in native and transplanted kidneys. In the transplantation field, immunosuppressive drugs may play an additional role in acute graft dysfunction. Rapamycin may impair renal regeneration post IRI. Heme oxygenase 1 (HO-1) is a protective gene with anti-inflammatory and anti-apoptotic actions. We investigated whether HO-1 played a role in rapamycin-induced renal dysfunction in an established model of IRI. Rapamycin (3 mg/kg) was administered to mice before being subjected to 45 min of ischemia. Animals subjected to IRI presented with impaired renal function that peaked at 24 h (2.05+/-0.23 mg/dl), decreasing thereafter. Treatment with rapamycin caused even more renal dysfunctions (2.30+/-0.33 mg/dl), sustained up to 120 h after reperfusion (1.54+/-0.4 mg/dl), when compared to the control (0.63+/-0.09 mg/dl, P<0.05). Rapamycin delayed tubular regeneration that was normally higher in the control group at day 5 (68.53+/-2.30 vs 43.63+/-3.11%, P<0.05). HO-1 was markedly upregulated after IRI and its expression was even enhanced by rapamycin (1.32-fold). However, prior induction of HO-1 by cobalt protoporphyrin improved the renal dysfunction imposed by rapamycin, mostly at later time points. These results demonstrated that rapamycin used in ischemic-injured organs could also negatively affect post-transplantation recovery. Modulation of HO-1 expression may represent a feasible approach to limit rapamycin acute toxicity.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 ± 5.85 percent (from 162 ± 10 to 73 ± 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 ± 2.7 vs 91.2 ± 0.9 percent, P < 0.02; 35.96 ± 11.64 vs 22.19 ± 9.65 percent, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 æM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.