Inhibition of intercellular adhesion molecule-1 with antisense deoxynucleotides prolongs renal isograft survival in the rat

Inhibition of intercellular adhesion molecule-1 with antisense deoxynucleotides prolongs renal isograft survival in the rat.BackgroundDelayed graft function from ischemia-reperfusion injury has a negative impact on long-term renal graft survival. We tested the utility of antisense oligodeoxynucleotide (ODN) against intercellular adhesion molecule-1 (ICAM-1) in the pretransplant treatment of renal isografts in improving long-term graft survival.MethodsThree groups of 16 inbred Lewis rats each underwent unilateral nephrectomy and were then transplanted with a kidney from a Lewis donor rat, which had received antisense ODN, reverse sense ODN, or saline vehicle six hours prior to nephrectomy. The kidneys were subjected to one hour of warm ischemia and 30minutes of cold ischemia, which when untreated results in delayed graft function. The remaining native kidney was removed 10days later. Serum creatinine and urinary protein excretion were measured in surviving rats at weeks 2, 4, 6, 8, 12, 16, and 20 after native nephrectomy.ResultsA Kaplan-Meier analysis revealed that by week 6 one half of the animals receiving reverse sense ODN and saline vehicle treatment had died, while all but 2 rats in the antisense ODN-treatment group survived to 20weeks. Serum creatinine concentrations and urine protein excretion of surviving reverse sense and saline vehicle-treated rats were significantly higher than antisense treated rats at every time point. Histology at week 20 revealed marked interstitial fibrosis, focal glomerular sclerosis, vascular intimal and medial thickening and tubular atrophy in reverse sense and saline vehicle-treated kidneys, while antisense ODN-treated kidneys showed only modest changes. Immunohistochemistry showed macrophage and lymphocyte infiltration, as well as substantial up-regulation of MHC class II, in reverse sense and saline vehicle-treated kidneys compared to antisense ODN-treated kidneys.ConclusionsThese results suggest that by ameliorating acute nonimmunological renal isograft injury, the long-term chronic nonimmunologic processes are improved as well. Furthermore, the data suggest that an antisense ODN strategy directed against ICAM-1 may have utility in human kidney transplantation

ICAM-1 antisense oligodesoxynucleotides prevent reperfusion injury and enhance immediate graft function in renal transplantation

BACKGROUND: Ischemia-reperfusion injury after organ transplantation is a major cause of delayed graft function. We showed earlier that antisense oligodesoxynucleotides (ODN) for intercellular adhesion molecule-1 (ICAM-1) ameliorate reperfusion injury after acute ischemia. This study tested the hypothesis that antisense ODN for ICAM-1 prevents ischemia-reperfusion injury and facilitates immediate graft function in a rat autotransplantation model. METHODS: Both kidneys were removed from male Lewis rats and re-implanted the left kidney after 30 minutes of cold ischemia time. The warm ischemia time was 60 minutes. Sham operated, uninephrectomized animals served as controls for renal function and histology. ICAM-1 antisense ODN (5 mg/kg), reverse ODN, or saline-vehicle were administered to donor animals i.v. six hours before autotransplantation. Glomerular filtration rate (insulin clearance), and serum creatinine concentrations were measured 24 hours post-transplantation. Tubular necrosis severity was assessed by histological grading scale. ICAM-1 expression was determined by immunohistochemistry and Western blot. RESULTS: Antisense ODN decreased ICAM-1 expression and leukocyte infiltration significant. Antisense ODN-treated animals showed significantly less tubular necrosis, than controls. Serum creatinine of antisense ODN-treated animals (N = 6) was 0.55 +/- 0.02 mg/dl compared to 1.92 +/- 0.07 mg/dl in reverse ODN-treated controls (N = 6; P < 0.01), 24 hours after transplantation. Antisense ODN-treated animals had normal GFR (0.93 +/- 0.07 ml/min/kidney wt) compared to sham-operated animals (0.95 +/- 0.09 ml/min/kidney wt), while autotransplanted animals treated with reverse ODN or saline-vehicle were all anuric. The ischemia-reperfusion-induced up-regulation of MHC class II was totally prevented by antisense ODN. CONCLUSIONS: ICAM-1 inhibition ameliorates ischemia-reperfusion injury and prevents delayed graft function. Antisense ODN-treatment of donors or donor organs for ICAM-1 may be useful for the prevention of reperfusion injury in human renal transplantation

Renal ischemia and transplantation predispose to vascular constriction mediated by angiotensin II type 1 receptor-activating antibodies

BACKGROUND: We previously described angiotensin II type 1 receptor-activating antibodies (AT1R-Abs) in renal transplant recipients with vascular rejection and malignant hypertension. In this study, we tested the hypothesis that AT1R-Abs can cause renal artery contraction by AT1R activation with renal ischemia representing a key permissive factor and therefore contribute to renal pathologic condition. METHODS: Isolated renal and mesenteric arteries from Lewis rats were incubated with purified AT1R-Abs from patients with human leukocyte antigen antibody-negative vascular rejection. Vascular contraction was measured using small vessel myography. The measurements were repeated with renal arteries derived from native kidneys subjected to ischemia-reperfusion or after transplantation in a low-responder Fischer 344-to-Lewis rat kidney-transplantation model. RESULTS: AT1R-Abs acted in a vascular bed-specific manner and caused small contractions only in native rat renal arteries but not in mesenteric arteries. AT1R-Abs did not alter the vascular reactivity to phenylephrine, angiotensin II, or acetylcholine in native renal arteries. In contrast, AT1R-Abs caused a pronounced (>10-fold) contraction of renal arteries after ischemia and after allogeneic transplantation. Pretreatment with pharmacologic AT1R blocker only partially inhibited the AT1R-Abs-induced contraction, which was almost completely abolished by neutralizing peptides targeting epitopes of AT1R-Abs on the second loop of AT1R. CONCLUSIONS: These data demonstrate that AT1R-Abs can induce renal vascular contraction under predisposing conditions such as in ischemic or transplanted kidneys. Neutralizing antibodies against specific epitopes in the AT1R can ameliorate this contraction

Inhibition of 20-HETE synthesis and action protects the kidney from ischemia/reperfusion injury

20-Hydroxyeicosatetraenoic acid (20-HETE) production is increased in ischemic kidney tissue and may contribute to ischemia/reperfusion (I/R) injury by mediating vasoconstriction and inflammation. To test this hypothesis, uninephrectomized male Lewis rats were exposed to warm ischemia following pretreatment with either an inhibitor of 20-HETE synthesis (HET0016), an antagonist (20-hydroxyeicosa-6(Z),15(Z)-dienoic acid), an agonist (20-hydroxyeicosa-5(Z),14(Z)-dienoic acid), or vehicle via the renal artery and the kidneys were examined 2 days after reperfusion. Pretreatment with either the inhibitor or the antagonist attenuated I/R-induced renal dysfunction as shown by improved creatinine clearance and decreased plasma urea levels, compared to controls. The inhibitor and antagonist also markedly reduced tubular lesion scores, inflammatory cell infiltration, and tubular epithelial cell apoptosis. Administering the antagonist accelerated the recovery of medullary perfusion, as well as renal medullary and cortical re-oxygenation, during the early reperfusion phase. In contrast, the agonist did not improve renal injury and reversed the beneficial effect of the inhibitor. Thus, 20-HETE generation and its action mediated kidney injury due to I/R. Whether or not these effects are clinically important will need to be tested in appropriate human studies

Involvement of functional autoantibodies against vascular receptors in systemic sclerosis

BACKGROUND: Systemic sclerosis (SSc) features autoimmunity, vasculopathy and tissue fibrosis. The renin-angiotensin and endothelin systems have been implicated in vasculopathy and fibrosis. A role for autoantibody-mediated receptor stimulation is hypothesised, linking three major pathophysiological features consistent with SSc. METHODS: Serum samples from 478 patients with SSc (298 in the study cohort and 180 from two further independent cohorts), 372 healthy subjects and 311 control-disease subjects were tested for antibodies against angiotensin II type 1 receptor (AT(1)R) and endothelin-1 type A receptor (ET(A)R) by solid phase assay. Binding specificities were tested by immunoprecipitation. The biological effects of autoantibodies in microvascular endothelial cells in vitro were also determined, as well as the quantitative differences in autoantibody levels on specific organ involvements and their predictive value for SSc-related mortality. RESULTS: Anti-AT(1)R and anti-ET(A)R autoantibodies were detected in most patients with SSc. Autoantibodies specifically bound to respective receptors on endothelial cells. Higher levels of both autoantibodies were associated with more severe disease manifestations and predicted SSc-related mortality. Both autoantibodies exert biological effects as they induced extracellular signal-regulated kinase 1/2 phosphorylation and increased transforming growth factor beta gene expression in endothelial cells which could be blocked with specific receptor antagonists. CONCLUSIONS: Functional autoimmunity directed at AT(1)R and ET(A)R is common in patients with SSc. AT(1)R and ET(A)R autoantibodies could contribute to disease pathogenesis and may serve as biomarkers for risk assessment of disease progression

Vascular receptor autoantibodies in pulmonary arterial hypertension associated with systemic sclerosis

Objective: Systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) portends worse outcome than other forms of PAH. Vasoconstrictive and vascular remodeling actions of endothelin-1 (ET-1) and angiotensin II (Ang II) via endothelin receptor type A (ETAR) and angiotensin receptor type-1 (AT1R) activation are implicated in PAH pathogenesis. We hypothesized that stimulating autoantibodies (Abs) targeting and activating AT1R and ETAR may contribute to SSc-PAH pathogenesis and tested their functional and biomarker relevance. Methods and Results: Anti-AT1R and -ETAR Abs detected by ELISA were significantly higher and more prevalent in patients with SSc-PAH (n = 81) and connective tissue disease (CTD)-associated PAH (n=110) compared to other forms of PAH/pulmonary hypertension (n=106). High anti-AT1R and anti-ETAR Abs predicted development of SSc-PAH and SSc-PAH-related mortality in a prospective analysis. Both Abs increased endothelial cytosolic Ca2+ concentrations in isolated perfused rat lungs which could be blocked by respective specific receptor antagonists. Ab-mediated stimulation of third to fourth-generation intralobar pulmonary rat artery ring segments in a myograph increased vasoconstrictive responses to Ang II and ET-1 and implicated cross-talk between both pathways demonstrated by reciprocal blockade with respective antagonists. Transfer of SSc-IgG containing both autoantibodies into healthy C57Bl/6J mice led to more abundant vascular and airway alpha-smooth muscle actin expression and inflammatory pulmonary vasculopathy. Conclusions: Anti-AT1R and -ETAR Abs are more frequent in SSc-PAH/CTD-PAH compared to other forms of PH and serve as predictive and prognostic biomarkers in SSc-PAH. Both antibodies may contribute to SSc-PAH via increased vascular endothelial reactivity and induction of pulmonary vasculopathy