Csaba Révész

Complement activation by polyethoxylated pharmaceutical surfactants: Cremophor-EL, tween-80 and tween-20

Immunosafety analysis of pharmaceutical surfactants is an important step in understanding the complex mechanisms by which they induce side effects in susceptible patients. This paper provides experimental evidences that polyethoxylated surfactants, Cremophor-EL and Tween-80, also known as Polysorbate-80, activate the complement system in vitro, in normal human serum and plasma. They appeared to be more efficient reactogens than their structural homolog, Tween-20. Cremophor-EL and Tween-80 promoted the generation of biologically active complement products, C3a, C5a and C5b-9. Consistently, Paclitaxel and Taxotere (Docetaxel), pharmaceuticals formulated in Cremophor-EL and Tween-80, activated the complement system in similar extent. Moreover, comparison of serum reactivity against the drug-loaded and drug-free formulations exhibited a significant linear correlation. Taken together, these results are consistent with the hypothesis that therapeutic side effects, such as acute hypersensitivity and systemic immunostimulation, caused by intravenous nanomedicines containing polyethoxylated detergents such as Cremophor-EL and Tween-80, can be attributed to complement activation-derived inflammatory mediators.

Proteomic identification of vanin-1 as a marker of kidney damage in a rat model of type 1 diabetic nephropathy

At present, the urinary albumin excretion rate is the best noninvasive predictor for diabetic nephropathy (DN) but major limitations are associated with this marker. Here, we used in vivo perfusion technology to establish disease progression markers in an animal model of DN. Rats were perfused with a reactive ester derivative of biotin at various times after streptozotocin treatment. Following homogenization of kidney tissue and affinity purification of biotinylated proteins, a label-free mass spectrometry-based proteomic analysis of tryptic digests identified and relatively quantified 396 proteins. Of these proteins, 24 and 11 were found to be more than 10-fold up- or downregulated, respectively, compared with the same procedure in vehicle-treated rats. Changes in the expression of selected differentially regulated proteins were validated by immunofluorescence detection in kidney tissue from control and diabetic rats. Immunoblot analysis of pooled human urine found that concentrations of vanin-1, an ectoenzyme pantetheinase, distinguished diabetic patients with macroalbuminuria from those with normal albuminuria. Uromodulin was elevated in the urine pools of the diabetic patients, regardless of the degree of albuminuria, compared with healthy controls. Thus, in vivo biotinylation facilitates the detection of disease-specific changes in the abundance of potential biomarker proteins for disease monitoring and/or pharmacodelivery applications.

Activation of the miR-17 family and miR-21 during murine kidney ischemia-reperfusion injury.

BACKGROUND Ischemia-reperfusion (I/R) is the main cause of acute kidney injury (AKI) in patients. We investigated renal microRNA (miRNA) expression profiles and the time course of changes in selected miRNA expressions after renal I/R to characterize the miRNA network activated during development and recovery from AKI. METHODS AND RESULTS One day after lethal (30 minutes) and sublethal (20 minutes) renal ischemia, AKI was verified by renal histology (tubular necrosis, regeneration), blood urea nitrogen (BUN) level, renal mRNA expression, and plasma concentration of neutrophil gelatinase-associated lipocalin (NGAL) in C57BL/6J mice. On the first day after 30-minute, lethal I/R miR-21, miR-17-5p, and miR-106a were elevated out of the 21 miRNAs successfully profiled on the Luminex multiplex assay. After 20-minute, sublethal I/R, renal miR-17-5p and miR-106a expressions were elevated on the first and second days of reperfusion, while miR-21 expression increased later and lasted longer. Renal miR-17-5p and miR-21 expressions correlated with each other. Renal function returned to normal on the fourth day after sublethal I/R. CONCLUSIONS Our results demonstrate that besides miR-21, miR-17-5p, and miR-106a are additionally activated during the maintenance and recovery phases of renal I/R injury. Furthermore, a correlation between renal miR-17-5p and miR-21 expressions warrants further investigation of how they may influence each other and the outcome of renal ischemia-reperfusion injury.

Immune Activation and Target Organ Damage Are Consequences of Hydrodynamic Treatment but Not Delivery of Naked siRNAs in Mice

Short-interfering RNAs (siRNAs), key mediators of RNA interference comprise a promising therapeutic tool, although side effects such as interferon (IFN) response are still not perfectly understood. Further, delivery to target organs is a major challenge, possibly associated with side effects including immune activation or organ damage. We investigated whether immune activation as a consequence of double-stranded RNA induced IFN response (Jak/STAT pathway activation or cytokine production) or target organ damage is induced by in vivo low-volume (LV) or high-volume (HV) hydrodynamic delivery or treatment with naked siRNA. NMRI mice were injected with naked siRNAs or saline by hydrodynamic injection (HDI) and positive control mice received polyinosinic-polycytidilic acid (poly I:C). LV (1 mL/mouse) and HV (10% of body weight) HDI were compared. After LV HDI, STAT1 and OAS1 gene expression inflammatory cytokine plasma levels and target organ injury were assessed. LV HDI induced slight alanine aminotransferase elevation and mild hepatocyte injury, whereas HV HDI resulted in high ALAT level and extensive hepatocyte necrosis. STAT1 or OAS1 was not induced by LV siRNA; however, HV saline led to a time-dependent slight increase in gene expression. Inflammatory cytokine plasma level and organ histology and functional parameters demonstrated no damage following LV HDI with or without siRNA. Our data demonstrate that naked siRNAs may be harnessed, without the induction of IFN response or immune activation, and that LV HDI is preferable, because HV HDI may cause organ damage.

Ezrin Is Down-Regulated in Diabetic Kidney Glomeruli and Regulates Actin Reorganization and Glucose Uptake via GLUT1 in Cultured Podocytes

Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes.

Oxidative/Nitrative Stress and Inflammation Drive Progression of Doxorubicin-Induced Renal Fibrosis in Rats as Revealed by Comparing a Normal and a Fibrosis-Resistant Rat Strain

Chronic renal fibrosis is the final common pathway of end stage renal disease caused by glomerular or tubular pathologies. Genetic background has a strong influence on the progression of chronic renal fibrosis. We recently found that Rowett black hooded rats were resistant to renal fibrosis. We aimed to investigate the role of sustained inflammation and oxidative/nitrative stress in renal fibrosis progression using this new model. Our previous data suggested the involvement of podocytes, thus we investigated renal fibrosis initiated by doxorubicin-induced (5 mg/kg) podocyte damage. Doxorubicin induced progressive glomerular sclerosis followed by increasing proteinuria and reduced bodyweight gain in fibrosis-sensitive, Charles Dawley rats during an 8-week long observation period. In comparison, the fibrosis-resistant, Rowett black hooded rats had longer survival, milder proteinuria and reduced tubular damage as assessed by neutrophil gelatinase-associated lipocalin (NGAL) excretion, reduced loss of the slit diaphragm protein, nephrin, less glomerulosclerosis, tubulointerstitial fibrosis and matrix deposition assessed by periodic acid–Schiff, Picro-Sirius-red staining and fibronectin immunostaining. Less fibrosis was associated with reduced profibrotic transforming growth factor-beta, (TGF-β1) connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL-1a1) mRNA levels. Milder inflammation demonstrated by histology was confirmed by less monocyte chemotactic protein 1 (MCP-1) mRNA. As a consequence of less inflammation, less oxidative and nitrative stress was obvious by less neutrophil cytosolic factor 1 (p47phox) and NADPH oxidase-2 (p91phox) mRNA. Reduced oxidative enzyme expression was accompanied by less lipid peroxidation as demonstrated by 4-hydroxynonenal (HNE) and less protein nitrosylation demonstrated by nitrotyrosine (NT) immunohistochemistry and quantified by Western blot. Our results demonstrate that mediators of fibrosis, inflammation and oxidative/nitrative stress were suppressed in doxorubicin nephropathy in fibrosis-resistant Rowett black hooded rats underlying the importance of these pathomechanisms in the progression of renal fibrosis initiated by glomerular podocyte damage.

Effects of vitamin D3 derivative – calcitriol on pharmacological reactivity of aortic rings in a rodent PCOS model

BACKGROUND The aim of this study was to examine the effects of the hyperandrogenic state in dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), the vascular responses to different vasoactive agents, and the modulatory role of vitamin D3. METHODS APCOS model was induced by DHT application in 20 female Wistar rats. Ten of the DHT treated rats simultaneously received calcitriol treatment. After 10 weeks, myographs were used to test the reactivity of isolated thoracic aortic rings to norepinephrine and acetylcholine. Thereafter, the vascular rings were incubated with the NO-synthase blocker (nitro-L-arginine methyl ester) or the cyclooxygenase inhibitor (indomethacin) for 20 min, and the effects of norepinephrine and acetylcholine were re-evaluated. RESULTS Norepinephrine-induced vasoconstriction was enhanced after DHT treatment, but this effect was attenuated by calcitriol administration. Vasorelaxation of DHT-treated thoracic aortic rings was impaired, but this could be partly reversed by calcitriol application. Impaired NO-dependent vasorelaxation in DHT-treated animals was mostly reversed by concomitant calcitriol administration, but this effect was diminished by prostanoid-dependent vasoconstriction. CONCLUSIONS These studies show that the enhanced sensitivity to vasoconstrictors and impaired NO-dependent vasorelaxation in hyperandrogenic PCOS rats could be partially reversed by calcitriol treatment.

Urine/Plasma Neutrophil Gelatinase Associated Lipocalin Ratio Is a Sensitive and Specific Marker of Subclinical Acute Kidney Injury in Mice

Background Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia. Methods Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression. Results A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups. Conclusions These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.

Altered insulin-induced relaxation of aortic rings in a dihydrotestosterone-induced rodent model of polycystic ovary syndrome

OBJECTIVE To clarify the effects of dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS) on the insulin-dependent vasodilatation of the thoracic aorta and the role of vitamin D in a rat model. DESIGN Controlled experimental animal study. SETTING Laboratory. ANIMAL(S) Thirty adolescent female Wistar rats. INTERVENTION(S) The PCOS model was induced by 10 weeks of DHT treatment (83 μg/d). One-half of the DHT-treated animals also received vitamin D (120 ng/kg/wk). MAIN OUTCOME MEASURE(S) The aortic rings of the control, DHT, and DHT plus vitamin D-treated animals were isolated. The insulin-dependent vasodilation of the isolated aortic rings was compared in Krebs-Ringer solution and under blockade of nitric oxide (NO) synthase or cyclooxygenase. RESULT(S) The insulin-dependent vasorelaxation decreased in both DHT-treated groups independently from the vitamin D treatment; NO-dependent and -independent relaxations were both impaired. In response to prostanoid, vasoconstriction was increased after DHT treatment. The NO-independent relaxation was partially improved by vitamin D treatment, which was neutralized by increased prostanoid-dependent vasoconstriction. CONCLUSION(S) Previously, we found that vitamin D treatment prevented systemic insulin resistance; however, in this study, we did not detect any influence on the vascular insulin resistance of the aorta that was induced by DHT treatment. Consequently, controlling insulin resistance with vitamin D alone did not resolve the aortic endothelial dysfunction caused by the hyperandrogenic state.

LPS-Induced Delayed Preconditioning Is Mediated by Hsp90 and Involves the Heat Shock Response in Mouse Kidney

Introduction We and others demonstrated previously that preconditioning with endotoxin (LPS) protected from a subsequent lethal LPS challenge or from renal ischemia-reperfusion injury (IRI). LPS is effective in evoking the heat shock response, an ancient and essential cellular defense mechanism, which plays a role in resistance to, and recovery from diseases. Here, by using the pharmacological Hsp90 inhibitor novobiocin (NB), we investigated the role of Hsp90 and the heat shock response in LPS-induced delayed renal preconditioning. Methods Male C57BL/6 mice were treated with preconditioning (P: 2 mg/kg, ip.) and subsequent lethal (L: 10 mg/kg, ip.) doses of LPS alone or in combination with NB (100 mg/kg, ip.). Controls received saline (C) or NB. Results Preconditioning LPS conferred protection from a subsequent lethal LPS treatment. Importantly, the protective effect of LPS preconditioning was completely abolished by a concomitant treatment with NB. LPS induced a marked heat shock protein increase as demonstrated by Western blots of Hsp70 and Hsp90. NB alone also stimulated Hsp70 and Hsp90 mRNA but not protein expression. However, Hsp70 and Hsp90 protein induction in LPS-treated mice was abolished by a concomitant NB treatment, demonstrating a NB-induced impairment of the heat shock response to LPS preconditioning. Conclusion LPS-induced heat shock protein induction and tolerance to a subsequent lethal LPS treatment was prevented by the Hsp90 inhibitor, novobiocin. Our findings demonstrate a critical role of Hsp90 in LPS signaling, and a potential involvement of the heat shock response in LPS-induced preconditioning.