Mária Godó

Elevated systemic TGF-β impairs aortic vasomotor function through activation of NADPH oxidase-driven superoxide production and leads to hypertension, myocardial remodeling, and increased plaque formation in apoE−/− mice

The role of circulating, systemic TGF-beta levels in endothelial function is not clear. TGF-beta(1) may cause endothelial dysfunction in apolipoprotein E-deficient (apoE(-/-)) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-mo-old control (C57Bl/6), apoE(-/-), TGF-beta(1)-overexpressing (TGFbeta(1)), and crossbred apoE(-/-) x TGFbeta(1) mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-mo-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-beta(1) levels (ELISA) were elevated in TGFbeta(1) mice without downregulation of TGF-beta-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in apoE(-/-) x TGFbeta(1) mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the apoE(-/-) x TGFbeta vs. C57Bl/6 mice. Our results demonstrate that elevated circulating TGF-beta(1) causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in apoE(-/-) mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGFbeta(1).

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.

Increased renoprotection with ACE inhibitor plus aldosterone antagonist as compared to monotherapies—the effect on podocytes

BACKGROUND Blockade of the renin-angiotensin-aldosterone system (RAAS) does not completely prevent progression of renal disease. Mineralocorticoid receptor blockade provides additional renoprotection over ACE-inhibition monotherapy. We examined the mechanisms underlying superior renoprotection in the subtotal nephrectomy (SNX) model. METHODS Sprague-Dawley rats were randomized into six groups: (1) sham-op, (2) SNX without treatment, (3) SNX + quinapril (Q), (4) SNX + spironolactone (S), (5) SNX + combination therapy (Q+S), (6) SNX + combination hydrochlorothiazide + reserpin + hydralazine (HRH). Albuminuria and blood pressure were monitored, and kidneys were examined by morphometric and molecular methods. RESULTS In SNX rats, albumin excretion was significantly higher than in sham-op rats. Blood pressure reduction was not significantly different between the treatment groups. All therapies (S, Q, Q+S and HRH) reduced albuminuria; the values were lowest in animals treated with Q+S. The volume density of glomerular matrix and the number of mesangial cells were significantly increased in SNX and were lowest in SNX treated with Q+S. The number of podocytes was reduced in SNX, but was normalized in SNX treated with Q+S. Glomerular volumes and podocyte volumes were significantly higher in SNX than in sham-op. Both volumes were reduced by all interventions, but almost normalized by treatment with Q+S. Expression of collagen IV, TGF-beta(1) and desmin was increased after SNX and significantly reduced by treatment with Q and Q+S. CONCLUSIONS In subtotally nephrectomized rats, mineralocorticoid blockade provided additional renoprotection over and above ACE inhibition. Such benefit was paralleled by major changes in podocyte number and morphology and was not blood pressure dependent.

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.

Role of invariant natural killer T (iNKT) cells in systemic lupus erythematosus

Natural killer T (NKT) cells represent a unique T cell lineage. The NKT cells bearing an invariant TCR (iNKT cells) recognize a small variety of glycolipid antigens in the context of CD1d (non-classical MHC-I) presentation. CD1d-restricted iNKT cells play a regulatory role during an immune response by producing cytokines (IFN-gamma, and IL-4). The identification of alpha-galactosyl-ceramide (alpha-GalCer), a marine sponge derivative as a potent stimulator of iNKT cells has raised the potential of therapeutic iNKT cell activation. Invariant NKT cells have been implicated in several different autoimmune diseases in mice and humans, including systemic lupus erythematosus (SLE). Abnormalities in the number and functions of NKT cells have been observed in SLE patients and mouse strains genetically predisposed to lupus (MRL/lpr, NZB/W F1). Moreover, inverse correlation between the frequency of NKT cells and IgG levels has been observed. Elevated IgG levels in relatives of patients with lupus as well as in patients with lupus were associated with low frequencies of NKT cells. This review focuses on the potential roles of NKT cells in the pathogenesis of SLE. It summarizes recent advances in glycolipid therapy for murine lupus. First, it has been demonstrated, that repeated administration of alpha-GalCer to MRL/lpr mice alleviated inflammatory dermatitis but did not influence kidney disease. Treatment of NZB/W mice with alpha-GalCer resulted in amelioration of SLE symptoms in young mice, but treatment of older animals resulted in disease exacerbation. The effects of NKT cell activation using alpha -GalCer, on disease progression, were influenced by a variety of parameters, including the genetic background of mice, the alpha -GalCer dose, number of injections and the stage of the disease process when treatment was performed. Manipulation of NKT cells in the human system may be a promising treatment alternative for the future, however possible deleterious effects have to be carefully investigated first.

Skin disease is prevented but nephritis is accelerated by multiple pregnancies in autoimmune MRL/LPR mice

The role of pregnancy in the progression of systemic lupus erythematosus (SLE) is still poorly understood. We analysed the effect of repeated pregnancies in MRL/lpr mice, a murine model of SLE. Seven-week old female mice were used: multiparous mice underwent three consecutive pregnancies (M); age-matched virgin mice served as controls (V). Animals were harvested at 20 weeks of age. Skin lesions were characterized by hair loss and scabs in the dorsum of the neck. Virgin skins showed thickened dermis, fibrosis and mononuclear cell infiltrates, which were practically absent in M. This was accompanied by higher IFN-γ and lower IL-10 mRNA expression levels in V compared to M skin. Plasma IFN-γ protein levels were also upregulated in V versus M. However, survival and kidney function were dramatically reduced and accompanied by hypertension after multiple pregnancies. Kidney histology also showed markedly increased renal lesions in M. In contrast to plasma and skin levels, both IL-10 and IFN-γ mRNA were lower in the kidneys of V versus M mice. Concluding our findings, the pathomechanisms of lupus kidney and skin disease may be regulated differently at the organ level during pregnancy. Both IFN-γ and IL-10 may be important regulatory cytokines at the local level. Lupus (2007) 16, 465—477.

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.

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.

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.