Valentina Campean

The Lysyl Oxidases LOX and LOXL2 Are Necessary and Sufficient to Repress E-cadherin in Hypoxia INSIGHTS INTO CELLULAR TRANSFORMATION PROCESSES MEDIATED BY HIF-1

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

Donor treatment with a PHD-inhibitor activating HIFs prevents graft injury and prolongs survival in an allogenic kidney transplant model.

Long-term survival of renal allografts depends on the chronic immune response and is probably influenced by the initial injury caused by ischemia and reperfusion. Hypoxia-inducible transcription factors (HIFs) are essential for adaptation to low oxygen. Normoxic inactivation of HIFs is regulated by oxygen-dependent hydroxylation of specific prolyl-residues by prolyl-hydroxylases (PHDs). Pharmacological inhibition of PHDs results in HIF accumulation with subsequent activation of tissue-protective genes. We examined the effect of donor treatment with a specific PHD inhibitor (FG-4497) on graft function in the Fisher–Lewis rat model of allogenic kidney transplantation (KTx). Orthotopic transplantation of the left donor kidney was performed after 24 h of cold storage. The right kidney was removed at the time of KTx (acute model) or at day 10 (chronic model). Donor animals received a single dose of FG-4497 (40 mg/kg i.v.) or vehicle 6 h before donor nephrectomy. Recipients were followed up for 10 days (acute model) or 24 weeks (chronic model). Donor preconditioning with FG-4497 resulted in HIF accumulation and induction of HIF target genes, which persisted beyond cold storage. It reduced acute renal injury (serum creatinine at day 10: 0.66 ± 0.20 vs. 1.49 ± 1.36 mg/dL; P < 0.05) and early mortality in the acute model and improved long-term survival of recipient animals in the chronic model (mortality at 24 weeks: 3 of 16 vs. 7 of 13 vehicle-treated animals; P < 0.05). In conclusion, pretreatment of organ donors with FG-4497 improves short- and long-term outcomes after allogenic KTx. Inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves clinical evaluation.

Atherosclerosis and Vascular Calcification in Chronic Renal Failure

Cardiovascular complications are a major clinical problem in patients with chronic kidney disease and end-stage renal failure; cardiac death accounts for approximately 40–50% of all deaths in these patients. Death from cardiovascular causes is up to 20 times more common in uremic patients than in the general population with the risk being even higher than in patients with diabetes mellitus. A high rate of myocardial infarction and excessive cardiac mortality have repeatedly been documented in patients with kidney disease and renal failure. Not only is the prevalence of myocardial infarction high, but also the case fatality rate is significantly higher in uremic patients with and without diabetes, respectively, compared to nonuremic patients. This is of particular interest since the prevalence of coronary atheroma in uremic patients was shown to be approximately 30% by autopsy and coronary angiography studies. Thus, coronary factors, i.e. atherosclerosis, and non-coronary factors may play an important role in the genesis of cardiac complications in the renal patient. In addition, renal failure recently has also be identified as a predictor of mortality in different stages of peripheral vascular disease. In particular, marked differences in the pathogenesis, morphology and course of atherosclerosis and arteriosclerosis under the conditions of renal failure have been documented. Among others increased plaque formation and particularly higher proportion and intensity of vascular calcification have been found in clinical and autopsy studies. In addition to the so-called classical or traditional risk factors, an important role for nonclassical risk factors such as microinflammation, hyperphosphatemia and oxidative stress has been documented in patients with renal failure and is discussed in detail.

Urinary Adiponectin Excretion: A Novel Marker for Vascular Damage in Type 2 Diabetes

OBJECTIVE Markers reliably identifying vascular damage and risk in diabetic patients are rare, and reports on associations of serum adiponectin with macrovascular disease have been inconsistent. In contrast to existing data on serum adiponectin, this study assesses whether urinary adiponectin excretion might represent a more consistent vascular damage marker in type 2 diabetes. RESEARCH DESIGN AND METHODS Adiponectin distribution in human kidney biopsies was assessed by immunohistochemistry, and urinary adiponectin isoforms were characterized by Western blot analysis. Total urinary adiponectin excretion rate was measured in 156 patients with type 2 diabetes who had a history of diabetic nephropathy and 40 healthy control subjects using enzyme-linked immunosorbent assay. Atherosclerotic burden was assessed by common carotid artery intima-media-thickness (IMT). RESULTS A homogenous staining of adiponectin was found on the endothelial surface of glomerular capillaries and intrarenal arterioles in nondiabetic kidneys, whereas staining was decreased in diabetic nephropathy. Low-molecular adiponectin isoforms (∼30–70 kDa) were detected in urine by Western blot analysis. Urinary adiponectin was significantly increased in type 2 diabetes (7.68 ± 14.26 vs. control subjects: 2.91 ± 3.85 μg/g creatinine, P = 0.008). Among type 2 diabetic patients, adiponectinuria was associated with IMT (r = 0.479, P < 0.001) and proved to be a powerful independent predictor of IMT (β = 0.360, P < 0.001) in multivariable regression analyses. In a risk prediction model including variables of the UK Prospective Diabetes Study coronary heart disease risk engine urinary adiponectin, but not the albumin excretion rate, added significant value for the prediction of increased IMT (P = 0.007). CONCLUSIONS Quantification of urinary adiponectin excretion appears to be an independent indicator of vascular damage potentially identifying an increased risk for vascular events.

The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

BackgroundChondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro.MethodsResected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed.ResultsExpression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment.ConclusionsThese findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma.

High-Dose Enalapril Treatment Reverses Myocardial Fibrosis in Experimental Uremic Cardiomyopathy

Aims Patients with renal failure develop cardiovascular alterations which contribute to the higher rate of cardiac death. Blockade of the renin angiotensin system ameliorates the development of such changes. It is unclear, however, to what extent ACE-inhibitors can also reverse existing cardiovascular alterations. Therefore, we investigated the effect of high dose enalapril treatment on these alterations. Methods Male Sprague Dawley rats underwent subtotal nephrectomy (SNX, n = 34) or sham operation (sham, n = 39). Eight weeks after surgery, rats were sacrificed or allocated to treatment with either high-dose enalapril, combination of furosemide/dihydralazine or solvent for 4 weeks. Heart and aorta were evaluated using morphometry, stereological techniques and TaqMan PCR. Results After 8 and 12 weeks systolic blood pressure, albumin excretion, and left ventricular weight were significantly higher in untreated SNX compared to sham. Twelve weeks after SNX a significantly higher volume density of cardiac interstitial tissue (2.57±0.43% in SNX vs 1.50±0.43% in sham, p<0.05) and a significantly lower capillary length density (4532±355 mm/mm3 in SNX vs 5023±624 mm/mm3 in sham, p<0.05) were found. Treatment of SNX with enalapril from week 8–12 significantly improved myocardial fibrosis (1.63±0.25%, p<0.05), but not capillary reduction (3908±486 mm/mm3) or increased intercapillary distance. In contrast, alternative antihypertensive treatment showed no such effect. Significantly increased media thickness together with decreased vascular smooth muscles cell number and a disarray of elastic fibres were found in the aorta of SNX animals compared to sham. Both antihypertensive treatments failed to cause complete regression of these alterations. Conclusions The study indicates that high dose ACE-I treatment causes partial, but not complete, reversal of cardiovascular changes in SNX.

Characterisation of renal immune cell infiltrates in children with nephrotic syndrome

There is increasing evidence that not only T cells but also B cells may play an important role in the pathogenesis of idiopathic nephrotic syndrome (NS). We have evaluated the infiltrating immune cells found in renal biopsies from 38 children with NS using immunohistochemistry techniques involving antibodies against T cells (CD3, CD4, CD8, FoxP3), B cells (CD20), macrophages (CD68) and follicular dendritic cells (CD21). Kidney biopsies with thin basement membrane disease were used as controls. We found higher numbers of interstitial CD3-positive T cells and macrophages in patients with focal segmental glomerulosclerosis (FSGS) than in those with minimal change glomerulopathy (MCGN) and in the controls, and significantly lower FoxP3-positive cells in patients with FSGS, MCGN and steroid-dependent NS than in the controls. Significantly higher numbers of glomerular B cells were found in FSGN patients than in MCGN patients and controls. Of note, in three patients who were later successfully treated with anti-CD20 antibody rituximab, the number of renal B cells was negligible in the preceding biopsy. In conclusion, the higher numbers of interstitial CD3-positive T cells in renal biopsies of pediatric patients with FSGS argue for a higher inflammatory activity. The significantly higher number of glomerular B cells in FSGS patients may indicate a particular pathogenetic role or epiphenomenon in this disease. However, patients with no interstitial or glomerular B cells could also benefit from rituximab treatment.

Changes in 11β-hydroxysteroid dehydrogenase type 2 expression in a low-protein rat model of intrauterine growth restriction

BACKGROUND Intrauterine growth restriction (IUGR) is associated with systemic hypertension of the offspring later in life. The exact mechanisms are still incompletely understood. 11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) in the distal renal tubule protects the mineralocorticoid receptor from cortisol. As we did not find a suppression of 11β-HSD2 in total kidney of IUGR animals, our objective was to investigate whether a suppression of 11β-HSD2 could be detected on a more sophisticated level such as in situ protein and gene expression of 11β-HSD2 in mildly hypertensive IUGR offspring. METHODS IUGR rats after maternal low-protein diet (n = 17) were compared with controls (n = 18). At 70 and 120 days of age, in situ distribution of 11β-HSD2 gene and protein expression was investigated by RT-PCR of microdissected tubules and immunohistochemistry. For in situ localization studies, double staining for 11β-HSD2 and calbindin was used. Serum levels of corticosterone and dehydrocorticosterone were measured by tandem mass spectrometry. RESULTS In IUGR rats, intra-arterial blood pressure significantly increased at Day 120 of life. Serum corticosterone/dehydrocorticosterone ratios and 11β-HSD2 mRNA in total kidney were not altered in IUGR animals. However, 11β-HSD2 mRNA concentration was significantly lower in microdissected tubuli of IUGR animals (Day 120: 0.18 ± 0.14 vs 1.00 ± 0.32 rel. units in controls; P < 0.05). In IUGR animals, immunostaining scores for 11β-HSD2 were significantly lower than in controls (P < 0.05). Double staining with calbindin showed lower expression of 11β-HSD2 in distal segments of the distal tubule. CONCLUSIONS Our data indicate lower gene and protein expression of the pre-receptor enzyme 11β-HSD2 in IUGR animals when looking at specific renal compartments, but not in total kidney extracts. Thus, lower 11β-HSD2 as a mechanism for hypertension later in life might be missed without methods for in situ detection.

Vascular inflammation and media calcification are already present in early stages of chronic kidney disease

BACKGROUND While patients with chronic kidney disease (CKD) have a high prevalence of classical coronary risk factors, there is increasing evidence that atherosclerosis is different in renal compared to nonrenal patients. Therefore, the present study compares changes in different vessels obtained at cardiac surgery between patients with early and advanced CKD and nonrenal control patients. METHODS AND RESULTS Fifty patients undergoing cardiac bypass surgery were divided into three groups: (i) 24 control patients with creatinine <1.3mg/dl, (ii) 14 patients with early CKD (creatinine 1.3-2.0mg/dl), and (iii) 12 patients with advanced CKD (creatinine >2.0mg/dl). Aorta, arteria mammaria interna, and vena saphena (V. saphena) were analyzed using morphometry, Kossa stain for vascular calcification, and immunohistochemistry for markers of inflammation and proosteogenic differentiation of vascular smooth muscle cells (VSMCs). Thereby, aortic wall thickness and calcification score of aortic intima and of V. saphena were significantly higher in advanced CKD patients than in nonrenal control patients, whereas significant vascular inflammation and proosteogenic dedifferentiation of VSMC and calcification of the aortic media were already present in early CKD. Interestingly, marked calcification of the V. saphena magna was seen in advanced CKD. Of note, calcium-phosphate product correlated well with markers of inflammation, but not with calcification itself. CONCLUSIONS Early stages of CKD are already associated with local up-regulation of proinflammatory and proosteogenic molecules in the vascular wall and calcification of the aortic media. These findings point to the importance of local microinflammation in CKD and may shed new light on the potentially overestimated role of the calcium-phosphate product for vessel calcification.

Sympathetic blockade prevents the decrease in cardiac VEGF expression and capillary supply in experimental renal failure

Uremic cardiomyopathy of men and rodents is characterized by lower myocardial capillary supply that in rats could be prevented by central and peripheral blockade of the sympathetic nervous system. The underlying pathomechanisms remain largely unknown. We investigated whether alterations of cardiac vascular endothelial growth factor (VEGF) gene and protein expression were involved. In our long-term experiment, we analyzed whether VEGF gene and protein expression was altered in the heart of male Sprague-Dawley rats with either sham operation (sham, n=10) or subtotal nephrectomy (SNX, n=10). In our short-term experiment (17 sham, 24 SNX), the effect of a putative downregulation of sympathetic nervous activity by surgical renal denervation (interruption of renal afferent pathways) on cardiac gene expression of VEGF, flt-1, and flk-1 and on myocardial capillary supply was analyzed. In the long-term study, cardiac capillary supply and vascular endothelial growth factor gene and protein expression were significantly lower in SNX than in sham. In the short-term experiment, cardiac VEGF mRNA expression was significantly lower in untreated SNX (4,258±2,078 units) than in both sham groups (11,709±4,169 and 8,998±4,823 units); this decrease was significantly prevented by renal denervation (8,190±3,889, P<0.05). We conclude that cardiac VEGF gene and protein expression is reduced in experimental renal failure, and this may be considered as one potential reason for impaired myocardial adaptation under the situation of cardiac hypertrophy. The beneficial effect of sympathetic downregulation on cardiac structure and function in renal failure may be at least in part explained by increased cardiac VEGF gene expression.