Hypoxia response and VEGF-A expression in human proximal tubular epithelial cells in stable and progressive renal disease.

Proteinuria, inflammation, chronic hypoxia, and rarefaction of peritubular capillaries contribute to the progression of renal disease by affecting proximal tubular epithelial cells (PTECs). To study the transcriptional response that separates patients with a stable course from those with a progressive course of disease, we isolated PTECs by laser capture microdissection from cryocut tissue sections of patients with proteinuric glomerulopathies (stable n=20, progressive n=11) with a median clinical follow-up of 26 months. Gene-expression profiling and a systems biology analysis identified activation of intracellular vascular endothelial growth factor (VEGF) signaling and hypoxia response pathways in progressive patients, which was associated with upregulation of hypoxia-inducible-factor (HIF)-1alpha and several HIF target genes, such as transferrin, transferrin-receptor, p21, and VEGF-receptor 1, but downregulation of VEGF-A. The inverse expression levels of HIF-1alpha and VEGF-A were significantly superior in predicting clinical outcome as compared with proteinuria, renal function, and degree of tubular atrophy and interstitial fibrosis at the time of biopsy. Interactome analysis showed the association of attenuated VEGF-A expression with the downregulation of genes that usually stimulate VEGF-A expression, such as epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and HIF-2alpha. In vitro experiments confirmed the positive regulatory effect of EGF and IGF-1 on VEGF-A transcription in human proximal tubular cells. Thus, in progressive but not in stable proteinuric kidney disease, human PTECs show an attenuated VEGF-A expression despite an activation of intracellular hypoxia response and VEGF signaling pathways, which might be due to a reduced expression of positive coregulators, such as EGF and IGF-1.

Bortezomib-induced survival signals and genes in human proximal tubular cells.

Bortezomib has been introduced recently in the therapy of multiple myeloma (MM), a disease that is frequently associated with progressive renal failure. Because bortezomib-based therapy has been reported to lead to a rapid recovery of kidney function in patients with MM, we decided to study its direct effects in proximal tubular epithelial cells (PTCs) compared with glomerular mesangial cells (GMCs). After 24 h of stimulation, 50 nM bortezomib led to a 6.37-fold induction of apoptosis and markedly activated caspase-9 and -3 in GMCs but not in PTCs. In PTCs but not in GMCs, bortezomib led to a strong time-dependent degradation of IkappaB-alpha and to a long-lasting phosphorylation of both NF-kappaBp65 and extracellular signal-regulated kinase 1/2. Microarray analysis in bortezomib-treated PTCs revealed a time-dependent predominance of antiapoptotic genes compared with proapoptotic genes. Bortezomib (50 nM) induced heat shock protein (Hsp) 70 mRNA and protein levels in PTCs, whereas basal and bortezomib-stimulated Hsp70 protein expression was much weaker in GMCs. Moreover, bortezomib induced Bcl-2-associated athanogene (BAG) 3 mRNA and protein expression but inhibited BAG5 mRNA levels in PTCs. These data suggest that the reduced susceptibility of PTCs to bortezomib-induced cell apoptosis is because of cell type-specific effects of this compound on apoptosis/survival genes and pathways. The concept of bortezomib representing a blocker of both NF-kappaB activation and cell survival should be carefully examined in particular renal cell types.

Copper-induced formation of reactive oxygen species causes cell death and disruption of calcium homeostasis in trout hepatocytes.

We have previously shown that copper is acutely toxic for trout hepatocytes, inducing enhanced influx of Ca(2+) and a loss of cell viability. The aim of the present study was to elucidate the pathways of Ca(2+) entry into the cells, the hypothetical role of reactive oxygen species (ROS) in copper toxicity, and the interaction of ROS formation and the disruption of Ca(2+) homeostasis. We found that, acutely, copper-induced cell death occurred independently from an increase of intracellular free Ca(2+) (Ca(2+)i), but could be prevented by addition of agents interfering with ROS production. Addition of the Ca(2+) channel blocker verapamil did not affect the Ca(2+)i increase evoked by copper, whereas in the presence of LaCl(3), an inhibitor of both Ca(2+) channels and Na(+)/Ca(2+)-exchange, this increase was significantly delayed. ROS formation, estimated by use of the fluorescence indicator 2',7'-dichlorofluorescin diacetate, was significantly enhanced by copper. Omission of extracellular Ca(2+) or addition of either verapamil or LaCl(3) did not diminish ROS formation induced by copper. In contrast, the hydroxyl radical scavenger dimethyl sulfoxide and the ferric ion chelator deferoxamine inhibited radical production. In addition, these agents either partially (dimethyl sulfoxide) or completely (deferoxamine) prevented an increase of Ca(2+)i. Altogether our results indicate that ROS formation is the crucial event leading to cell death during acute exposure to copper, whereas the increase of Ca(2+)i is a secondary, acutely less toxic, phenomenon. Furthermore, these findings suggest that Ca(2+) entry occurs via a LaCl(3)-sensitive pathway, presumably representing Na(+)/Ca(2+)-exchange, and non-specific membrane leaks induced by lipid peroxidation in the presence of copper.