Herbert Schramek

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)-1α 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-1α 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-2α. 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 IκB-α and to a long-lasting phosphorylation of both NF-κBp65 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-κB activation and cell survival should be carefully examined in particular renal cell types.

Oncostatin M is a novel inhibitor of TGF-β1-induced matricellular protein expression.

Matricellular proteins in the kidney have been associated with the development of tubulointerstitial fibrogenesis and the progression of renal disease. This study investigated potential antifibrotic effects of the cytokine oncostatin M (OSM) in human proximal tubule cells (PTC), particularly with regard to inhibition of profibrotic events initiated by TGF-β1. In human PTC, OSM diminished transforming growth factor (TGF)-β1-induced expression of the transcriptional epithelial-mesenchymal transition mediator FoxC2. Furthermore, exposure to OSM attenuated basal and TGF-β1-induced expression of the matricellular proteins SPARC, TSP-1, TNC, and CTGF regardless of the sequence of ligand administration. OSM was shown to result in rapid and sustained phosphorylation of both Stat1 and Stat3 and also in transient phosphorylation of Smad2/3 in contrast to TGF-β1, which demonstrated a gradually building phosphorylation of Smad2/3 and a brief phosphorylation of Smad1/5/8. Utilizing receptor-blocking molecules, we found the inhibitory effect of OSM on TGF-β1-induced CTGF mRNA expression occurs independently of Smad2/3 signaling and present evidence that this effect may be partially driven by OSM receptor-mediated Stat1 and/or Stat3 signaling pathways, thereby providing a mechanism whereby OSM can contribute to tubulointerstitial protection.

In Vitro Selection of Cell-Internalizing DNA Aptamers in a Model System of Inflammatory Kidney Disease

Chronic kidney disease (CKD) is a progressive pathological condition marked by a gradual loss of kidney function. Treatment of CKD is most effective when diagnosed at an early stage and patients are still asymptomatic. However, current diagnostic biomarkers (e.g., serum creatinine and urine albumin) are insufficient for prediction of the pathogenesis of the disease. To address this need, we applied a cell-SELEX (systematic evolution of ligands by exponential enrichment) approach and identified a series of DNA aptamers, which exhibit high affinity and selectivity for cytokine-stimulated cells, resembling some aspects of a CKD phenotype. The cell-SELEX approach was driven toward the enrichment of aptamers that internalize via the endosomal pathway by isolating the endosomal fractions in each selection cycle. Indeed, we demonstrated co-localization of selected aptamers with lysosomal-associated membrane protein 1 (LAMP-1), a late endosomal and lysosomal marker protein, by fluorescence in situ hybridization. These findings are consistent with binding and subsequent internalization of the aptamers into cytokine-stimulated cells. Thus, our study sets the stage for applying selected DNA aptamers as theragnostic reagents for the development of targeted therapies to combat CKD.

Ultrapure Polymerized Bovine Hemoglobin Improves Structural and Functional Integrity of the Isolated Perfused Rat Kidney

Since it became evident that organ dysfunctions after acute hemolysis are not induced by hemoglobin per se, but by stroma-contaminated hemoglobin, solutions of ultrapure stroma-free hemoglobins were regarded to be possible substitutes for blood in transfusion medicine. We tested one of the recently developed modified bovine hemoglobins (Ultrapure polymerized bovine hemoglobin 1; UPPBHb1) in the isolated perfused rat kidney (IPRK) model, using a recirculating system. Control kidneys were perfused with a substrate-enriched Ringer solution containing hydroxyethyl starch (HES) to produce isoncotic conditions. In the experimental group HES was substituted in part by UPPBHb1 (34 g/l). For determination of functional parameters, the kidneys were perfused for 180 min. A separate set of kidneys of both groups was perfusion fixed after 80 min of perfusion which is the period of optimal function. Light and electron microscopic analysis revealed major alterations only for the outer medulla of HES kidneys. Only these suffered from a considerable extent of proximal tubular S3 damage, exhibiting condensed tubular epithelia. In the inner stripe of the outer medulla, which is the zone of greatest sensitivity to damage in the isolated perfused kidney, severe hydropic degeneration, cell detachment, and necrotic destruction of the medullary thick ascending limb were seen in the HES-perfused group, too. In the UPPBHb1 group, the medullary thick ascending limb was well preserved, and S3 showed only a minor degree of damage. UPPBHB1 kidneys were further characterized by the occurrence of intracapillary and interstitial precipitates of UPPBHb1 in inner stripe of the outer medulla and inner medulla. The glomerular filtration rate was significantly higher in UPPBHb1-perfused kidneys (870 +/- 80 vs. 630 +/- 55 microliters/min/g kidney weight for HES). Absolute reabsorption of sodium paralleled the behavior of the glomerular filtration rate. The values for renal perfusate flow and urinary flow rate did not differ significantly between both groups. Renal autoregulation was better preserved in UPPBHb1-perfused kidneys (74 +/- 6% of full autoregulatory response) than in HES-perfused controls (42 +/- 4%). Our results suggest that perfusion of isolated rat kidneys with UPPBHb1 improves kidney function and morphology, providing better oxygenation than in control kidneys. UPPBHb1 does not exert additional nephrotoxic effects on the IPRK that will exceed the noxious potential of the method itself. Thus, it must be concluded that UPPBHb1 may be an oxyphoretic blood substitute with nephroprotective characteristics when compared with nonoxyphoretic substitutes. At least, UPPBHb1 seems to be a promising candidate as oxyphoretic additive to perfusates for the IPRK model.