Markus Pirklbauer

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.

FGF23 is associated with disease severity and prognosis in chronic heart failure

Elevated levels of fibroblast growth factor 23 (FGF23) are associated with incident heart failure in individuals with or without chronic kidney disease. We aimed to investigate the association between serum FGF23 concentrations and disease severity and long‐term outcome in patients with stable heart failure.

The exchangeable calcium pool: physiology and pathophysiology in chronic kidney disease

Excessive soft tissue and vascular calcifications are typical complications of chronic kidney disease (CKD) and disorders of phosphate homeostasis are considered to be a major contributor to the pathogenesis. However, at least in some individuals, calcium administration also increases the risk, and furthermore, it is widely accepted that there is a link between bone disease and vascular pathology. In this review, we discuss the role of the bone exchangeable calcium pool (ECP) in the acute regulation of the serum calcium concentration (Ca(s)) in health and CKD. This pool is able to buffer an acute calcium load as well as to maintain a stable Ca(s) during acute calcium deprivation. Indeed, the minute-to-minute regulation of Ca(s) appears to depend exclusively on this mechanism without any obvious contribution of other factors like parathyroid hormone, which nonetheless define the Ca(s) steady state set point. It is tempting to speculate that a reduction of the bone ECP plasticity in some patients with CKD leads to short-lasting increases in Ca(s) above the individual mid- to long-term set point as observed during haemodialysis or after the ingestion of calcium-containing phosphate binders. This could contribute to and partially explain the propensity of these subjects to develop extraosseous calcifications. An improved understanding of the processes involved and the availability of new techniques to assess the capacity of this pool, at least in dialysis patients, will make this area an attractive target for new investigations.

Neuropilin-1 and neuropilin-2 are differentially expressed in human proteinuric nephropathies and cytokine-stimulated proximal tubular cells.

Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are transmembrane glycoproteins with large extracellular domains that interact with class 3 semaphorins, vascular endothelial growth factor (VEGF) family members, and ligands, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-β1 (TGF-β1), and fibroblast growth factor2 (FGF2). Neuropilins (NRPs) have been implicated in tumor growth and vascularization, as novel mediators of the primary immune response and in regeneration and repair; however, their role in renal pathophysiology is largely unknown. Here, we report upregulation of tubular and interstitial NRP2 protein expression in patients with focal segmental glomerulosclerosis (FSGS). In an additional cohort of patients with minimal change disease (MCD), membranous nephropathy (MN), and FSGS, elevated NRP2 mRNA expression in kidney biopsies inversely correlated with estimated glomerular filtration rate (eGFR) at the time of biopsy. Furthermore, upregulation of NRP2 mRNA correlated with post-bioptic decline of kidney function. Expression of NRP1 and NRP2 in human proximal tubular cells (PTCs) was differentially affected after stimulation with TGF-β1, interleukin-1β (IL-1β), and oncostatin M (OSM). Although the pro-fibrotic mediators, TGF-β1 and IL-1β, induced upregulation of NRP2 expression but downregulation of NRP1 expression, OSM stimulated the expression of both NRP1 and NRP2. Basal and OSM-induced NRP1 mRNA expression, as well as TGF-β1-induced NRP2 mRNA and protein expression were partially mediated by MEK1/2-ERK1/2 signaling. This is the first report suggesting a differential role of NRP1 and NRP2 in renal fibrogenesis, and TGF-β1, IL-1β, and OSM represent the first ligands known to stimulate NRP2 expression in mammalian cells.

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.

Oncostatin M inhibits TGF-β1-induced CTGF expression via STAT3 in human proximal tubular cells.

Matricellular proteins play a critical role in the development of tubulointerstitial fibrosis and renal disease progression. Connective tissue growth factor (CTGF/CCN2), a CCN family member of matricellular proteins, represents an important mediator during development of glomerular and tubulointerstitial fibrosis in progressive kidney disease. We have recently reported that oncostatin M (OSM) is a potent inhibitor of TGF-β1-induced CTGF expression in human proximal tubular cells (PTC). In the present study we examined the role of TGF-β1- and OSM-induced signaling mechanisms in the regulation of CTGF mRNA expression in human proximal tubular HK-2 cells. Utilizing siRNA-mediated gene silencing we found that TGF-β1-induced expression of CTGF mRNA after 2h of stimulation at least partially depends on SMAD3 but not on SMAD2. In contrast to TGF-β1, OSM seems to exert a time-dependent dual effect on CTGF mRNA expression in these cells. While OSM led to a rapid and transient induction of CTGF mRNA expression between 15 min and 1h of stimulation it markedly suppressed basal and TGF-β1-induced CTGF mRNA levels thereafter. Silencing of STAT1 or STAT3 attenuated basal CTGF mRNA levels indicating that both STAT isoforms may be involved in the regulation of basal CTGF mRNA expression. However, knockdown of STAT3 but not STAT1 prevented OSM-mediated suppression of basal and TGF-β1-induced upregulation of CTGF mRNA expression. Together these results suggest that the inhibitory effect of OSM on TGF-β1-induced CTGF mRNA expression is mainly driven by STAT3, thereby providing a signaling mechanism whereby OSM may contribute to tubulointerstitial protection.

Synergistic induction of CCL2/MCP-1 expression driven by oncostatin M and IL-1β in human proximal tubular cells depends on STAT3 and p65 NFκB/RelA

In response to tubular injury, production, and secretion of cytokines, chemokines or extracellular matrix components by human proximal tubular epithelial cells (PTC) directly contribute to the development of tubulointerstitial inflammation and fibrosis. Here, we report a novel stimulatory and synergistic effect of oncostatin M (OSM) on proinflammatory CCL2/MCP‐1 mRNA expression in human PTC. Although OSM inhibited IL‐1β‐ and TNF‐α‐mediated mRNA expression of matricellular proteins TSP‐1 and tenascin C (TNC), it acted synergistically with these two proinflammatory cytokines to induce CCL2 mRNA expression for up to 24 h. Stimulation of two independent human PTC lines with OSM alone led to a rapid and strong induction of this chemokine within the first hour of ligand administration, which subsequently returned toward basal levels in between 3 and 24 h and finally switched into a significant OSM‐mediated 70% inhibition of basal CCL2 mRNA expression after 48 h of incubation. In contrast to OSM, which stimulated both STAT1/3 and ERK1/2 signaling, IL‐1β led to a strong phosphorylation of p65 NFκB/RelA, SMAD2/3, and p38 MAPK in human PTC. Selective silencing of these signaling molecules revealed that p65 NFκB/RelA is involved in IL‐1β‐mediated stimulation of CCL2 mRNA, and that superinduction of CCL2 mRNA expression in the presence of both OSM and IL‐1β at least partially depends on STAT3 signaling. Thus, with respect to the expression of the proinflammatory chemokine CCL2, OSM may stimulate acute inflammation via its synergistic effect with other proinflammatory cytokines early after injury.

Acute calcium kinetics in haemodialysis patients

To avoid excessive calcium loading in haemodialysis (HD) patients, current guidelines suggest a dialysate calcium concentration (dCa) of 2·5 mEq/L based on relatively stable intradialytic serum calcium levels. However, the latter do not account for possible calcium storage in acutely accessible pools. A rapidly exchangeable calcium pool located at the bone level has been previously proposed to be involved in acute (minute‐to‐minute) extracellular calcium regulation.