Rare Diseases: don't stop the research
Project location: ITALY
Project start date: January 2001 - Project end date: January 2002
Project number: 2001-15
Intracellular signals involved in complement activation by protein overload in proximal tubular cells
Accumulating evidence indicates a causal association between proteinuria and tubulointerstitial disease that correlates with the rate of declining renal function in progressive nephropathies. However, to date, the exact mechanisms by which proteinuria induces tubulointerstitial damage have not been fully elucidated. Among individual components of proteinuria, ultrafiltered complement molecules can be activated to exert chemotactic and proinflammatory effects and cell cytotoxicity. The local synthesis of complement C3 by tubular epithelial cells may also occur and play role in tissue damage in response to protein overload. By in vitro studies on cultured human proximal tubular epithelial cells (PTEC) we will evaluate whether exposure to excess proteins upregulates mRNA expression of complement components, and the mechanisms/intracellular signals underlying such upregulation (transcription factors as NF-kB and C/EBP, protein kinase C (PKC), radical oxygen species (ROS), ERK 1/2).
PTEC will be incubated with medium alone or high concentrations of human albumin, transferrin or IgG. RNA will be extracted for the evaluation of C3 mRNA expression by real-time PCR. Since there is evidence that a region within the C3 promoter contains two CCAAT/enhancer binding protein (C/EBP) consensus sequence and data strongly suggest that C/EBP, which is functionally and physically associated with NF-kB, regulates the acute phase expression of C3 gene, we will evaluate by electrophoretic mobility shift assay C/EBP and NF-kB activation in nuclear extracts from PTEC exposed to proteins. Then, the role of C/EBP and NF-kB in complement expression (measured by real time PCR) will be assessed in PTEC exposed to proteins in the presence of specific C/EBP and NF-kB inhibitors. In other experimental settings PKC activation and ROS production have been described as intracellular signals for C3 synthesis; moreover ROS induces phosphorylation of extracellular signal-regulated kinase (ERK) implicated in the activation of NF-kB. Based on this evidence, the involvement of PKC and ROS via the activation of ERK 1/2 (Western blot) in the regulation of transcription factors and complement expression will be investigated by EMSA and real time PCR experiments in protein overloaded PTEC in the presence or absence of specific inhibitors of PKC and ERK 1/2 or antioxidants.