Kallikrein gene 'knock-down' by small interfering RNA transfection induces a profibrotic phenotype in rat mesangial cells.
BACKGROUND
Emerging evidence suggests that kallikrein exerts renoprotective effects independent of its haemodynamic actions. The aim of the current investigation was to delineate the role of kallikrein in the regulation of fibrosis, by 'knocking down' its expression using specific small interfering RNAs (siRNA).


METHODS
Rat mesangial cells were treated with 12, 60, 120 nmol/l kallikrein-specific siRNAs. The consequent cellular genotypes and phenotypes were analysed.


RESULTS
Western blotting demonstrated that mesangial cells produced a kallikrein protein, which was of a different molecular weight to urinary kallikrein from rats of the same species. Treatment of cells with siRNA resulted in a dose-dependent decrease in kallikrein mRNA levels, which impacted on other components of the kallikrein-kinin system, dose-dependently reducing bradykinin B2 receptor mRNA expression. Kallikrein suppression resulted in significant increases in fibronectin and transforming growth factor-beta protein levels in culture supernatants over control levels. Gelatin zymography demonstrated a siRNA dose-dependent decrease in active MMP-2 enzyme levels. Bradykinin, an effector molecule of the kallikrein system, is known to stimulate tissue plasminogen activator production. Paradoxically, however, tissue plasminogen activator protein levels were augmented with increasing kallikrein mRNA silencing. This was accompanied by a dose-dependent decrease in low-density lipoprotein receptor-related protein mRNA levels, indicating that increased tissue plasminogen activator levels were due to an attenuation of receptor-mediated protease clearance.


CONCLUSIONS
These data lend strong support to the hypothesis that kallikrein exerts antifibrotic, renoprotective effects that are independent of its classical haemodynamic actions.
Pawluczyk
Izabella Z A
IZ
Department of Infection, Immunity and Inflammation, University of Leicester, UK. izap1@le.ac.uk
Tan
Eddie K C
EK
Lodwick
David
D
Harris
Kevin  P G
K
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D005353:Fibronectins / Q000187:drug effects / Q000378:metabolism; D005355:Fibrosis / Q000139:chemically induced / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000187:drug effects / Q000378:metabolism; D050527:Mesangial Cells / Q000187:drug effects* / Q000378:metabolism; D010641:Phenotype; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D043782:Receptor, Bradykinin B2 / Q000187:drug effects / Q000235:genetics; D020840:Tissue Kallikreins / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000187:drug effects / Q000378:metabolism
D017077:Culture Media, Conditioned; D005353:Fibronectins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D043782:Receptor, Bradykinin B2; D016212:Transforming Growth Factor beta; D020840:Tissue Kallikreins; D020778:Matrix Metalloproteinase 2