C3a is made by proximal tubular HK-2 cells and activates them via the C3a receptor

Background Some individual components of complement are synthesized by the kidney. However, it is not known whether these form functional pathways tha t are able to mediate more fundamental cellular events. We examined the abi lity of HK-2 tubular cells to produce an intact alternative pathway of comp lement and to respond to the C3a fragment thus produced through the C3a rec eptor. Methods. The production of mRNA for alternative pathway components was dete cted by reverse transcription-polymerase chain reaction, whereas protein sy nthesis was investigated by probing Western blots of concentrated culture s upernatants with polyclonal antisera. Levels of C3a and inositol phosphate produced by HK-2 cells were determined by radioimmunoassay, whereas those o f transforming growth factor-beta 1 (TGF-beta 1) were measured by ELISA. In tracellular tyrosine phosphorylation in response to C3a was evaluated by We stern blotting and chemiluminescence. Results. HK-2 cells produce the complement polypeptides C3a, C3, and factor s B and H. They also contain mRNA for all components of the alternative pat hway and the C3a receptor. mRNA levels were up-regulated by interleukin-1 a lpha, interleukin-1 beta, and tumor necrosis factor-alpha. Incubation of HK -2 cells with C3a led to an increase in intracellular inositol phosphate an d to tyrosine phosphorylation of at least two proteins in a pertussis-toxin -sensitive fashion. C3a and C3a desarg also up-regulated the secretion of T GF-beta 1 by these cells. Conclusion. HK-2 cells produce an intact alternative pathway of complement. In addition, both locally produced and urinary C3a have the potential to a ctivate these cells, resulting in inflammatory events such as TGF-beta 1 pr oduction.