BACKGROUND: Some individual components of complement are synthesized by the kidney. However, it is not known whether these form functional pathways that are able to mediate more fundamental cellular events. We examined the ability of HK-2 tubular cells to produce an intact alternative pathway of complement and to respond to the C3a fragment thus produced through the C3a receptor. METHODS: The production of mRNA for alternative pathway components was detected by reverse transcription-polymerase chain reaction, whereas protein synthesis was investigated by probing Western blots of concentrated culture supernatants with polyclonal antisera. Levels of C3a and inositol phosphate produced by HK-2 cells were determined by radioimmunoassay, whereas those of transforming growth factor-beta1 (TGF-beta1) were measured by ELISA. Intracellular tyrosine phosphorylation in response to C3a was evaluated by Western blotting and chemiluminescence. RESULTS: HK-2 cells produce the complement polypeptides C3a, C3, and factors B and H. They also contain mRNA for all components of the alternative pathway and the C3a receptor. mRNA levels were up-regulated by interleukin-1alpha, interleukin-1beta, and tumor necrosis factor-alpha. Incubation of HK-2 cells with C3a led to an increase in intracellular inositol phosphate and to tyrosine phosphorylation of at least two proteins in a pertussis-toxin-sensitive fashion. C3a and C3a desarg also up-regulated the secretion of TGF-beta1 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 activate these cells, resulting in inflammatory events such as TGF-beta1 production.
BACKGROUND: Some individual components of complement are synthesized by the kidney. However, it is not known whether these form functional pathways that are able to mediate more fundamental cellular events. We examined the ability of HK-2 tubular cells to produce an intact alternative pathway of complement and to respond to the C3a fragment thus produced through the C3a receptor. METHODS: The production of mRNA for alternative pathway components was detected by reverse transcription-polymerase chain reaction, whereas protein synthesis was investigated by probing Western blots of concentrated culture supernatants with polyclonal antisera. Levels of C3a and inositol phosphate produced by HK-2 cells were determined by radioimmunoassay, whereas those of transforming growth factor-beta1 (TGF-beta1) were measured by ELISA. Intracellular tyrosine phosphorylation in response to C3a was evaluated by Western blotting and chemiluminescence. RESULTS: HK-2 cells produce the complement polypeptides C3a, C3, and factors B and H. They also contain mRNA for all components of the alternative pathway and the C3a receptor. mRNA levels were up-regulated by interleukin-1alpha, interleukin-1beta, and tumor necrosis factor-alpha. Incubation of HK-2 cells with C3a led to an increase in intracellular inositol phosphate and to tyrosine phosphorylation of at least two proteins in a pertussis-toxin-sensitive fashion. C3a and C3a desarg also up-regulated the secretion of TGF-beta1 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 activate these cells, resulting in inflammatory events such as TGF-beta1 production.
Authors: Hongmei Gu; Amanda J Fisher; Elizabeth A Mickler; Frank Duerson; Oscar W Cummings; Marc Peters-Golden; Homer L Twigg; Trent M Woodruff; David S Wilkes; Ragini Vittal Journal: FASEB J Date: 2016-03-08 Impact factor: 5.191