The experiments in this study highlight the dual role of factor H in regulating CAP activation. Circulating factor H prevents consumption of C3 in the fluid phase, thus maintaining adequate levels of C3 to support complement activation on cell surfaces. Consequently, little tubulointerstitial complement activation was observed in fH-/- mice subjected to renal I/R. On the other hand, the interaction of the carboxy terminus of factor H with host cells limits CAP mediated inflammation on tissue surfaces. When we blocked this interaction in wild-type mice by treating them with rH19-20, the mice displayed greater tubulointerstitial complement activation and more severe injury. Therefore, although factor H does not fully prevent CAP activation on ischemic tubules, interaction of factor H with the TEC surface is critical for limiting complement activation after renal I/R.
The efficacy of factor H at regulating CAP activation on a given surface is a function of its affinity with that surface (8
). High concentrations of factor H did not prevent CAP activation on zymosan particles (an “unreceptive” surface) but did prevent CAP activation on TECs in culture. CR2-fH, which specifically targets the complement regulatory region of factor H to sites of complement activation, effectively prevented complement activation on both unreceptive (zymosan) and receptive (TEC) surfaces. CR2-fH also effectively prevented CAP activation in the renal tubulointerstitium after I/R. This strategy can therefore prevent CAP activation on tissues where endogenous factor H does not fully prevent CAP activation due to insufficient quantity or affinity for the surface, effectively overcoming the limitations of the native protein.
Mutations and polymorphisms in the tissue binding regions of factor H have been linked to several diseases, including aHUS and AMD. Conversely, CAP mediated injury in a host with normal factor H structure and normal circulating concentrations suggests a limited affinity of factor H for the target tissue. It is noteworthy that the CAP has been implicated in models of several types of renal disease, including renal I/R, aHUS, DDD, FSGS, ANCA associated vasculitis and lupus nephritis (4
). The predilection of the kidney to be a primary site of injury may be a consequence of the particular repertoire of surface complement regulatory proteins expressed by the different renal cell types, and it may also be a consequence of the affinity of factor H for the various structures within the kidney. Complement activation on ischemic tubules, for example, may be the result of inadequate complement inhibition by factor H in the setting of decreased expression of Crry by the TECs (13
The concentration of complement proteins in interstitial fluid, vascular permeability, and local production of complement proteins may also be important determinants of complement activation in the renal tubulointerstitium. These factors likely contribute to the local balance of activation. It is possible, for example, that circulating factor H has only limited access to the TEC cell surface. In that case in vitro
assays, such as those shown in , would not accurately represent the balance of complement proteins (activators and inhibitors) present on the TEC surface in vivo
. The optimal design of tissue-targeted therapeutics might, therefore, have to consider tissue penetration of a molecule as equally important to target affinity. Local production of C3 by the tubules has been identified as an important factor in tubulointerstitial complement activation after renal I/R (24
). The absence of tubular C3 deposition in the fH-/-
mice subjected to I/R, however, suggests that intact circulating C3 (which is reduced in the fH-/-
mice) may be more important than locally produced C3 in our model.
Targeted inhibitors such as CR2-fH offer great promise as therapeutics. Because they specifically bind at sites of inflammation they may offer favorable pharmacokinetics and also may cause less systemic immunosuppression than untargeted agents (21
). A therapeutic such as CR2-fH may be particularly useful for patients carrying factor H mutations, or for patients with pathologic CAP activation in tissues for which circulating factor H has limited affinity. It is notable that the administration of CR2-fH considerably reduced the degree of CAP activation in the tubulointerstitium, even in the presence of circulating factor H. It is also possible that the administration of additional purified factor H would have been equally effective, although the concentration of factor H needed to achieve inhibition on TECs (roughly 1 mg/mL in undiluted serum) would require a considerable amount of protein.
In conclusion, these studies demonstrate that endogenous factor H plays an important role in limiting CAP activation on the TEC surface after renal I/R. CAP activation still causes tissue injury in the ischemic kidney, however, likely due to insufficient affinity or access of circulating factor H to this tissue surface. Therapeutic complement inhibitors should be effective for the treatment of diseases in patients with mutations in the complement regulatory proteins. These agents should also be beneficial in diseases, such as ischemic AKI, in which the body’s normal complement regulatory mechanisms do not adequately control activation of the CAP. The expression of cell surface complement regulatory proteins and the affinity of factor H to the cell surface may be unique to each tissue. The common involvement of the CAP in many different types of renal disease indicates that complement regulation within the kidney is easily disrupted or overwhelmed, even in the absence of mutations in the complement regulatory proteins. A greater understanding of the function and limitations of the endogenous regulatory proteins within specific tissue sites may permit the development of more effective complement inhibitors, possibly even the development of tissue-specific therapeutic agents.