The kidney is particularly susceptible to complement mediated injury in a number of clinical settings, and congenital deficiency or defects in the complement regulatory proteins MCP and factor H are strongly associated with the development of renal disease. In the current study we demonstrated that Crry (the murine homologue of MCP in the kidney) is the only membrane bound regulatory of complement expressed by murine TECs. Crry is expressed on the cell membrane, and its expression is concentrated in the basolateral portion of the cell. Polarized TECs regulate complement more efficiently on the basolateral surface of the cells than on the apical surface, in part due to Crry expression at this site. As with renal ischemia/reperfusion (I/R) (21
), chemical hypoxia of the TECs causes a reduction in surface Crry levels, and the distribution within the cell is also altered.
Spontaneous complement activation on the surface of TECs is also controlled by endogenous factor H. When rH 19-20 was added to TECs it permitted increased spontaneous deposition of C3 on both the apical and the basal surfaces of the TECs. Thus, both factor H and Crry are important for regulating the complement system on the surface of TECs and preventing autologous injury. Polymorphisms in both factor H and MCP (9
) are associated with the development of aHUS. Although endothelial injury is regarded as the trigger of aHUS, tubular injury and cortical necrosis are also well described findings (34
), reflecting the vulnerability of the TEC to complement mediated injury. Our findings demonstrate that control of the complement system on the TEC requires proper functioning of both of these proteins and proper localization of the membrane inhibitor on the basolateral surface when it is exposed to serum complement proteins. Hypoxia of the cells disrupts the organization of the cell membrane and renders the cell susceptible to complement activation. This may be due to the overall reduction in surface Crry. It may also be due to increased access of serum complement proteins to the apical surface as the integrity of the monolayer is lost.
Tickover of the alternative pathway in the fluid phase deposits C3b on nearby surfaces, and the deposited C3b causes auto-activation unless effectively inhibited by complement regulatory proteins (35
). Several studies have indicated that the absence or functional loss of Crry renders the kidney susceptible to tubular injury (21
). The studies presented herein demonstrate that the loss of surface regulation by Crry is sufficient to permit alternative pathway activation on TECs, even without cellular ischemia or another cellular insult. The presence of sporadic C3 in the tubulointerstitium of wild-type mice demonstrates that this is a location of basal complement activation. This constitutive tubulointerstitial activation may help explain why the kidney is susceptible to spontaneous injury in the setting of inadequate complement regulation.
The susceptibility of the kidney to complement mediated injury is likely influenced by fact that expression of the membrane bound inhibitor by the TECs is restricted to the basal surface of the cells, and levels are decreased in response to cellular stress and injury. It has also recently been demonstrated that properdin binds to the apical surface of TECs and may catalyze alternative pathway activation on this surface (28
). Factor H regulates the alternative pathway on both the apical and the basal surface of TECs, but regulation of complement by factor H is inadequate to prevent spontaneous complement activation on the apical surface of the cells, and it cannot compensate for the loss of regulation by Crry on the basolateral surface of the cells. Likewise, expression of Crry on the basolateral surface of TECs does not fully prevent complement activation when the C-terminal membrane-interacting portion of factor H was blocked by rH 19-20. Although DAF and CD59 are not expressed in the interstitium of normal kidneys, they can be detected in the tubulointerstitium of some diseased kidneys (31
). Therefore, the in vivo dependence of TECs on protection by MCP may not be as stringent as our current results would suggest.
The reduction of surface Crry on hypoxic cells may be an active response to stress. Cervical epithelial cells have been demonstrated to down-regulate surface MCP after infection with piliated Neisseria gonorrhea (37
), and down-regulation of surface regulatory proteins may be a mechanism by which epithelial cells foster the immune response to invasive pathogens. Renal TECs form a barrier epithelium, and they may play an important role in the anti-microbial response to pathogens (38
). In aseptic diseases such as I/R, however, such a response to stress could be maladaptive. Our findings help explain why the TECs are a target for uncontrolled complement activation in several clinical settings, including proteinuric states and ischemic injury. The mechanisms we have described may also apply to other diseases in which the TECs sustain cellular stress or injury.
Recent studies have examined complement regulation in Crry−/−
). Although maternal complement proteins injure the placentae of Crry−/−
), if the mother is deficient in alternative pathway proteins (either C3 or factor B) then Crry−/−
pups can be generated. Two groups have bred Crry−/−
mice that do not have genetic deletion of the C3 or factor B genes. The Crry−/−
mice displayed increased consumption of factor B and C3, and levels of these two proteins were decreased in plasma (39
). Although the alternative pathway activation was presumably occurring on tissue surfaces, no injury phenotype was described and pathologic complement activation in the glomeruli was not seen. Renal function was not reported in the studies, but the authors stated that the mice were followed for over a year without evidence of renal damage (39
). Just as the chronic, diffuse consumption of the complement proteins allows successful parturition in these mice, the extrarenal consumption of factor B and C3 may prevent complement activation on the tubules from reaching a level sufficient to cause renal injury.
Several previous studies have examined the role of Crry in protecting the kidney from complement-mediated injury of the tubules (21
). One of these studies demonstrated that basolateral expression of Crry in mice is disrupted by renal ischemia (21
). Another study demonstrated progressive complement mediated injury of Crry−/−
kidneys transplanted into wild-type hosts (22
). In the current study, the acute infusion of factor B restored sufficient activity to cause foci of complement deposition and tissue injury within the kidney. Thus, in a setting that is not potentially complicated by effects of warm ischemia or transplantation-related I/R, our results clearly show that the absence of Crry renders TECs susceptible to spontaneous complement mediated injury.
Although several different renal diseases are closely linked to uncontrolled activation of the alternative pathway, the molecular mechanisms appear to be distinct in several of the models. In DDD, for example, activation in the absence of adequate factor H function appears to occur primarily in the fluid phase (11
). The tropism of systemically generated C3 fragments for the glomerular basement membrane is not, as yet, explained. Atypical HUS involves defects in alternative pathway regulation that could potentially cause injury on many host cells but which usually manifests as renal injury (10
). The mechanisms of complement regulation on the TECs are distinct from those that protect the glomerular endothelial cells and the GBM, and yet it is striking that the kidney is such a common target of injury in patients with defects in complement regulatory proteins. Other factors, such as the large percentage of cardiac output that the kidneys receive, could possibly contribute to the development of renal injury as a consequence of global defects in complement regulation.
In summary, we have found that Crry is the only cell surface complement regulatory protein expressed by murine TECs, but factor H in the serum also contributes to complement regulation on the TEC surface. At baseline, Crry is concentrated on the basolateral surface, and complement regulation is more efficient on this surface than on the apical surface. Cellular hypoxia disrupts the TEC tight junctions, reduces surface levels of Crry, and potentiates complement activation on the TEC membrane. Finally, we have performed studies demonstrating that complement is spontaneously activated on TECs with genetic deletion of Crry when they are exposed to an intact alternative pathway, and reconstitution of Crry−/−fB−/− mice with purified factor B protein induced acute kidney injury. These results underscore the importance of Crry for protecting TECs from autologous injury. These findings also help to explain the particular vulnerability of TECs to complement mediated injury in several clinical settings, and indicate that therapies capable of restoring control of the complement system at the TEC surface may be beneficial in renal tubulo-interstitial disease.