This study aimed to clarify the role of AP regulation in renal tubular damage through both in vivo and in vitro examinations. First, we demonstrated that U-P, fH and MAC levels were significantly higher in patients with various renal diseases than in healthy controls and were correlated with U-protein levels and tubular damage markers. In addition, U-protein levels were correlated with the tubular damage markers, and the U-P, fH and MAC levels were independent of the histological diagnosis, so that the filtered plasma protein through glomerular barrier impacted as an important causative factor in renal tubular damage [2, 9, 23, 24]. Even in a patient with MCNS who had decreased renal function (eGFR: 60.4 mL/min), there was a tendency of high levels of U-tubular damage markers, B2MG 9014.28 ng/mg Cre (all patients: 1176 ± 3113) and NAG 5.41 U/mg Cre (all patients: 1.57 ± 2.08), U-protein 10.38 g/g Cre and U-complement components, U-P 44.10 ng/mg Cre, and U-fH 254.22 ng/mg Cre and U-MAC 62.59 ng/mg Cre. Although there were no high levels of tubular damage markers and U-complement components in patients without decreased eGFR in MCNS, these results suggested that relationships among high levels of U-protein, U-complement components and U-tubular damage markers existed, even in MCNS patients. Therefore, as U-complement components and tubular damage markers increase in patients with MCNS and advanced renal dysfunction, the levels of U-complement components and tubular damage markers may correlate with the levels of U-protein. Furthermore, multivariable analysis revealed that only U-P was correlated with both tubular damage markers, B2MG and NAG. In the context of renal disease, this study represents the first analysis, as per our knowledge, to clarify the role of AP regulation by measuring the combination of U-P, fH and MAC in the same samples from patients with various renal diseases.
The question arises whether these U-complement components play a crucial role in renal tubular damage. We examined complement AP activation on PTECs and could delineate a delicate balance between P and fH. From the single addition of P or fH, we could confirm dose-dependent depositions of P and fH on PTECs [14, 19, 23]. Zaferani et al.  suggested that P could bind dose-dependently to heparan sulfate proteoglycan (HSPG), which was thought to be one of the binding sites on PTECs. They also suggested  that fH could bind dose-dependently to HSPG and that P and fH could bind to different epitopes of HSPG on PTECs. Our results support these previous reports and indicated that P and fH could bind dose-dependently to PTECs. Although the depositions of fH were observed in a concentration (≥20 μg/mL) lower than the physiological serum concentration of fH (300–500 μg/mL), the bindings of P and fH are thought to be non-competitive in this experiment. Unfortunately, the individual binding site on PTECs could not be identified in this experiment, but it does suggest that the binding of fH does not have enough power to prevent the binding of P on PTECs.
We then recaptured the activation of complement AP on PTECs. NHS was used as a source of complements and a mimic of filtered plasma protein through the glomerular barrier. The depositions of P, fH, C3 and MAC on PTECs were observed to be dose-dependent of NHS [9, 25]. Next, to examine the enhancement effect of P in AP activation on PTECs, we preincubated PTECs with P before the addition of NHS. Preincubation with the fixed concentration of P before the addition of NHS increased not only the depositions of P but also those of C3 and MAC compared with controls; preincubation also increased the depositions of C3 and MAC in a serum-dependent manner from 5% to 25% NHS. Gaarkeuken et al.  also reported that the filtered P may bind to PTECs and act as a focal point for AP activation. In that report, P could be detected along the brush border of the proximal tubules in the patients with MN, but it was not detected in the tubules of healthy kidney tissues (pretransplant renal biopsies of living kidney donors). Additional P was shown to increase the depositions of C3 and MAC on PTECs in a P dose-dependent manner before incubation with 5% NHS. These findings could add new insight to the previous reports by suggesting that PTEC could initiate complement activation in a serum-dependent manner, with pre-exposure to a physiological concentration of P.
We previously focused on the importance of PDP activation in the pathogenesis of LN and reported that PDP activation in the glomeruli may lead to the early onset and progression of LN and reflect disease activity . In this study, we also confirmed that preincubation with P before the addition of PDS increased not only the deposition of P but also the deposition of C3, while it relatively increased the deposition of MAC; it was also suggested that P may act as an enhancer of complement AP activation referred to as PDP activation . A similar result was confirmed using flow cytometry in a previous report , where additional P restored complement activation of PDS on PTECs. We unexpectedly detected slight P staining on PTECs incubated with 25% PDS, but not 5% PDS. We considered the possibility that PDS was not completely depleted of P in the serum and that even a very small amount of P could bind to PTECs and activate AP and PDP, leading to the deposition of MAC. We could confirm that 5.77 ng/mL of P was present in the 25% PDS by ELISA (physiological serum concentration of P: 4–6 μg/mL).
In our study, although the activation of AP was seemingly ineffective in morphology, a functional effect was observed. After the addition of NHS to PTECs, cell viability was not significantly suppressed; however, preincubation with P before the addition of NHS significantly suppressed viability without causing morphological changes. Although diverse factors affected cell viability , the changes in this experiment were thought to reflect one possible result of complement AP activation enhanced by P. Therefore, we demonstrated the relationship between AP activation enhanced by P and tubular damage in experimental situations.
While there are numerous reports, including this study, that support the role of P in AP complement-mediated renal tubular damage, P-targeting therapy has also been evaluated [11, 27, 28]. P blockade may be a logical therapeutic target, and the accumulation of further research is required to decrease renal tubular damage.