Renin and renin blockade have no role in complement activity

Abstract

Renin, an aspartate protease, regulates the renin-angiotensin system by cleaving its only known substrate angiotensinogen to angiotensin. Recent studies have suggested that renin may also cleave complement component C3 to activate complement or contribute to its dysregulation. Typically, C3 is cleaved by C3 convertase, a serine protease that uses the hydroxyl group of a serine residue as a nucleophile. Here, we provide seven lines of evidence to show that renin does not cleave C3. First, there is no association between renin plasma levels and C3 levels in patients with C3 Glomerulopathies (C3G) and atypical Hemolytic Uremic Syndrome (aHUS), implying that serum C3 consumption is not increased in the presence of high renin. Second, in vitro tests of C3 conversion to C3b do not detect differences when sera from patients with high renin levels are compared to sera from patients with normal/low renin levels. Third, aliskiren, a renin inhibitor, does not block abnormal complement activity introduced by nephritic factors in the fluid phase. Fourth, aliskiren does not block dysregulated complement activity on cell surfaces. Fifth, recombinant renin from different sources does not cleave C3 even after 24 hours of incubation at 37 °C. Sixth, direct spiking of recombinant renin into sera samples of patients with C3G and aHUS does not enhance complement activity in either the fluid phase or on cell surfaces. And seventh, molecular modeling and docking place C3 in the active site of renin in a position that is not consistent with a productive ground state complex for catalytic hydrolysis. Thus, our study does not support a role for renin in the activation of complement.

Keywords: C3 cleavage; aliskiren; alternative pathway; complement activation; renin.

Figure 1.

Renin concentration in patients with complement-mediated renal diseases. a) Plasma renin levels were measured in normal individuals (n = 23) and patients with aHUS (n = 34) and C3G (n = 54). Renin levels are significantly higher in patients with C3G and aHUS; ~50% of patients have elevated renin levels (> 1 ng/ml). Patients with aHUS had higher total renin than those with C3G (P < 0.05); b) and c) C3 and sC5b-9 levels in patients with low (<1 ng/ml) and high (>1 ng/ml) renin levels (red: aHUS; blue: C3G). There are no significant differences between groups (P > 0.05), suggesting that high renin levels are not linked to increased C3 activation/consumption or higher C5 convertase activity. Patients on anti-C5 treatment are excluded in c; d) Fluid-phase activity in patients with C3G. The group with high renin (n = 30) is not enriched for patients with higher fluid phase activity as compared to the group with low renin (n =24). C3Nef-positive C3G patients with high renin levels show no greater fluid-phase C3 conversion than those with low renin levels (for all figures, horizontal dashed line, normal limit).

Figure 2.

The renin-specific inhibitor, aliskiren, does not block unregulated complement activity. a) In the fluid phase. In 4 patients, each having renin levels of 848, 1006, 2027 and 1074 pg/ml, respectively, and presenting varying degrees of complement dysregulation, aliskiren was added to a 1:1 mixture of pooled normal human serum and patient serum at increasing concentrations ranging from 0 to 280 nM (representing 2800-fold molar excess to renin). The mixture was incubated in buffer containing EGTA supplemented with MgCl₂ to allow for alternative pathway (AP) activation or EDTA to stop complement activity (circle, patient #1, second lane shows concentration at the periphery of a dot blot optimal for immunoprecipitation with purified C3 (0.5 mg/ml) for the purpose of quality control for the anti-C3 antibody; C3AP, C3 activation products); b) On cell surfaces. In a patient with high renin (3036 pg/ml), factor H and aliskiren were spiked in at concentrations indicated. The addition of FH at a final concentration of 0.03 μM significantly reduced C3b deposition, and at a final concentration of 0.17 μM, C3b deposition was completely prevented. However, when aliskiren was added at low (0.4 μM), medium (4 μM) or high (40 μM) concentrations, which are 4,000x, 40,000x and 400,000x molar excess relative to renin levels, respectively, there was no reduction in C3b deposition associated with renin blockade (blue = nuclei (DAPI); green = C3b; bar = 200 μm).

Figure 3.

Recombinant renin does not cleave purified C3. a) Renin (40 U) and C3 (250 ug/ml) were mixed and incubated at 37°C (pH = 7.4) for 1 h (top) or 24 h (bottom). Batches A, B and D are C-terminal His-tagged renin; batches E and F are N-terminal FLAG-tagged renin (Table 1). The respective mixtures were resolved on a 4–15% polyacrylamide gel and probed with an anti-C3 antibody, which targets the Factor I cleavage site on C3 or C3b (this antibody recognizes the intact C3 α chain (116 kDa) and the C3b α’ chain (106 kDa)). Control Lane: FB and FD added to form C3 convertase. With the exception of batch A, four renins (batches B, D, E, F) did not cleave C3 even after a 24-h incubation. Batch A did show signs of C3 cleavage, which was partial after 1 h of incubation (C3/C3b mixed band, top gel) and complete after 24 h of incubation (bottom); b) Untagged renin batches G (AnaSpec) and H (Cayman) at increasing concentrations of 5 to 40 U (left to right). No cleavage activity was detected using batch G in both 1-h (top) and 24-h (bottom) incubations at 37°C. However, dose-dependent cleavage of C3 with batch H was apparent in both 1-h and 24-h incubation studies.

Figure 4.

Dose-Dependent AngT and C3 Cleavage in Combined AngT-C3a Assay. a) AngT-C3 combination cleavage assay. AngT 14aa peptide and C3 were mixed in a single tube before adding renin at increasing concentrations ranging from 0.1 to 14 Units for a 1-h incubation at 37°C (pH = 7.4). The results showed a dose response in AngT cleavage for both renins (red bars). While batch G did not exhibit any noticeable increase in C3a, with batch H showed a dose-dependent increase (blue bars). FB/FD and C3 mixture served a control for C3a; b) Identification of trypsin contamination via Mass Spectrometry. Spectra: Distinct peaks in mass spectroscopy corresponding to bovine trypsin (M peaks, M+Xs peaks, marked by 3 arrows) were detected in batch H but absent in batch G.

Figure 5.

Recombinant renin does not induce complement activation. a) Renin does not increase complement activity in the fluid phase. Recombinant FLAG-tagged renin (batch F) was spiked in at >1000x higher than the concentration observed in patients. Three examples are shown – patient #6 with negative pre-spiking fluid-phase activity [no C3 activation products (C3AP) shown in the second lane] and no increase with spiking (third lane); patients #7 and #8 with positive pre-spiking fluid-phase activity (second lane) and no increase with renin spiking (third lane) (total 15 patients tested; D = EDTA, complement activity stopped; G = EGTA, supplemented with MgCl₂ making AP activation possible); b) Increased renin concentration does not increase complement deposition on cell surfaces. C3b deposition does not increase as renin concentration is increased in both pooled normal human serum (top) and patient serum (bottom). As a positive control, FD (instead of renin) was spiked in at a final concentration of 10 μg/ml (~10x above normal) (right). Renin low = final concentration 1.9 nM or ~19x above the highest renin observed in patient. Renin high = final concentration 19 nM or ~190x above the highest renin observed in patients. FD added to a final concentration of 10 ug/ml (~10x higher than controls) (blue = nuclei, DAPI; green = C3b; bar = 200 μm).

Figure 6.

Molecular docking of AngT and C3. a) The AngT tetradecapeptide, depicted in blue stick view, is docked into the active site of the renin enzyme (6i3f). The docking result closely resembles the co-crystal structure complex, which contains the AngT peptide, shown as the orange strand. The root-mean-square deviation (RSMD) between the docked and co-crystal structure complex is less than 0.1; b) A closeup of the enzymatic site, which cleaves the scissile bond between Leu10 and Val11 of AngT; c) The C3 tetradecapeptide is shown in green stick view, docked into the active site of renin. RSMD between the docked C3a and Renin receptor interface is 1.76 (the best fitting model among 100 docking possibility). For comparison, note the AngT tetradecapeptide again shown as the orange strand; d) A closeup of the enzymatic site in renin. This docking configuration does not result in a catalytically productive complex because there is a distance of 9.34 Å between the catalytic base (Asp38 and Asp226) and the carbonyl carbon of the scissile bond between Arg748 and Ser749, indicating that the necessary interactions for catalysis cannot be established. In figures b and d, the catalytic residues (Asp38 and Asp226) are depicted as green spheres and their oxygen atoms are colored red. The interaction surface between ligand and renin is shown with a transparent sheet view.