Using Aliskiren to help obliterate renal diseases caused by uncontrolled renin-mediated complement activation

Based on: Aliskiren inhibits renin-mediated complement activation, published 5 June 2018

Complement is a system of plasma proteins that induces a series of inflammatory responses in the immune system to fight off infection [1]. The complement system does this via one of three pathways that result in the formation of a membrane attack complex, which forms on the surface of the pathogen’s cell membrane, targeting it for destruction and cell death [2].

Although compliment activation is beneficial for the immune system, it can become harmful if left unregulated through the uncontrolled cleavage and subsequent deposition of complement components. For example, renal diseases are associated with the unregulated activation of the alternative pathway due to mutations in complement regulators, resulting in deposition of components of the alternative pathway [1]. The deposition of these components can create conditions that lead to problems such as dense deposit disease (DDD), which negatively impacts kidney function [5]. In this study, Bekassy et al showed that renin, an enzyme secreted by the kidney, activates the alternative pathway of complement by acting in the same manner as C3 convertase [3,4]. After verifying the role of renin as a complement mediator, the authors investigated whether aliskiren, a drug that treats high blood pressure, inhibits renin in order to regulate the uncontrolled complement cascade [6].

In order to verify the role of renin, 100 mg/ml of C3 was combined with plasma renin to see if C3 cleavage occurred by immunoblotting. The presence of renin resulted in C3 cleavage, as indicated by a band corresponding to C3b after 1.5 hours. C3 alone without the presence of renin did not undergo cleavage into C3a and C3b. Furthermore, C3a levels induced by renin were comparable to C3a levels induced by C3 convertase, and the N-terminal sequencing of the cleaved product confirmed that renin and C3 convertase cleaved C3 at the same site, suggesting that they cleave C3 by the same mechanism [3, 7].  

Figure 1 | C3 cleavage by renin and its inhibition. (a) C3 cleavage by renin. C3 (100 ug/ml, lane 1) incubated with plasma renin (4.5 pg/ml) for 1.5 hours and analyzed by immunoblotting showing cleavage to C3b (lane 2). Pure C3b, under the same conditions (100 ug/ml, lane 3).

To test the hypothesis that aliskiren inhibits renin, the authors preincubated C3, plasma renin (0.45 pg/ml), and aliskiren (0.04 M) for 3 hours to see if C3 cleavage was inhibited by immunoblotting. The results demonstrated that aliskiren inhibits renin-mediated C3 cleavage because there is no C3b band in the presence of aliskiren. Aliskiren alone with no renin had no effect on C3 cleavage, indicating that aliskiren works by specifically inhibiting renin [3].

Figure 1 | C3 cleavage by renin and its inhibition. (f) Aliskiren (0.04) and pepstatin (0.3 mM) preincubated with plasma renin (0.45 pg/ml) for 3 hours inhibited C3 cleavage by renin during a 2-hour incubation demonstrated silver staining (lanes 3 and 5, respectively). Aliskiren and pepstatin did not have an effect on C3 itself after 2 hours’ incubation in the absence of renin (lanes 4 and 10 aliskiren).

After demonstrating that renin cleaves C3 in a similar manner as C3 convertase and that aliskiren inhibits renin-mediated cleavage, the authors analyzed the interaction between C3 and renin by plasmon resonance [8]. They did this by using an anti-C3b antibody that specifically recognizes the C3b formed after C3 cleavage and found that binding was detected with the C3 incubated with plasma renin but not in the presence of C3 alone without renin. This experiment further confirmed that renin acts as a mediator for C3 cleavage.

The authors further confirmed the roles of renin and aliskiren by demonstrating Factor B (CFB) binding to cleaved C3 [9]. When Factor B was incubated overnight with C3, C3b, and C3 incubated with renin, there was considerably more binding of Factor B to C3b and C3 cleaved by renin than C3 alone. This result again demonstrates the similar mechanisms by which renin and C3 convertase cleave C3. Furthermore, Factor B did not bind to C3 in the presence of aliskiren, demonstrating the role of aliskiren as a renin inhibitor.

Aliskiren was also shown to decrease C3 deposition on renin-producing cells, which can occur when the alternative complement pathway is left unregulated. The authors incubated renin-producing cells with C3 for 4 hours with and without aliskiren. In the presence of aliskiren, C3 deposition was reduced. This finding is important because it suggests a potential mechanism for combating dense deposit disease (DDD). Thus, the authors next conducted an experiment that tested the effect of aliskiren on complement activation in three pediatric DDD patients. When treated with aliskiren, decreased complement activation was observed in all three patients by significantly increased C3 levels, indicating less C3 cleavage. Furthermore, decreased C3 deposition was demonstrated in all three patients.

In summary, Bekassy et al demonstrated that renin cleaves C3 into C3a and C3b, initiating the alternative pathway of the complement system. Renin activity was inhibited by aliskiren, a drug commonly used to treat high blood pressure. C3b binds Factor B, which forms the C3 convertase and thus triggers the alternative pathway that, when left unregulated, can become problematic and lead to diseases such as DDD. Furthermore, this complement activation cascade is specifically amplified in the kidney because renin concentrations are higher in the kidney, which can lead to other renal diseases. These problems occur when there are mutations in certain complement regulators, resulting in unregulated complement cascades. Therefore, the inhibitory role of aliskiren is important because it helps to regulate renin-mediated complement activation in order to help combat diseases such as DDD. However, no single treatment of aliskiren has been effective in all DDD patients. The success of aliskiren in the three DDD patients in this study is promising nonetheless, and it could set the stage for a larger clinical trial with the drug.

A future direction for this study may be in the form of a larger clinical trial with a larger sample size to see how beneficial the drug really is in vivo. Although aliskiren functioned to inhibit renin-mediated cleavage in DDD patients, the paper only shows success for three specific patients, which is a small sample size. Therefore, it would be beneficial to conduct a larger study and perhaps search for alternative renin inhibitor drugs that may work for patients who are not receptive to aliskiren and why this might be. 

References:

[1] Janeway CA Jr, Travers P, Walport M, et al. "The complement system and innate immunity." Immunobiology: The Immune System in Health and Disease 5 (2001). https://www.ncbi.nlm.nih.gov/books/NBK27100/. 

[2] Devine, Dana V. "Complement membrane attack complex. ScienceDirect, Elsevier, 2018, https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/complement-membrane-attack-complex. 

[3] Bekassy, Zivile D. et al. "Aliskiren inhibits renin mediated complement activation." Kidney Int (2018). https://www.ncbi.nlm.nih.gov/pubmed/29884545. 

[4] The Editors of Encyclopaedia Britannica. "Renin" Encyclopaedia Britannica, Encyclopaedia Britannica Inc, 23 November 2018, https://www.britannica.com/science/renin. 

[5] "Dense deposit disease." Genetic and Rare Diseases Information Center, 15 December 2014. https://rarediseases.info.nih.gov/diseases/8555/dense-deposit-disease. 

[6] "Aliskiren." Mayo Clinic, Truven Health Analytics Inc, 1 March 2017, https://www.mayoclinic.org/drugs-supplements/aliskiren-oral-route/description/drg-20070895.

[7] "Complement C3 Convertase." Sino Biological, Sino Biological Inc, https://www.sinobiological.com/complement-c3-convertase-lgf.html.

[8] Tang, Yijun et al. "Surface Plasmon Resonance: An Introduction to a Surface Spectroscopy Technique." J Chem Educ 87 (2010). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045209/. 

[9] "CFB Complement Factor B." NCBI, 5 August 2018, https://www.ncbi.nlm.nih.gov/gene/629.