Defining the pathophysiology of C3 glomerulopathy - an ultra-rare complement-mediated renal disease -

C3 glomerulopathy (C3G), is a complex ultra-rare complement-mediated renal disease affecting approximately two to three individuals per 1,000,000 per year, as a result of the dysregulation of the complement alternative pathway (AP) proteins in the fluid-phase breakdown products are deposited in the glomeruli of the kidney. C3G encompasses two major disease sub-types, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD). Many patients with C3G have abnormal levels of proteins of the AP in their bloodstream. In particular complement component C3, an essential protein of the AP has been demonstrated to be decreased in the circulation. The increased breakdown products are indicative of on-going complement activity. In measuring the complement protein levels, and their breakdown products, we can define patient-specific biomarker profiles that are characteristic of C3GN and DDD. These biomarker profiles are unique “signatures” that can be determined for each person. They have demonstrated the importance of evaluating complement proteins in the disease state. C3GN and DDD have commonalities and differences when a broad panel of alternative pathway complement proteins are compared. A pilot study evaluation of AP proteins indicated that C3 is decreased in both C3GN and DDD when compared to normal controls, and that reduction was greater in DDD patients. Additionally, the study also demonstrated greater terminal pathway activity, soluble C5b-9, in C3GN than in DDD. These tests localize the sites of on-going complement dysregulation is C3G. SOMAscan, a robust, multiplexed proteomic DNA microarray technology targeting >1300 proteins in patients’ plasma samples was used to validate the complement biomarkers currently used clinically for predicting complement dysregulation in C3G. Additionally, SOMAscan provides an opportunity for the discovery of novel biomarkers in association with C3G to further inform our understanding of C3G. In this study, C3G patient plasma samples (C3GN; N=25 and DDD; N=33) were analyzed employing SOMAscan technology compared to normal controls (no renal phenotype; N=34) and Anti-Neutrophil Cytoplasmic Antibodies (ANCA) glomerulonephritis pool (N=10). The C3G samples were retrospectively screened for additional clinical factors: dialysis, transplant, and Eculizumab treatment. Our SOMAscan study showed that renin was among the top differentially expressed proteins in C3G (p-value = 0.0013, one-way ANOVA) as well as in other diseases (e.g. hypertension) as a result of the treatments received and not the underlying disease. Renin is an aspartyl protease and key protein of the renin-angiotensin-aldosterone system (RAAS) playing a major role in blood pressure regulation. Whether renin may play a contributory role in the pathogenesis of C3G is unknown. Therefore, we sought to test renin cleavage of C3 using a broad range of renin concentrations using silver staining to detect cleavage activity. In conclusion, clinical data should be considered when analyzing biomarkers since therapies can alter protein expression. Renin’s increased expression in C3G was an incidental finding, secondary to non-disease specific treatments. The two recombinant renin (rRenin) proteins used in this study produced different results. One of which did not cleave C3 (BioVision) and one which cleaved C3 (AbCam). No C3 breakdown products were identified at biologically relevant concentrations of renin. The rRenin that cleaved C3 contained PRSS1, a serine protease, likely the reason for cleavage of C3. This finding supported previous findings of serine proteases cleaving C3. Although, we demonstrated that renin was elevated in patients with C3G our renin studies did not provide evidence to support a direct role for renin in cleaving C3. Uncovering novel biomarkers using SOMAscan may be of value as diagnostic markers, or for the development of disease-specific drug therapies, as well as aid in refining our understanding of C3G.