Urine Proteomics Link Complement Activation with Interstitial Fibrosis/Tubular Atrophy in Lupus Nephritis Patients

Abstract

Background: Intrarenal complement activation has been implicated in the pathogenesis of tubulointerstitial fibrosis in lupus nephritis (LN) based on prior animal studies. The assembly of the membrane attack complex (MAC) by complement C5b to C9 on the cell membrane leads to cytotoxic pores and cell lysis, while CD59 inhibits MAC formation by preventing C9 from joining the complex. We hypothesize that complement activation and imbalance between complement activation and inhibition, as defined by increased production of individual complement components and uncontrolled MAC activation relative to CD59 inhibition, are associated with interstitial fibrosis and tubular atrophy (IFTA) in LN and correlate with the key mediators of kidney fibrosis- transforming growth factor receptors beta (TGFRβ), platelet-derived growth factor beta (PDGFβ) and platelet-derived growth factor receptor beta (PDGFRβ).

Methods: We included urine samples from 46 adults and pediatric biopsy-proven lupus nephritis patients who underwent clinically indicated kidney biopsies between 2010 and 2019. We compared individual urinary complement components and the urinary C9-to-CD59 ratio between LN patients with moderate/severe IFTA and none/mild IFTA. IFTA was defined as none/mild (<25% of interstitium affected) versus moderate/severe (≥ 25% of interstitium affected). Proteomics analysis was performed using mass spectrometry (Orbitrap Fusion Lumos, Thermo Scientific) and processed by the Proteome Discoverer. Urinary complement proteins enriched in LN patients with moderate/severe IFTA were correlated with serum creatinine, TGFβR1, TGFβR2, PDGFβ, and PDGFRβ.

Results: Of the 46 LN patients included in the study, 41 (89.1%) were women, 20 (43.5%) self-identified as Hispanic or Latino, and 26 (56.5%) self-identified as Black or African American. Ten of the 46 (21.7%) LN patients had moderate/severe IFTA on kidney biopsy. LN patients with moderate/severe IFTA had an increased urinary C9-to-CD59 ratio [median 0.91 (0.83-1.05) vs 0.81 (0.76-0.91), p=0.01]. Urinary C3 and CFI levels in LN patients with moderate/severe IFTA were higher compared to those with none/mild IFTA [C3 median (IQR) 24.4(23.5-25.5) vs. 20.2 (18.5-22.2), p= 0.02], [CFI medium (IQR) 28.8 (21.8-30.6) vs. 20.4 (18.5-22.9), p=0.01]. Complement C9, CD59, C3 and CFI correlated with TGFβR1, PDGFβ, and PDGFRβ, while C9, CD59 and C3 correlated with TGFβR2.

Conclusion: This study is one of the first to compare the urinary complement profile in LN patients with moderate/severe IFTA and none/mild IFTA in human tissues. This study identified C3, CFI, and C9-to-CD59 ratio as potential markers of tubulointerstitial fibrosis in LN.

Keywords: Biomarkers; Complement; Kidney fibrosis; Lupus nephritis.

Figure 1:. Comparison of the C9-to-CD59 ratio between LN patients with moderate/severe IFTA vs. none/mild IFTA suggest increased terminal complement activation relative to its regulation among those with more advanced tubulointerstitial fibrosis.

The urinary C9-to-CD59 ratio was defined as the urinary C9 protein abundance divided by the urinary CD59 protein abundance on the log scale. If C9, CD59 or both of those values were missing for a subject, then the corresponding C9-to-CD59 ratio was omitted from analysis. The black line represents the median value in each group. The yellow line represents each individual LN patient.

Figure 2:. Comparison of the individual complement components and inhibitors between LN patients with moderate/severe IFTA [dark grey] and none/mild IFTA [white] showed increased urinary C3 and CFI complement products among those with more advanced tubulointerstitial fibrosis.

The relative and absolute abundance of the individual complement proteins was reported as median (interquartile range, IQR) [represented by black line in each bar] and compared between the groups on a logarithmic scale. The dots within the bar represent each individual patient. Only complement proteins with data available for greater than 50% of the LN patients are shown.

Figure 3:. Positive correlation between C3, CFI, C9 and CD59 with TGFβR1 [Fig. 3A] and TGFβR2 [Fig 3B] suggests a link between complement activation and TGFβ signaling, which is known to mediate kidney fibrosis.

Urinary complement proteins enriched in patients with moderate/severe IFTA were correlated with TGFβR1, TGFβR2 using the Spearman’s rho correlation to assess if there is any correlation with a known mediator of kidney fibrosis. The blue dot represents each individual patient.

Figure 3:. Positive correlation between C3, CFI, C9 and CD59 with TGFβR1 [Fig. 3A] and TGFβR2 [Fig 3B] suggests a link between complement activation and TGFβ signaling, which is known to mediate kidney fibrosis.

Urinary complement proteins enriched in patients with moderate/severe IFTA were correlated with TGFβR1, TGFβR2 using the Spearman’s rho correlation to assess if there is any correlation with a known mediator of kidney fibrosis. The blue dot represents each individual patient.

Figure 4:. Positive correlation between C3, CFI, C9 and CD59 with PDGFβ [Fig. 4A] and PDGFRβ [Fig 4B] is consistent with prior animal studies which showed that PDGFRβ positive pericytes secrete complement products.

Urinary complement proteins enriched in patients with moderate/severe IFTA were correlated with PDGFβ and PDGFRβ using the Spearman’s rho correlation to assess if there is any association with a known mediator of kidney fibrosis. The blue dot represents each individual patient.

Figure 4:. Positive correlation between C3, CFI, C9 and CD59 with PDGFβ [Fig. 4A] and PDGFRβ [Fig 4B] is consistent with prior animal studies which showed that PDGFRβ positive pericytes secrete complement products.

Urinary complement proteins enriched in patients with moderate/severe IFTA were correlated with PDGFβ and PDGFRβ using the Spearman’s rho correlation to assess if there is any association with a known mediator of kidney fibrosis. The blue dot represents each individual patient.