Methods
This cross-sectional study used deidentified clinically indicated kidney biopsy samples collected at New York Langone Medical Center between July 2014 and July 2016. Informed consent was obtained for using biopsy samples collected for clinical use. A renal pathologist (MW) selected 30 unstained paraffin embedded renal biopsies from specimens previously stored in pathology. Inclusion criteria were age ≥18 years old, confirmation of LN diagnosis on biopsy, lupus nephritis ISN/RPS class II, III, IV, V, III+V or IV+V and at least 10 glomeruli present in the renal biopsy. Patients with advance sclerosing LN Class VI (over 90% of global sclerosis) were excluded because of advance end-stage damage.
We assessed MAC deposition in the renal tubules by using a previously developed immunohistochemistry (IHC) protocol.21 We performed immunohistochemical staining for Complement C9, which is a part of the MAC and a proxy marker for MAC deposition.22
In brief, chromogenic IHC was performed on formalin-fixed, paraffin-embedded, 4 µm human renal biopsy sections using the Ventana Medical Systems Discovery XT instrument and unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197). The IHC protocol was optimised using C3 glomerulopathy as positive control and normal renal tissue as negative control.21 C9 staining was assessed in different locations: the tubular basement membrane, glomerulus and arterioles. Tubular C9 stain intensity was reviewed independently by three renal pathologists (MW, JP, DS) and scored on a standard semiquantitative scale of none (0), mild (1+), moderate (2+) and severe (3+) to add internal validity. Percentage of tubules with C9 staining was also analysed. Any discrepancy was resolved with re-evaluation of slides.
Tubular C9 staining was defined as positive if any staining was present (intensity score of 1 to 3) and negative if staining was absent (intensity score of 0). Figure 1 shows one of the patients with LN with positive C9 staining along the tubular basement membrane. To define IFTA, the main outcome of interest, the percentage of total tubules with fibrosis and/or atrophy was evaluated and categorised based on the NIH criteria as minimal (<10%), mild (10%–24%), moderate (25%–50%) and severe (>50%).6 7 For the main analysis, minimal and mild IFTA were combined. Additional analysis was done for all IFTA categories. The secondary outcome was proteinuria, which was analysed as a continuous variable. We recorded the 24-hour urine protein at the time of biopsy, and if not available, spot urine protein to creatine ratio was recorded. Nephrotic range proteinuria was defined as proteinuria >3.5 g/24 hours. IFTA and proteinuria were compared between C9 positive (+) and C9 negative (-) groups.
Figure 1C9 deposition in the tubular basement membrane of a patient with lupus nephritis Class V and moderate interstitial fibrosis/tubular atrophy.
As part of additional analyses, we evaluated tubular versus glomerular C9 staining to determine whether complement’s roles in the tubules and glomerulus are independent of one another. In addition, we compared tubular C9 staining to existing complement markers in assessing tubulointerstitial injury.23 Specifically, we assessed the association of low serum C3 and C4 with IFTA.
Clinical, demographic, laboratory and histopathological parameters were compared between C9 + and C9- groups. Medical records and pathology reports were reviewed by a rheumatologist (SW) who was blinded to C9 staining results to minimise bias. The following demographic and clinical data were collected at the time of biopsy: age, sex, self-reported race and ethnicity, systolic and diastolic blood pressure, and average disease activity as measured by the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K).24 Medications including hydroxychloroquine, prednisone and immunosuppression (use of mycophenolate mofetil, cyclophosphamide, tacrolimus, azathioprine, rituximab and belimumab) were recorded at the time of biopsy. Laboratory data included urinalysis, serum complement levels, dsDNA antibodies, creatinine and estimated glomerular filtration rate (eGFR). Low serum complement was categorised as serum C3 <70 mg/dL and/or serum C4 <14 mg/dL. eGFR was estimated using the Modification of Diet in Renal Disease (MDRD) equation in adults.25
As in the pathology reports, each case was classified based on the 2003 ISN/RPS LN classification.5 The NIH Activity Index and Chronicity Indices were used to characterise glomerular disease activity and chronic interstitial damage, respectively. Activity Index was categorised as mild (0 to 5) moderate (6 to 14) and high (≥15). Chronicity Index was categorised as mild (0 to 3), moderate (score 4 to 7) and high (≥8).5 Global glomerulosclerosis was categorised as ≤10% vs >10% of glomeruli with sclerosis. Number of crescents was counted. Routine immunofluorescence (IF) staining of C3, C4, C1q in the renal biopsy was recorded. The presence of concomitant C3, C4 and C1q deposition in renal biopsy was considered evidence for classical complement pathway activation.
Statistical analyses were performed using STATA (V.16). Fisher’s exact test for categorical data and Student’s t-test for continuous measurements were performed for comparison between tubular C9 + and C9- groups. The Wilcoxon Mann-Whitney test was used when the normality assumption was not met. A p value <0.05 was considered statistically significant and ≤0.20 was considered suggestive of possible association.
Patient and public involvement statement
Patients and the public were not directly involved in the design and conduct of this study. The research questions and outcomes were developed to better understand the role of complement-mediated tubulointerstitial injury in LN. The information learnt from this project will affect care of patients with LN including prognostication and treatment, to improve their survival and quality of life. Dissemination of study results will be done through online patient websites, lupus foundations and annual rheumatology meetings.