Study population and study design
This was a prospective observational study conducted at the renal clinic at Groote Schuur Hospital in Cape Town. The study was approved by the University of Cape Town human research ethics committee (HREC #332/2017) following which we enrolled consenting adult patients (≥ 18 years) diagnosed with biopsy proven active LN from June 2017 to December 2018. Patients received induction treatment using high dose corticosteroids with either oral mycophenolate mofetil (1 gram twice daily) or monthly pulsed cyclophosphamide using the protocol of the National Institute of Health (NIH).[16] A standardized data abstraction sheet to obtain socio-demographic and clinical information was administered at baseline and at end of induction therapy. Physical examinations and collection of biological specimens were performed, including serum, plasma and urine.
Clinical and laboratory measurements
We used the systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) score[17] to assess disease activity and the systemic lupus collaborating clinics / American college of rheumatology damage index (SLICC/ACR Damage Index) [18] to assess irreversible impairment. Standard of care blood tests were performed at the hospital’s accredited laboratory and included full blood count (FBC), serum creatinine, anti-nuclear antibodies (ANA), anti-Smith antibodies (anti-Sm), anti-double stranded deoxyribonucleic acid antibodies (anti-dsDNA) and serum complement (C3 and C4) at each visit. Estimation of glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology (CKD-EPI) equation.[19] A fresh mid-stream urine sample was used to assess urine protein to creatinine ratio (UPCR) at the laboratory and for the qualitative analysis of protein and blood (dipstick) in the clinic. Aliquots of urine were centrifuged to remove urinary sediment and stored frozen at -80 degrees Celsius until batched analysis for MCP-1 and TWEAK. Histological findings for the renal biopsies were reported using the International Society of Nephrology/Renal Pathology Society classification of 2003.[20] Activity and chronicity indices were assessed using previously described methods. [21] Proliferative LN was defined as histological classes III, IV, or if mixed with class V and interstitial fibrosis was reported as present or absent, irrespective of degree, if reported by the pathologist.
Measurement of Urinary MCP-1
Urine MCP-1 was measured using the Human MCP-1 Quantikine ELISA Kit by R&D Systems. Briefly, monoclonal antibody specific for human MCP-1 had been pre-coated onto a microplate. Standards, controls and samples were prepared, and 200 uL of each was pipetted into the wells in duplicate. The microplate was covered and left to incubate on a microplate shaker at room temperature for two hours. MCP-1 present in the sample bound the immobilized antibody. After washing away any unbound substances three times, an enzyme-linked polyclonal antibody specific for human MCP-1 was added to the wells and incubated for one hour. Following three washes a substrate solution was added to the wells and incubated for 30 minutes protected from light. Colour development was stopped, and the intensity of the colour was measured using a spectrophotometer to determine the optical density of each well at 450 nm, with wavelength correction at 540 nm.
Measurement of Urinary TWEAK
Urine TWEAK was measured using the TWEAK Human Instant ELISA Kit by eBioscience (Thermo Fisher Scientific). Briefly, an anti-human TWEAK antibody had been pre-coated onto a microplate, with a number of the reagents already present in the wells in a lyophilised pellet. The appropriate volume of distilled water was first added to each well, followed by 50 µL of standard, control or sample, in duplicate. The microplate was covered and incubated on a microplate shaker at room temperature for 3 hours, followed by six washes. Substrate solution was then added, and the colour development was monitored using the plate reader set to 620 nm. The substrate reaction was stopped as soon as Standard 1 reached an OD of 0.9 – 0.95. The absorbance of each microwell was measured at 450 nm as the primary wavelength and wavelength correction at 630 nm.
Determining the Concentration of uMCP-1 and uTWEAK
Colour development in both ELISAs occur in proportion to the concentration of analyte in the sample. The standard curves prepared during each run were used to determine the concentration of the other samples and were derived from an average of the duplicates performed. Samples with analyte concentrations above the measuring range of the kit were repeated in dilution to obtain an absolute value in picograms per millilitre (pg/mL). Both MCP-1 and TWEAK results were also mathematically standardised to the urine creatinine concentration present in the respective sample to account for variations in urine concentration, as per convention. These results were denoted “corrected” and expressed as picograms per milligram of creatinine (pg/mgCr). In this study we provide the results for both absolute and corrected concentrations of the respective biomarkers.
Definition of remission status
Complete remission was defined as return of serum creatinine to previous baseline, plus a decline in the UPCR to <0.05 g/mmol. Partial remission was defined as stabilization (±25%), or improvement of serum creatinine, but not to normal, plus a ≥50% decrease in UPCR from start of induction therapy. Alternatively, if there was nephrotic-range proteinuria then remission required a ≥50% reduction in UPCR, and a UPCR <0.300 g/mmol. Patients not meeting the above criteria were considered to not be in remission.[22] Early remission refers to those who achieved remission after completion of induction therapy.
Statistical Analysis
Statistical analysis was undertaken using Stata 15.1 (Stata Corp. Texas, USA). Data was assessed for normality using the Shapiro-Wilk test. Continuous variables were reported as means ± standard deviation if normally distributed and median (with interquartile ranges) if not normally distributed. Categorical variables were reported as frequencies (or percentages). The Spearman rank correlation was used to determine the degree of linear relationship between continuous variables. The Student t-test or its non-parametric equivalent, the Wilcoxon rank-sum test was used to compare means between two groups. The Wilcoxon sign rank test was used for comparing the pre and post treatment values of the corrected uMCP1 and uTWEAK levels. A p-value <0.05 was interpreted as statistically significant.