Identification of urinary metabolites that distinguish membranous lupus nephritis from proliferative lupus nephritis and focal segmental glomerulosclerosis

Abstract

Introduction: Systemic lupus erythematosus (SLE or lupus) is a chronic autoimmune disease, and kidney involvement with SLE, a.k.a. lupus nephritis (LN), is a frequent and severe complication of SLE that increases patient morbidity and mortality. About 50% of patients with SLE encounter renal abnormalities which, if left untreated, can lead to end-stage renal disease. Kidney biopsy is considered the criterion standard for diagnosis and staging of LN using the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification, which was developed to help predict renal outcomes and assist with medical decision-making. However, kidney biopsy-based classification of LN is highly invasive and impractical for real-time monitoring of LN status. Here, nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling was used to identify urinary metabolites that discriminated between proliferative and pure membranous LN as defined by the ISN/RPS classification, and between LN and primary focal segmental glomerulosclerosis (FSGS).

Methods: Metabolic profiling was conducted using urine samples of patients with proliferative LN without membranous features (Class III/IV; n = 7) or pure membranous LN (Class V; n = 7). Patients with primary FSGS and proteinuria (n = 10) served as disease controls. For each patient, demographic information and clinical data was obtained and a random urine sample collected to measure NMR spectra. Data and sample collection for patients with LN occurred around the time of kidney biopsy. Metabolic profiling analysis was done by visual inspection and principal component analysis.

Results: Urinary citrate levels were 8-fold lower in Class V LN compared to Class III/IV patients, who had normal levels of urinary citrate (P < 0.05). Class III/IV LN patients had > 10-fold lower levels of urinary taurine compared to Class V patients, who had mostly normal levels (P < 0.01). Class V LN patients had normal urinary hippurate levels compared to FSGS patients, who completely lacked urinary hippurate (P < 0.001).

Conclusions: This pilot study indicated differences in urinary metabolites between proliferative LN and pure membranous LN patients, and between LN and FSGS patients. If confirmed in larger studies, these urine metabolites may serve as biomarkers to help discriminate between different classes of LN, and between LN and FSGS.

Figures

Figure 1

Box and whisker plots. The plots shown are comparing citrate concentration (mM), taurine concentration (mM), SLE Disease Activity Index (SLEDAI), chronicity index (CI), and urine creatinine (mg/mL) for class III/IV versus class V LN patients.

Figure 2

Principal component analysis of urine samples from patients with class III/IV LN and class V LN. (a) Two-dimensional principal component analysis scores plot of urine samples from patients with class III/IV LN (green) and class V LN (black) for peaks in the region from δ 3.40 to 4.50 ppm calculated using the first two principal components. Each point in the scores plot represents the NMR spectrum of an individual patient projected onto the two-dimensional space defined by the first two principal components. The dashed lines encircling the points define the 95% confidence intervals for each group. The color-matched stars indicate the centroid of each group and the line connecting the stars represents the Mahalanobis distance between the group centroids. (b) The loadings plot corresponding to the scores plot shown in Figure 1a. The labeled bucket (point) corresponds to the triplet belonging to taurine in the 1H NMR spectra. The coordinates of each point indicate the PC loadings for that bucket, and represent how strongly that bucket is weighted in the eigenvector defining either the first or second principal component. The loadings plot points are heat map color-coded according to bucket P value: Black (> 1.25 × 10-2), Blue (1.25 × 10-2-10-5). The Bonferroni corrected α-value was 0.0125.

Figure 3

Principal component analysis of urine samples from class V LN patients and focal segmental glomerulosclerosis patients. (a) Two-dimensional principal component analysis scores plot of urine samples from patients with class V LN patients (black) and focal segmental glomerulosclerosis patients (red) using the first two principal components. The dashed lines encircling the points define the 95% confidence intervals for each group. The color-matched stars indicate the centroid of each group and the line connecting the stars represents the Mahalanobis distance between the group centroids. (b) The loadings plot corresponding to the scores plot in Figure 2a. The buckets shown are in the region from δ 0.02 to 10.0 ppm. The loadings plot is heat map color-coded according to bucket P values: Black (> 1.730 × 10-4), Blue 1.730 × 10-4-10-5). The Bonferroni corrected α-value was 1.730 × 10-4.

Figure 4

NMR urine spectra in the region from 7.650 to 7.550 ppm of class V LN patients (black) and focal segmental glomerulosclerosis patients (red). The triplets at δ7.64 and δ7.55 belong to the metabolite hippurate, as indicated in the inset.