Volume 95, Issue 3, Pages 708-716 (March 2019) Development of a novel cell-based assay to diagnose recurrent focal segmental glomerulosclerosis patients  Pankaj Srivastava, Ashish K. Solanki, Ehtesham Arif, Bethany J. Wolf, Michael G. Janech, Milos N. Budisavljevic, Sang-Ho Kwon, Deepak Nihalani  Kidney International  Volume 95, Issue 3, Pages 708-716 (March 2019) DOI: 10.1016/j.kint.2018.10.030 Copyright © 2019 International Society of Nephrology Terms and Conditions

Kidney International 2019 95, 708-716DOI: (10.1016/j.kint.2018.10.030) Copyright © 2019 International Society of Nephrology Terms and Conditions

Figure 1 (a) Schematic of assay development: Total RNA was isolated from podocytes treated with recurrent focal segmental glomerulosclerosis (rFSGS) and control patient plasma and subjected to mRNA profiling. Candidate-upregulated genes with proapoptotic function were selected from the list of differential expression genes (Padj < 0.05). Promoter regions from the candidate genes were cloned in a luciferase reporter vector, and stable cell lines were constructed using puromycin as a selection marker. Stable cell lines expressing the promoter-driven reporter were treated with plasma from control or rFSGS patients, and the reporter activity was measured using ONE-Glo EX Luciferase Assay System (Promega). Fold changes were calculated after normalizing with the control patient plasma. (b) Quantitative reverse transcriptase polymerase chain reaction of upregulated candidate genes: Quantitative polymerase chain reaction analysis using BMF, IL1β, and IGFBP3 gene-specific primers showed upregulation of candidate genes in podocytes treated with rFSGS plasma (from patients rF A–J), whereas minimal or no upregulation was noted in podocytes treated with FSGS plasma (from patients Fs A–E). The unpaired t test was performed (*P < 0.05, **P < 0.01, and ***P < 0.01). bp, base pairs; HEK293, human embryonic kidney 293; MCS, multi-cloning site; UTR, untranslated region. Kidney International 2019 95, 708-716DOI: (10.1016/j.kint.2018.10.030) Copyright © 2019 International Society of Nephrology Terms and Conditions

Figure 2 Luciferase reporter assay. The constructed reporter-based podocyte cell lines selectively responded to recurrent focal segmental glomerulosclerosis (rFSGS) patient plasma but not to control patient plasma or plasma derived from other nephropathy patients. Treatment of reporter cell lines BMF (a), IL1β (b), and IGFBP3 (c) with plasma from the majority of rFSGS patients (rF A–N) showed elevated reporter activity (∼1.5- to 2.5-fold increase over the control patient plasma), whereas the plasma from membranous nephropathy patients (MGN A, B) and myasthenia gravis patient (MG1) showed no response. (d) The negative control, LAMP3 gene promoter reporter cell line was tested with plasma from control, rFSGS (rF A, D, E), FSGS (Fs A–E), and MGN (MGN A, B) patients. CD63 antibody was used as a positive control (PC), to induce LAMP3 promoter reporter activity, and untreated control cells were used as a negative control sample. (e) The reporter cell lines were evaluated for their specificity by treating them with plasma from non-rFSGS patients (Fs A–P), and the maximal response was noted only from patient rF A, which was used as a positive control subject. The results are expressed as fold change in light units over control patient plasma, which is the ratio of absolute light units of the experimental samples to that of control patient plasma–treated cell lines. Results represent 3 independent biological repeats and 2 technical repeats. Kidney International 2019 95, 708-716DOI: (10.1016/j.kint.2018.10.030) Copyright © 2019 International Society of Nephrology Terms and Conditions

Figure 3 The BMF, IL1β, and IGFBP3 reporter cell lines detect recurrent focal segmental glomerulosclerosis (rFSGS) patients with high specificity. (a) To evaluate the specificity of this assay, differences in mean fold-change response from BMF, IL1β, and IGFBP3 cell lines between rFSGS patient samples and other nephropathies were statistically analyzed after pooling the data, and they were categorized into 3 groups (black, FSGS; red, rFSGS; and blue, membranous nephropathy, myasthenia gravis, or control patient plasmas). The fold change for BMF, IL1β, and IGFBP3 promoter cell lines treated with rFSGS plasma were significantly higher when compared with plasma from a non-rFSGS/MGN and membranous nephropathy patient. Points are values averaged within the subject for each group, and the lines represent the 95% confidence interval for mean fold change. The horizontal dashed line represents 1-fold change (control). P values beside the points for each group represent the P value for comparison to the control patient group. Significant differences for pairwise group comparisons are signified by the vertical lines where **P < 0.01 and ***P < 0.001. All P values are corrected for multiple comparisons using Tukey’s honestly significant difference. In contrast, no significant change in the expression of LAMP3 relative in rFSGS samples versus other kidney diseases was observed (P = 0.999). Additionally, no differences were observed between samples from patients with FSGS or other nephropathies in this cell line. (b–d) To further evaluate the discriminative performance for each promoter, sensitivity and specificity values for IL1β, BMF, and IGFBP3 promoter cell lines were estimated using receiver-operator characteristic curves. The area under the curves (AUCs) for model fits discriminating between rFSGS and all other nephropathies and between rFSGS and non-rFSGS ranged from 0.81 to 0.86, respectively, for IL1β and BMF reporter cell lines, whereas these were slightly lower for the IGFBP3 cell line (70% and 64%, respectively). Kidney International 2019 95, 708-716DOI: (10.1016/j.kint.2018.10.030) Copyright © 2019 International Society of Nephrology Terms and Conditions