Clinical usefulness of anti-M-type phospholipase-A-receptor antibodies in patients with membranous nephropathy and the comparison of three quantification methods

Figures & data

Table 1. Demographic and clinical characteristics of the patients with primary MN.

	Mean (standard deviation)	Median (range)	Number
Age	57.4 (13.2)	62 (21–77)
Gender, woman/man			14/9
Disease duration (months)	14.5 (22.3)	5 (1–100)
Urine protein (g/day)	2.85 (2.14)	1.96 (0.2–7.13)
Nephrotic/non-nephrotic			7/16
Serum albumin (g/dl)	2.8 (0.6)	2.8 (1.9–4.4)
Serum creatinine (mg/dl)	0.79 (0.26)	0.72 (0.50–1.61)
Creatinine clearance (ml/min)	93.3 (26.9)	86 (21–142)

MN: membranous nephropathy.

Figure 1. Stable expression of PLA2R in the HEK293T cells. (A) Flow cytometry showed the expression of PLA2R on the surface of HEK293T cells. The mean fluorescence intensity of PLA2R (red) and of an isotype control (white) are shown. (B) Western blot showed that the PLA2R transfected HEK293T cells expressed the PLA2R transgene between 150 and 250 kD (right lane), while in the mock-transfected cell line (left lane) no positive signal could be detected.

Figure 2. Western blot of anti-PLA2R in the serum of patients with primary MN. By western blot, anti-PLA2R antibodies were detected in 12 out of 23 (52%) patients with primary MN. ‘M’s represent protein molecular weight markers. ‘P’s represent positive controls (i.e., rabbit polyclonal antibodies against human PLA2R).

Figure 3. Association between the three measurement methods of anti-PLA2R antibodies. (A) Quantified western blot and cell-based ELISA (Pearson's correlation coefficient, r = 0.94). (B) Quantified western blot and commercial solid-phase ELISA (r = 0.84). (C) Cell-based ELISA and commercial solid-phase ELISA (r = 0.85). (D) The titres of anti-PLA2R antibodies by cell-based ELISAs were significantly higher in western blot-positive samples than those in western blot-negative samples among primary MN patients (p = .048). (E) The titres of anti-PLA2R antibodies by commercial solid-phase ELISAs were significantly higher in western blot-positive samples than those in western blot-negative samples among primary MN patients (p = .015). Correlations were evaluated by Pearson's correlation coefficient. p values were determined by Welch's t-test. Horizontal bars represent mean levels of each group in D and E.

Table 2. Clinical characteristics and the results of anti-PLA2R antibodies in each patient with primary membranous nephropathy.

										Anti-PLA2R antibodies
Case No.	Age	Gender	Disease duration (months)	Proteinuria (g/day)	Nephrotic syndrome	Serum albumine (g/dl)	Serum creatinine (mg/dl)	Creatinine clearance (ml/min)	Treatment	Western blot	Cell-based ELISA (AU/ml)	Commercial solid-phase ELISA (RU/ml)
1	67	F	12	2.65	Non-nephrotic	2.9	0.55	78	Supportive	Positive	5.3	334.5
2	63	F	5	4.23	Nephrotic	2.8	0.68	125	Immunosuppressive	Positive	3.4	39.8
3	60	F	14	5.61	Nephrotic	2.3	0.97	86	Immunosuppressive	Positive	107.1	287.6
4	63	M	1	6.30	Nephrotic	2.4	0.91	79	Immunosuppressive	Positive	137.7	990.6
5	63	F	2	1.47	Non-nephrotic	3.0	0.65	91	Supportive	Negative	4.7	4.5
6	57	M	100	5.99	Nephrotic	2.0	1.02	126	Immunosuppressive	Positive	15.0	86.0
7	32	F	5	7.13	Nephrotic	1.9	0.71	104	Immunosuppressive	Negative	15.8	50.5
8	62	M	4	4.25	Nephrotic	2.9	1.03	81	Immunosuppressive	Positive	49.9	347.8
9	61	M	60	0.96	Non-nephrotic	4.4	0.83	108	Supportive	Negative	0.0	0.0
10	47	M	13	1.16	Non-nephrotic	3.5	0.83	112	Supportive	Negative	13.3	0.0
11	74	F	7	2.99	Non-nephrotic	2.8	0.61	108	Supportive	Negative	5.7	0.0
12	64	F	1	1.31	Non-nephrotic	2.2	0.52	142	Immunosuppressive	Positive	13.8	29.0
13	64	M	5	7.12	Nephrotic	2.3	1.17	65	Supportive	Negative	6.6	8.0
14	46	F	14	0.20	Non-nephrotic	3.3	0.50	69	Supportive	Positive	8.2	25.4
15	77	F	1	2.81	Non-nephrotic	2.2	0.59	78	Immunosuppressive	Positive	60.4	634.2
16	21	F	5	0.95	Non-nephrotic	3.6	0.61	123	Supportive	Negative	15.0	0.0
17	67	M	13	2.26	Non-nephrotic	1.9	0.92	69	Immunosuppressive	Negative	7.0	35.0
18	69	F	26	1.66	Non-nephrotic	2.6	1.61	21	Immunosuppressive	Positive	29.9	304.8
19	54	M	6	1.96	Non-nephrotic	2.3	0.83	82	Supportive	Positive	0.0	14.2
20	55	F	4	1.15	Non-nephrotic	3.2	0.52	73	Supportive	Negative	1.2	0.0
21	36	F	1	1.05	Non-nephrotic	2.9	0.72	82	Supportive	Negative	4.9	2.9
22	52	M	30	1.28	Non-nephrotic	3.3	0.86	123	Immunosuppressive	Negative	14.2	6.5
23	66	F	4	1.08	Non-nephrotic	3.5	0.54	122	Immunosuppressive	Positive	10.8	16.2

Table 3. Comparisons of measurement methods about associations between anti-PLA2R antibodies and clinical characteristics among patients with primary MN.

Clinical characteristics	Western blot	Cell-based ELISA	Commercial solid-phase ELISA
	Pearson's correlation coefficients (r) (95% confidence interval)
Urine protein	0.42 (0.01 to 0.71)	0.47 (0.07 to 0.74)	0.39 (−0.03 to 0.69)
Serum albumin	−0.28 (−0.62 to 0.15)	−0.29 (−0.63 to 0.13)	−0.30 (−0.63 to 0.13)
Serum creatinine	0.34 (−0.09 to 0.66)	0.26 (−0.17 to 0.61)	0.21 (−0.22 to 0.57)
Creatinine clearance	−0.32 (−0.64 to 0.11)	−0.20 (−0.57 to 0.23)	−0.35 (−0.67 to 0.07)
	p values determined by Welch's t-test (anti-PLA2R positive vs. negative)
Urine protein	.48	.039	.048
Serum albumin	.19	.02	<.01
Serum creatinine	.68	.16	.41
Creatinine clearance	.64	.09	.35

ELISA: enzyme-linked immunosorbent assay; MN: membranous nephropathy; PLA2R: M-type phospholipase A2 receptor.

Figure 4. Titres of anti-PLA2R antibodies at baseline and thereafter in patients with primary MN by commercial solid-phase ELISA. Repeat sera were available in nine patients with primary MN. Patients who were treated with and without immunosuppressive treatment were indicated with red circles and blue triangles, respectively. In all of the six patients who were treated with immunosuppressive treatment (red circles), titres of anti-PLA2R antibodies significantly declined (p = .03 by Wilcoxon signed-rank test). By contrast, in two of the three patients in whom immunosuppressive treatment was not administered (blue triangles), the titres of anti-PLA2R antibodies increased.