PLA₂R Antibody Levels and Clinical Outcome in Patients with Membranous Nephropathy and Non-Nephrotic Range Proteinuria under Treatment with Inhibitors of the Renin-Angiotensin System

<div><p>Patients with primary membranous nephropathy (MN) who experience spontaneous remission of proteinuria generally have an excellent outcome without need of immunosuppressive therapy. It is, however, unclear whether non-nephrotic proteinuria at the time of diagnosis is also associated with good prognosis since a reasonable number of these patients develop nephrotic syndrome despite blockade of the renin-angiotensin system. No clinical or laboratory parameters are available, which allow the assessment of risk for development of nephrotic proteinuria. Phospholipase A₂ Receptor antibodies (PLA₂R-Ab) play a prominent role in the pathogenesis of primary MN and are associated with persistence of nephrotic proteinuria. In this study we analysed whether PLA₂R-Ab levels might predict development of nephrotic syndrome and the clinical outcome in 33 patients with biopsy-proven primary MN and non-nephrotic proteinuria under treatment with blockers of the renin-angiotensin system. PLA₂R-Ab levels, proteinuria and serum creatinine were measured every three months. Nephrotic-range proteinuria developed in 18 (55%) patients. At study start (1.2±1.5 months after renal biopsy and time of diagnosis), 16 (48%) patients were positive for PLA₂R-Ab. A multivariate analysis showed that PLA₂R-Ab levels were associated with an increased risk for development of nephrotic proteinuria (HR = 3.66; 95%CI: 1.39–9.64; p = 0.009). Immunosuppressive therapy was initiated more frequently in PLA₂R-Ab positive patients (13 of 16 patients, 81%) compared to PLA₂R-Ab negative patients (2 of 17 patients, 12%). PLA₂R-Ab levels are associated with higher risk for development of nephrotic-range proteinuria in this cohort of non-nephrotic patients at the time of diagnosis and should be closely monitored in the clinical management.</p></div

Hazard ratios with 95% confidence intervals and p values as estimated by univariate (A) and multivariate (B) Cox regression analysis.

<p>The explanatory variable PLA₂R-Ab levels was ln-transformed prior to analysis. Effects of age and PLA₂R-Ab levels are significant at α = 0.05. The corresponding hazard ratios indicate increasing risks for development of nephrotic range proteinuria with increasing age and PLA₂R-Ab levels (high PLA₂R-Ab levels).</p

Survival analysis by PLA₂R-Ab positivity.

<p><i>4A) Time when PLA₂R-Ab positive or negative patients developed nephrotic range proteinuria. 4B) Kaplan-Meier curves.</i> P values are from log rank tests. N = number of patients at risk at the different time points.</p

Proteinuria during the study follow-up.

<p>Proteinuria significantly increased in PLA₂R-Ab positive patients (solid line) but remained low in PLA₂R-Ab negative patients (dashed line). The bars show the SD-values of proteinuria for PLA₂R-Ab positive patients (up) and PLA₂R-Ab negative patients (down). “N” gives the number of patients for whom data were available at these specific times of follow-up. “*” shows a statistically significant difference (p<0.05) between the single time point and the start of the study. “§” shows a statistically significant difference (p<0.05) between PLA₂R-Ab positive patients and PLA₂R-Ab negative patients.</p

Flow chart of patients included in the study and their follow-up.

<p>Of the 33 patients included in the study 16 were PLA₂R-Ab positive and 17 were PLA₂R-Ab negative at the start of the study. Nephrotic proteinuria developed in 13 PLA₂R-Ab positive and five PLA₂R-Ab negative patients. Immunosuppressive treatment was used in 13 PLA₂R-Ab positive patients and two PLA₂R-Ab negative patients. At the end of the follow-up eight patients were still PLA₂R-Ab positive, two of them had a remission of proteinuria (both partial remission) and five of them had a significant increase in serum creatinine. PLA₂R-Ab were negative in 25 patients at the end of the follow-up, 24 of them had a remission of proteinuria (16 complete remission, eight partial remission) and one of them had a significant increase in serum creatinine. CR = complete remission; PR = partial remission; NR = no remission. ^a = Significantly more PLA₂R-Ab positive patients developed nephrotic-range proteinuria compared to PLA₂R-Ab negative patients (Fisher’s exact test: p<0.005). ^b = Significantly more PLA₂R-Ab positive patients received immunosuppressive therapy compared to PLA₂R-Ab negative patients (Fisher’s exact test: p<0.001). ^c = Significantly less patients who were still positive for PLA₂R-Ab at the end of the study follow-up reached remission of proteinuria compared to patients who were negative for PLA₂R-Ab at the end of the follow-up (Fisher’s exact test: p<0.001). ^d = Significantly more patients who were still positive for PLA₂R-Ab at the end of the study follow-up had a significant increase of serum creatinine compared to patients who were negative for PLA₂R-Ab at the end of the follow-up (Fisher’s exact test: p = 0.001).</p

Clinical baseline characteristics of the patients at the time of inclusion in the study.

<p>At the time of inclusion in the study 16 (48%) patients had detectable PLA₂R-Ab in the serum. Proteinuria at inclusion in the study was higher in PLA₂R-Ab positive patients compared to PLA₂R-Ab negative patients but this difference did not reach statistical significance. Serum creatinine and age were not different between PLA₂R-Ab positive and PLA₂R-Ab negative patients. Significantly more PLA₂R-Ab positive patients received immunosuppressive treatment during the follow-up time.</p><p>Clinical baseline characteristics of the patients at the time of inclusion in the study.</p

Control of secondary <i>L. monocytogenes</i> infection in CXCR6-deficient mice.

<p>Wt and CXCR6^GFP/GFP mice were primary infected with 5×10³<i>Lm</i> i.v. and 60 days later challenged with 1×10⁵<i>Lm</i> i.v. Two days after secondary infection, listeria titers in spleen and liver were determined. Colony forming units (CFU) for individual mice and the median of one representative experiment of two are shown, n≥7. Detection limit was 20 CFU. *, p<0.05.</p

The Function of the Chemokine Receptor CXCR6 in the T Cell Response of Mice against <i>Listeria monocytogenes</i>

<div><p>The chemokine receptor CXCR6 is expressed on different T cell subsets and up-regulated following T cell activation. CXCR6 has been implicated in the localization of cells to the liver due to the constitutive expression of its ligand CXCL16 on liver sinusoidal endothelial cells. Here, we analyzed the role of CXCR6 in CD8⁺ T cell responses to infection of mice with <i>Listeria monocytogenes</i>. CD8⁺ T cells responding to listerial antigens acquired high expression levels of CXCR6. However, deficiency of mice in CXCR6 did not impair control of the <i>L. monocytogenes</i> infection. CXCR6-deficient mice were able to generate listeria-specific CD4⁺ and CD8⁺ T cell responses and showed accumulation of T cells in the infected liver. In transfer assays, we detected reduced accumulation of listeria-specific CXCR6-deficient CD8⁺ T cells in the liver at early time points post infection. Though, CXCR6 was dispensable at later time points of the CD8⁺ T cell response. When transferred CD8⁺ T cells were followed for extended time periods, we observed a decline in CXCR6-deficient CD8⁺ T cells. The manifestation of this cell loss depended on the tissue analyzed. In conclusion, our results demonstrate that CXCR6 is not required for the formation of a T cell response to <i>L. monocytogenes</i> and for the accumulation of T cells in the infected liver but CXCR6 appears to influence long-term survival and tissue distribution of activated cells.</p></div

CXCR6 expression on CD8⁺ T cells during <i>L. monocytogenes</i> infection.

<p>CXCR6^+/GFP mice were infected with 1×10⁴<i>Lm</i>OVA i.v. and cells from spleens and livers were analyzed at indicated time points. (A) Representative histogram of CXCR6 expression by CD8⁺ T cells. (B) Expression of CXCR6 by CD8⁺ T cells. (C) Representative dot plots of CXCR6 and IFN–γ expression by CD8⁺ T cells. Upper row: spleen, lower row: liver (D) Expression of CXCR6 by IFN–γ⁺ listeria-specific CD8⁺ T cells. Symbols in (B) and (D) give the mean ± SEM, n≥5 and are representative for two experiments.</p

CXCR6 controls early accumulation of activated CD8⁺ T cells in the liver.

<p>Purified CD8⁺ T cells from wt OT–I and CXCR6^GFP/GFP OT–I mice were mixed in a ratio of 1∶1 and a total of 5×10⁴ cells were injected into congenic wt mice i.v. infected with 1×10⁵<i>Lm</i>OVA. Transferred cells from spleens and livers of recipient mice were analyzed at day 3 and day 5 p.i. (A) Representative CD90.1 and CD90.2 staining of purified and mixed OT–I cells before (dc, donor cells) and three and five days after transfer. Numbers show percentages of positive cells. (B) Normalized ratio of transferred OT–I cells in spleens and livers of recipient mice on indicated time points. (C, D) Activation status of transferred OT–I cells. (C) Bars show distribution of CD44⁻CD62L⁺, CD44⁺CD62L⁺, and CD62L⁻ cells within wt and CXCR6^GFP/GFP OT–I cells. (D) Mean fluorescence intensity (MFI) of KLRG1 staining on transferred OT–I cells. Bars give mean ± SEM, n≥3. The experiment was repeated twice with consistent results. **, p<0.01; ***, p<0.001.</p