RhoA vesicle trafficking–mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy
Zhong Zhong, Zhijian Li, Yanjie Li, Lanping Jiang, Qingyu Kong, Wei Chen, Shaozhen Feng
Zhong Zhong, Zhijian Li, Yanjie Li, Lanping Jiang, Qingyu Kong, Wei Chen, Shaozhen Feng
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Research Article Nephrology

RhoA vesicle trafficking–mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy

Abstract

Transglutaminase 2 (TGase2) has been shown to contribute to the mesangial IgA1 deposition in a humanized mouse model of IgA nephropathy (IgAN), but the mechanism is not fully understood. In this study, we found that inhibition of TGase2 activity could dramatically decrease the amount of polymeric IgA1 (pIgA1) isolated from patients with IgAN that interacts with human mesangial cells (HMC). TGase2 was expressed both in the cytosol and on the membrane of HMC. Upon treatment with pIgA1, there were more TGase2 recruited to the membrane. Using a cell model of mesangial deposition of pIgA1, we identified 253 potential TGase2-associated proteins in the cytosolic fraction and observed a higher concentration of cellular vesicles and increased expression of Ras homolog family member A (RhoA) in HMC after pIgA1 stimulation. Both the amount of pIgA1 deposited on HMC and membrane TGase2 level were decreased by inhibition of the vesicle trafficking pathway. Mechanistically, TGase2 was found to be coprecipitated with RhoA in the cellular vesicles. Membrane TGase2 expression was greatly increased by overexpression of RhoA, while it was reduced by knockdown of RhoA. Our in vitro approach demonstrated that TGase2 was transported from the cytosol to the membrane through a RhoA-mediated vesicle-trafficking pathway that can facilitate pIgA1 interaction with mesangium in IgAN.

Authors

Zhong Zhong, Zhijian Li, Yanjie Li, Lanping Jiang, Qingyu Kong, Wei Chen, Shaozhen Feng

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Figure 5

Polymeric IgA1 deposition on HMC and the membrane transfer of cytosol TGase2 are reduced by vesicular trafficking inhibition.

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Polymeric IgA1 deposition on HMC and the membrane transfer of cytosol TG...
(A) Exponentially growing HMC were cultured in serum-free medium with Exo1 (30 μM) or dissolve for 24 hours. Cellular vesicles were purified by centrifugation (5,000g for 5–8 minutes at room temperature) and filtration as shown in Figure 4. The microparticle size distribution of cellular vesicles was obtained using nanoparticle tracking analysis (n = 3). (B) HMC (1.6 × 105 cells in 200 μL 0.5% FBS-1640 medium) was preincubated with 30 μM Exo1 or dissolve (Ctl) at 37°C overnight and then treated with pIgA1 (20 μg/mL) for 1 hour. Cells were washed with 0.5% BSA-PBS and then stained with goat anti–human IgA antibody. Cells were washed with 0.5% BSA-PBS and then stained with goat anti–human IgA antibody, followed by secondary staining with Alexa Fluor 647–conjugated donkey anti-goat antibody. Cells stained without anti-IgA antibody were used as negative control. The stained cells were analyzed on FACScan. (C) The results were expressed as mean ± SD of the percentage of stained cells from 3 individual experiments. Data were analyzed by 1-way ANOVA. (D) Exponentially growing HMC planted in a 6-well plate were treated with Exo1 (30 μM), dissolve (Ctl), or blank (NC) in serum-free medium. After 24 hours, Western blot was used to detect the expression of TGase2 in the cytosolic (C) and membrane (M) fraction. (E) Quantification of D is shown in the bar graph; data are shown as mean ± SD (n = 3, 1-way ANOVA). *P < 0.05, ***P < 0.001.