Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy

Abstract

Background: Gas6 is a growth factor that causes proliferation of mesangial cells in the development of glomerulonephritis. Gas6 can bind to three kinds of receptors; Axl, Dtk, and Mer. However, their expression and functions are not entirely clear in the different glomerular cell types. Meanwhile, representative cell cycle regulatory protein p27 has been reported to be expressed in podocytes in normal glomeruli with decreased expression in proliferating glomeruli, which inversely correlated with mesangial proliferation in human IgA nephropathy (IgAN).

Methods: The aim of this study is to clarify Gas6 involvement in the progression of IgAN. Expression of Gas6/Axl/Dtk was examined in 31 biopsy proven IgAN cases. We compared the expression levels with histological severity or clinical data. Moreover, we investigated the expression of Gas6 and its receptors in cultured podocytes.

Results: In 28 of 31 cases, Gas6 was upregulated mainly in podocytes. In the other 3 cases, Gas6 expression was induced in endothelial and mesangial cells, which was similar to animal nephritis models. Among 28 podocyte type cases, the expression level of Gas6 correlated with the mesangial hypercellularity score of IgAN Oxford classification and urine protein excretion. It also inversely correlated with p27 expression in glomeruli. As for the receptors, Axl was mainly expressed in endothelial and mesangial cells, while Dtk was expressed in podocytes. In vitro, Dtk was expressed in cultured murine podocytes, and the expression of p27 was decreased by Gas6 stimulation.

Conclusions: Gas6 was uniquely upregulated in either endothelial/mesangial cells or podocytes in IgAN. The expression pattern can be used as a marker to classify IgAN. Gas6 has a possibility to be involved in not only mesangial proliferation via Axl, but also podocyte injury via Dtk in IgAN.

Figure 1. Gas6 expression in human IgA nephropathy.

Biopsy samples were immunostained using indirect immunohistochemistry procedure with anti-Gas6 antibody. Representative images are shown in (A) from control patients, (B) from IgA nephropathy cases immunostained in epithelial cells, and (C) from IgA nephropathy cases immunostained in endothelial and mesangial cells. The original magnification was X200. Endo/mes, Endothelial and mesangial.

Figure 2. Gas6 stained area in human IgA nephropathy.

Gas6 stained area is expressed as a percentage of total glomerular area occupied by Gas6 immunostained area. For each sample, at least eight glomerular profiles per patient were measured. IgA epi, IgA epithelial type.

Figure 3. Correlation of Gas6 stained area with prognostic factors.

Gas6 stained area in human IgA nephropathy correlated with (A) Oxford mesangial hypercellularity score and (B) urine protein excretion. N = 28. Oxford M score, Oxford mesangial hypercellularity score.

Figure 4. Inverse correlation of Gas6 stained area with p27 positive cell number.

(A) Biopsy samples were immunostained using indirect immunohistochemistry procedure with anti-p27 antibody. p27 positive cell number was counted. The result was divided by glomeruli number. For each sample, at least eight glomerular profiles per patient were measured. p27 positive cell number correlated with Oxford mesangial hypercellularity score. (B) Gas6 stained area inversely correlated with p27 positive cell number. N = 28. Oxford M score, Oxford mesangial hypercellularity score.

Figure 5. Expression of Dtk and Axl in control glomeruli.

(A,B) Photographs of isolated glomeruli. The glomeruli were isolated from the uninvolved portion of tumor nephrectomy specimens by sieving method from three patients. The relative purity of isolated glomeruli was shown. The original magnification was (A) X100. (B) X50. (C) Ten µg of each glomerular lysate was analyzed by westernblotting with the antibodies indicated. Dtk and Axl were expressed in normal glomeruli.

Figure 6. Double immunohistochemistry of Dtk and Axl with cell specific markers or Gas6.

Biopsy samples were immunostained using indirect immunohistochemistry procedure with (A) anti-Dtk, anti-Nephrin (a podocyte marker), (B) anti-Dtk, anti-Gas6 or (C) anti-Axl, anti-CD34 (an endothelial cell marker), (D) anti-Axl, anti-Gas6 antibody. (A,B) Dtk immunostaining was detected in both control and IgA nephropathy, mostly merged with Nephrin. It was also merged with Gas6 in epithelial type IgA nephropathy. (C,D) Axl immunostaining was detected in both control and IgA nephropathy, mostly merged with CD34. It was also merged with Gas6 in endothelial and mesangial type IgA nephropathy. X200. IgAN, IgA nephropathy. IgA epi, IgA epithelial type. IgA endo/mes, IgA endothelial and mesangial type.

Figure 7. Effect of Gas6 on Dtk and p27 expression in podocytes.

Podocytes were differentiated and stimulated with recombinant Gas6 or TGFβ1 for 24 hours. Cell lysates were subjected to immunoblotting with the antibodies indicated. Then, podocytes expressed Dtk. Gas6 could increase the expression of Dtk and reduce p27, as well as TGFβ1. Representative data were shown from four independent experiments. Quantitative examinations of the band density for (B) Dtk and (C) p27 were shown. The columns and error bars are the mean ± SD of four independent experiments.