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. 2023 Nov 13;21(1):324.
doi: 10.1186/s12964-023-01218-w.

Effects of a novel ANLN E841K mutation associated with SRNS on podocytes and its mechanism

Li Lin  1 , Yuhong Ye  1 , Haidong Fu  1 , Weizhong Gu  2 , Manli Zhao  2 , Jingmiao Sun  1 , Zhongkai Cao  1 , Guoping Huang  1 , Yi Xie  1 , Fei Liu  1 , Lu Li  1 , Qiuyu Li  1 , Jianhua Mao  3 , Lidan Hu  4
Affiliations

Effects of a novel ANLN E841K mutation associated with SRNS on podocytes and its mechanism

Li Lin et al. Cell Commun Signal. .

Abstract

Background: Steroid-resistant nephrotic syndrome (SRNS) is characterized by unrelieved proteinuria after an initial 4-8 weeks of glucocorticoid therapy. Genes in podocytes play an important role in causing SRNS.

Objective: This study aimed to report a pathogenic mutation in SRNS patients and investigate its effects on podocytes, as well as the pathogenic mechanism.

Methods: We screened out a novel mutation by using whole-exon sequencing in the SRNS cohort and verified it via Sanger sequencing. Conservative analysis and bioinformatic analysis were used to predict the pathogenicity of the mutation. In vitro, stable podocyte cell lines were constructed to detect the effect of the mutation on the function of the podocyte. Moreover, an in vivo mouse model of podocyte ANLN gene knockout (ANLNpodKO) was used to confirm clinical manifestations. Transcriptome analysis was performed to identify differential gene expression and related signaling pathways.

Results: ANLN E841K was screened from three unrelated families. ANLN E841K occurred in the functional domain and was predicted to be harmful. The pathological type of A-II-1 renal biopsy was minimal change disease, and the expression of ANLN was decreased. Cells in the mutation group showed disordered cytoskeleton, faster cell migration, decreased adhesion, increased endocytosis, slower proliferation, increased apoptosis, and weakened interaction with CD2 association protein. ANLNpodKO mice exhibited more obvious proteinuria, more severe mesangial proliferation, glomerular atrophy, foot process fusion, and increased tissue apoptosis levels than ANLNflox/flox mice after tail vein injection of adriamycin. Upregulated differentially expressed genes in cells of the mutation group were mainly enriched in the PI3K-AKT pathway.

Conclusion: The novel mutation known as ANLN E841K affected the function of the ANLN protein by activating the PI3K/AKT/mTOR/apoptosis pathway, thus resulting in structural and functional changes in podocytes. Our study indicated that ANLN played a vital role in maintaining the normal function of podocytes. Video Abstract.

Keywords: ANLN mutation; Podocyte; Podocyte-specific knockout mouse; SRNS.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Identification of ANLN c.2521 G > A mutation in pedigrees with SRNS. a Three probands related to SRNS were screened, and DNA samples were available from individuals labeled S, which were analyzed by WES. The black arrow indicates proband. ●: female patient. ■: male patient. b Representative HE and TEM images of kidney biopsy histology from proband 1 showed an area of minimal-change disease. The scale bar was 50 μm for HE and 1 μm for TEM. c Missense heterozygous mutation ANLN c.2521 G > A (p. E841K) occurred in exon 15. Top, mutant sequence; bottom, wildtype sequence. d Representative immunofluorescence staining of Control and proband A-II-1 kidney tissue. The co-localization of ANLN and WT1 in the kidney tissue of the proband A-II-1 was similar to that of the negative control without ANLN primary antibody incubation. Red represents ANLN, green WT1, and blue DAPI. The scale bar was 50 μm. e The mutated nucleotide is located in the 15th exon and mutated amino acid is located in the RhoA domain indicated by the black arrow. f The E841 residue is conserved in evolution between different species. The mutated residue is labeled with red triangles. Conserved residues (*); highly similar residues (:); similar residues (.)
Fig. 2
Fig. 2
The effect of overexpressed ANLN E841K on the phenotype of podocytes. a Gene expression quantification of ANLN in three groups of cells were tested by RT‒qPCR. b-c Representative WB plots and WB quantification showed that podocyte gene overexpression cell lines were successfully constructed. d Representative immunofluorescence images of phalloidin. Red stands for Phalloidin, green for GFP, and blue for DAPI. The cytoskeleton of the mutant group was clustered and arranged disorderly. e, g Representative pictures of three group cells at the same position on plate at 0, 12, 24, and 36 h, respectively. The cell migration speed of E841K group was significantly faster than that of the WT group. f, h Representative immunofluorescence images of endocytosis. Red stands for Dextran, green for GFP, and blue for DAPI. The fluorescence intensity of podocytes with dextran in E841K was significantly higher than the other two groups. i, j Representative pictures of Vector, WT and E841K cells adhering to the 6-well plate. The adhesion ability of E841K cells was significantly reduced compared with that of the WT cells. k-m ANLN E841K interacted with CD2AP. k Representative WB plots of protein samples from the three groups of cells. l Representative fluorescence images of the three group cells. Red stands for CD2AP, green for ANLN, and blue for DAPI. m WB quantification of Flag protein pulled down by CD2AP. The amount of Flag protein pulled down by CD2AP in the mutant group was significantly less than that in the WT group. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001
Fig. 3
Fig. 3
The effect of overexpressed ANLN E841K on function of podocytes. a, c Flow cytometry of cell cycle showed that the cells of E841K group were arrested in G2 phase. d The cell proliferation rate of the E841K group was significantly lower than that of the WT at 96 and 120 h. b, e Flow cytometry of apoptosis analysis revealed that percentage of total apoptosis of E841K group was significantly higher than that of the WT. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001
Fig. 4
Fig. 4
Kidney changes in glomerular ANLNpodKO mice. a Flow chart of the construction of ANLNpodKO mice. loxP indicates the site of gRNA insertion. F1, R1, F2, and R2 represent primer. b Sanger sequencing of the mouse ANLN gene inserted gRNA. c Representative agarose gel electrophoretogram of PCR products of mouse tail and kidney tissue DNA. M indicates Marker, + / + indicates no loxP site, flox/ + indicates that one allele is inserted with gRNA, flox/flox indicates that both alleles are inserted with gRNA, WT indicates that the fragment in the middle of the two loxP sites is not deleted, and KO indicates that the fragment in the middle of the two loxP sites is deleted. d Representative immunofluorescence images of glomerulus. Red stands for ANLN, green for WT1, and blue for DAPI. The scale bar was 10 μm. e Representative images of HE, PAS, Masson staining and TEM observation in four groups. The scale bar was 20 μm for HE and 1 μm for TEM. f The urine Albumin/Creatinine ratio of ANLNflox/flox, ANLNpodKO, ANLNflox/flox + ADR and ANLNpodKO + ADR mice. g Quantification of glomerular injury in four groups. h Quantification of slit diaphragm number on GBM in four groups. i Representative WB plots of kidney tissue in ANLNflox/flox and ANLNpod−KO mice after ADR treated. j-m WB quantification of Bax/Bcl2, C-Caspsase3/Caspsase3, CHOP in ANLNflox/flox + ADR and ANLN.podKO + ADR mice groups. n.s. indicates P > 0.05, * indicates P < 0.05, and ** indicates P < 0.01
Fig. 5
Fig. 5
RNA seq and pathway verification. a Statistics of pathway enrichment between WT and E841K cell samples showed that up regulated differential expression gene enriched in PI3K/AKT pathway. b Relative gene expression of the cells of Vector, WT, and E841K groups. c Representative WB plots and quantification of the three group cells. * indicates P < 0.05 and ** indicates P < 0.01
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