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Dysregulated Dynein-Mediated Trafficking of Nephrin Causes INF2-related Podocytopathy - PubMed

Dysregulated Dynein-Mediated Trafficking of Nephrin Causes INF2-related Podocytopathy

Hua Sun et al. J Am Soc Nephrol. 2021 Feb.

Abstract

Background: FSGS caused by mutations in INF2 is characterized by a podocytopathy with mistrafficked nephrin, an essential component of the slit diaphragm. Because INF2 is a formin-type actin nucleator, research has focused on its actin-regulating function, providing an important but incomplete insight into how these mutations lead to podocytopathy. A yeast two-hybridization screen identified the interaction between INF2 and the dynein transport complex, suggesting a newly recognized role of INF2 in regulating dynein-mediated vesicular trafficking in podocytes.

Methods: Live cell and quantitative imaging, fluorescent and surface biotinylation-based trafficking assays in cultured podocytes, and a new puromycin aminoglycoside nephropathy model of INF2 transgenic mice were used to demonstrate altered dynein-mediated trafficking of nephrin in INF2 associated podocytopathy.

Results: Pathogenic INF2 mutations disrupt an interaction of INF2 with dynein light chain 1, a key dynein component. The best-studied mutation, R218Q, diverts dynein-mediated postendocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport can rescue this effect. Augmented dynein-mediated trafficking and degradation of nephrin underlies puromycin aminoglycoside-induced podocytopathy and FSGS in vivo.

Conclusions: INF2 mutations enhance dynein-mediated trafficking of nephrin to proteolytic pathways, diminishing its recycling required for maintaining slit diaphragm integrity. The recognition that dysregulated dynein-mediated transport of nephrin in R218Q knockin podocytes opens an avenue for developing targeted therapy for INF2-mediated FSGS.

Keywords: cytoskeleton; genetic renal disease; podocyte.

Copyright © 2021 by the American Society of Nephrology.

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Figures

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Graphical abstract
Figure 1.
Figure 1.

INF2 interacts with Dynll1. (A) The interaction of INF2-DID and Dynll1 is disrupted by FSGS-causing mutations (E184K, S186P, R218Q), as demonstrated by yeast mating. (B) An endogenous interaction of INF2 and Dynll1 in mouse kidney lysates (WT) was confirmed by co-IP, using kidney lysate of INF2 knockout (KO) mice as a negative control. (C) Mouse podocytes were cotransfected with flag full–length INF2 and Myc-Dynll1. The cell lysates immunoprecipitated with anti-Myc (Dynll1) were subjected to immunoblotting with anti-Flag (INF2). (D) Mouse podocytes were cotransfected with Myc-Dynll1 and GFP FL, d-DID, or DID of INF2. The cell lysates were immunoprecipitated with Myc (Dynll1) and subjected to immunoblotting with anti-GFP (INF2–d-DID or INF2-DID truncations). Coimmunofluorescent stain of INF2 and Dynll1 in WT mouse podocytes (E) and mouse kidney (F) showed a limited focal colocalization at the edge of podocytes (arrows). (G) Coimmunostaining of overexpressed Dynll1 and INF2 showed a decreased colocalization in cells expressing mutant INF2. EV, empty vector; EK, E184K; SP, S186P; RQ, R218Q; KO, knockout; FL, full length; d-DID, delta-DID.

Figure 2.
Figure 2.

Bioinformatic analysis suggests INF2 participates in the dynein trafficking pathway. (A and B) PPI generated in GeneMANIA (13 CMT-causing genes ARHGEF10, DCTN1, DCTN2, DNM2, DYNC1H1, FGD4, INF2, KIF1B, KIF5A, NEFH, NEFL, PLEKHG5, RAB7A, and Dynll1 are drawn in the inner cycle, whereas 20 predicted genes by the molecular function–based weighting method are located in the outer circle. Distinct colors of the network edge indicate the bioinformatics methods applied: physical interactions, coexpression, predicted, colocalization, and pathway. The different colors used for the nodes indicate the biologic functions of the sets of enrichment genes: cytoplasmic dynein (orange), cytoskeletal-based intracellular transport (purple), organelle fission (red), and surface protein processing and presenting (green). (C) STRING generated a PPI with significant enrichment (PPI enrichment P<0.001). The network was further categorized into two subnetworks (in red and in green) by K-means clustering. The intercluster edges are represented by dashed lines.

Figure 3.
Figure 3.

Enhanced dynein involvement in postendocytic sorting of nephrin. (A) Schematic of antibody-mediated nephrin crosslink and endocytosis. Podocytes overexpressing nephrin are incubated in media containing anti-nephrin antibody followed by Alexa Fluor 488–labeled secondary antibody to induce crosslinking and endocytosis of nephrin. (B) Hypothetic schematic model of dynein-mediated nephrin trafficking along the microtubule, coupled to UPS by the bridging of HDAC6. (L, Dynll1; M, dynein intermediate chains; H, dynein heavy chains; blue tubulin, deacetylated alpha tubulin; pink tubulin, acetylated alpha tubulin). (C) WT podocytes or R218Q KI podocytes with or without antibody-mediated nephrin crosslink were lysed and subjected to IP with anti-nephrin (using IgG as a negative control). The Dynll1, Dynactin 1, HDAC6, and the Lysine acetylated alpha tubulin in nephrin pulldown were analyzed by immunoblotting, quantified in Image J, and normalized to nephrin. The ratios of these normalized levels in cells with crosslink to those without crosslink were calculated. For statistical analysis, ratios of normalized Dynactin 1, HDAC6, and the Lysine acetylated alpha tubulin in cells +/- crosslink from three independent experiments were compared using independent sample t test. *P<0.05 R218Q versus WT. (D) Immunofluorescent-based colocalization illustrated the recruitment of Dynactin 1 and Dynll1 (red) to endocytosed nephrin (green) after antibody-mediated crosslinking.

Figure 4.
Figure 4.

Time-lapse imaging and single-molecule tracking of nephrin using KymographClear, KymographDirect, and TrackMate. (A) The endocytic, recycling, and static events are labeled in red, green, and blue, respectively. Single-molecule tracking of nephrin was performed five times × five cells × three independent experiments. (B) The fractions and (C) average velocities of trafficking events in cells treated with Ciliobrevin (50 μM) and control cells treated DMSO (0.3%) were analyzed by using one-way ANOVA and the difference between groups analyzed by Student–Newman–Keuls (SNK) q test. *P<0.05 versus WT control (DMSO).

Figure 5.
Figure 5.

Immunostaining. Coimmunostaining of the nephrin signalosome (red) with Rab 5, Rab7, Rab 11, HDAC6, and ubiquitin (green) in WT podocytes and R218Q KI cells.

Figure 6.
Figure 6.

Antagonizing dynein trafficking pathway rescued impaired-nephrin recycling in R218Q KI cells. Nephrin recycling in WT and R218Q KI cells was visualized using (A) a fluorescent-based nephrin recycling assay and (C) quantified by surface biotinylation–based recycling assay. (A) As shown schematically, nephrin expressed on podocytes was crosslinked by anti-nephrin antibody, followed by Alexa Fluor 405–labeled second antibody. After endocytosis and acid strip, cells stained with Alexa Fluor 594–labeled second antibody showed successful removal of uninternalized anti-nephrin (dotted frame). Then the recycled nephrin was chased by Alexa Fluor 594–labeled second antibody (Alexa Fluor 488–labeled second antibody in cells transfected with DesRed DN-DCTN1) (solid frame). (B) A schematic of potential targets in the dynein transport complex. (1) Ciliobrevin D that inhibits the ATPase of the heavy chain (HC); (2) overexpression of GFP-WT INF2-DID that binds to and trap Dynll1; (3) Dynll1 siRNA; (4) dominant negative dynactin 1 (DesRed-DN-DCTN1); and (5) HDAC6 inhibitor (Ricolinostat). (C) Schematics of surface biotinylation–based recycling assay: cells went through a workflow from surface biotinylation of the surface nephrin, endocytosis, and recycling of the biotinylated nephrin, incubation with mesna to strip biotin off the recycled nephrin. Cells collected before (degradation control, DC) and after the mesna strip (recycling, R) were lyzed. The biotinylated nephrin in DC and in R were analyzed by streptavidin IP and normalized to total nephrin. The recycled nephrin is quantitated as biotinylated nephrin ([DC-R]/DC) ×100%. Data were collected from three independent experiments and analyzed using one-way ANOVA, and the differences between groups were analyzed by SNK q test. *P<0.05 versus WT control (DMSO). The impaired nephrin recycling in R218Q KI cells was rescued by coexpression of dominant negative dynactin 1(DN DCTN1), Dynll1 siRNA, WT INF2-DID, Ciliobrevin D (50 μM), or Ricolinostat (5 μM). (D) Knocking down of Dynll1 in R218Q KI cells using Dynll1 siRNA was demonstrated by western blotting, compared with cells treated with control siRNA with no targetable mRNA (sequences of these siRNA duplexes are listed in Methods).

Figure 7.
Figure 7.

PAN of R218Q KI mice. PA-induced proteinuria in R218Q KI mice (wt/ki and ki/ki, but not in wt/wt mice) as demonstrated by Coomassie blue stain of a urine gel (A). (B) The trend of urine albumin to creatinine ratio in PAN of INF2-transgenic mice. Data were analyzed using repeated-measures ANOVA and the differences among wt/wt, wt/ki, and ki/k mice were analyzed by SNK q test. P<0.05 versus wt/wt mice at the same time point (n=3 for each genotype). (C) Kidney function was assessed by measured serum creatinine levels in blood samples collected at the time of animal sacrifice (D21). Data were analyzed using independent sample t test (n=5). P<0.05, PAN versus control mice of the same genotype without PA injection. (D) Masson trichrome and periodic acid–Schiff stain of mouse kidney sections showed PA induced segmental and global glomerulosclerosis in R218Q KI mice, and increased tubulointerstitial fibrosis. (E) Electron microscopy showed podocytopathy of PAN in R218Q KI mice characterized by foot process effacement with clusters of active vesiculation (orange arrows). (F) The glomerulosclerosis was quantified as the ratio of sclerotic glomeruli/total glomeruli, and tubulointerstitial fibrosis (TIF) was quantified as the ratio of TIF area to the whole section area. The podocytopathy was quantified by measuring the number of SDs per μm and capillary length covered by healthy appearing podocytes with intact foot processes and SDs (G). Data were analyzed using independent sample t test (n=5). P<0.05 PAN versus control mice of the same genotype without PA injection.

Figure 8.
Figure 8.

Enhanced dynein-mediated trafficking and degradation of nephrin in PAN model in R218Q KI mice. (A) Dynll1 and ubiquitinated nephrin were detected in the nephrin pulldown of kidney lysates by immunoblotting, and biotinylated nephrin was detected in streptavidin pulldown. Semiquantitative analysis was performed to compare Dynll1/total nephrin pulldown, biotinylated nephrin/total nephrin, ubiquitinated nephrin/total nephrin in mice (wt/wt, wt/ki and ki/ki), with PA or without PA injection using an independent sample t test (n=5). P<0.05 PAN versus control mice of the same genotype without PA injection. (B) Immunofluorescent stain showed increased colocalization of Dynll1 with nephrin in PAN of R218 KI mice (wt/ki and ki/ki) compared with the wt/wt mice. (C) Increased Dynll1 and decreased nephrin expression at the GBM of sclerosing glomeruli of the wt/ki and ki/ki mice with PAN.

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