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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2023 May 3;34(7):1222–1239. doi: 10.1681/ASN.0000000000000150

Inhibition of Importin-α–Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis

Maulana A Empitu 1,2, Mitsuhiro Kikyo 3,4, Naritoshi Shirata 3,4, Hiroyuki Yamada 1,4,5, Shin-ichi Makino 1,4,5, Ika N Kadariswantiningsih 1,2, Masashi Aizawa 1, Jaakko Patrakka 6,7, Katsuhiko Nishimori 8, Katsuhiko Asanuma 1,4,✉
PMCID: PMC10356163  PMID: 37134307

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Keywords: dendrin, nuclear translocation, podocyte loss, anoikis, apoptosis, nephropathy, CKD, JNK phosphorylation, MAGI2

Abstract

Significance Statement

Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N-terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment–induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin-α. Inhibition of importin-α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin-α–mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis.

Background

Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes.

Methods

The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 (MAGI2) podocyte-specific knockout (MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1–deleted dendrin. Ivermectin was used to inhibit importin-α.

Results

Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N-terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin-α mediate nuclear translocation of dendrin. The inhibition of importin-α/β reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin-α3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients.

Conclusions

Nuclear translocation of dendrin promotes cell detachment–induced apoptosis in podocytes. Therefore, inhibiting importin-α–mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

Introduction

In CKD, podocyte injury is a critical factor for the progression toward ESKD.1,2 Podocytes are a subset of specialized cells that form a slit diaphragm (SD), a structure responsible for blood filtration in the kidney. Mutation in genes encoding SD proteins, such as membrane-associated guanylate kinase inverted 2 (MAGI2), CD2-associated protein (CD2AP), and nephrin, caused congenital glomerular diseases in humans.

Dendrin is a protein abundantly expressed in the cytoplasm of podocytes near SD. Dendrin was initially characterized in the telencephalic dendrites of sleep-deprived rats, in which cellular apoptosis was implicated.3 In the synapse, dendrin forms a complex with Cbl-interacting 85-kDa protein and MAGI2.4 In normal podocytes, dendrin is located in the cytoplasm and interacts with SD proteins, such as MAGI2, CD2AP, nephrin, and podocin.5–8

In injured podocyte, dendrin translocates to the nuclei. Nuclear translocation of dendrin was found in the glomeruli of individuals with FSGS, IgA nephropathy, and lupus nephritis (LN).9,10 Dendrin also enhances TGF-β–mediated apoptosis.11 Mechanistically, dendrin was involved in regulating podocyte survival by interacting with kidney–brain protein (KIBRA) and yes-associated protein (YAP) signaling.12,13 Given that dendrin deficiency does not cause any pathologic consequence in mice, we hypothesized that dendrin would be an attractive therapeutic target.

In this study, we investigated the mechanism of nuclear translocation of dendrin and its implication for podocyte survival. Dendrin carries nuclear localization signal (NLS), which may interact with nuclear trafficking cargo. Using stable overexpression (OE) of full-length and NLS1-deleted dendrin, we investigated the influence of dendrin's subcellular localization to the podocyte's survival. In vivo, we generated adriamycin (ADR)–induced nephropathy, MAGI2 podocyte-specific knockout (MAGI2 podKO), and MAGI2 podKO with dendrin deficiency (MAGI2 podKO/dendrin KO) mice to investigate the role of dendrin in nephropathy.

Methods

Animal Experiment

ADR-induced nephropathy was performed in male BALB/c dendrin-null (dendrin−/−) and wild type (WT) (dendrin+/+) mice aged 8 weeks (body weight, 20–25 g) by injection of ADR (doxorubicin hydrochloride; Wako, Osaka, Japan; 10 mg/kg, retro-orbital), as described previously.14 Ivermectin (Sigma-Aldrich, Saint Louis, MO) was diluted in 1.5% DMSO in sterile saline. ADR-injected mice and MAGI2 podKO (age 6 weeks) mice received ivermectin 2.5 mg/kg body wt per day i.p. or 1.5% DMOS for 2 weeks. All animal experiments and its protocol were approved by the Animal Research Committee of Chiba University and performed according to the Chiba University Guidelines for Animal Experiments.

Generation of MAGI2 podKO Mice and Dendrin-KO/MAGI2 podKO Mice

Conditional (flox) type knockout (KO) mice, which lacked MAGI2 exons 6–22, were generated as previously described.5 MAGI2flox/flox mice were crossed with Podocin-cre (+); MAGI2flox/+ mice to generate podocyte-specific Nphs2-cre (+); MAGI2flox/flox (MAGI2 podKO) mice. Nphs2-cre (−)/MAGI2flox/+ mice served as the control. The primers used for MAGI2 flox genotyping were forward (F1) 5′-AATAAAAATAGCTGCTTTGAGGACAGGGAG-3′, reverse (R1) 5′-GTCAAATAGAACCCACAGGGATGACAAAGA-3′, and reverse (R2) 5′-CATCGATTTTTTCCCAGCCATATGGAAGCT-3′. The primers used for Nphs2-cre genotyping were forward 5′-TTTGCCTGCATTACCGGTCGATGCAAC-3′ and reverse 5′-TGCCCCTGTTTCACTATCCAGGTTACGGA-3′.

The C57BL/6 dendrin−/− mice were generated as previously described.15 The C57BL6 dendrin−/− male mouse was mated with female BALB/c mouse to generate heterozygous N1. The male heterozygous N1 mice were backcrossed to BALB/c background for eight generations. Backcrossed dendrin−/− mice were crossed with Nphs2-cre (+); MAGI2flox/+ to generate dendrin+/−; P Nphs2-cre (+); MAGI2flox/+ and also dendrin+/−; Nphs2-cre (−); MAGI2flox/+. Then, dendrin+/−; Nphs2-cre (+); MAGI2flox/+ were crossed with dendrin+/−; Nphs2-cre (−)/MAGI2 flox/+ to generate dendrin−/−, Nphs2-cre (+); MAGI2flox/flox (MAGI2 podKO/dendrin KO) mice. The dendrin+/+; Nphs2-cre (+); MAGI2flox/flox served as a control.

Immunofluorescence Analysis

The immunostaining and the immunofluorescence analyses of human kidney, mouse, and cultured podocytes were performed as previously described.9 The immunofluorescence images were taken using a Zeiss LSM780 (Zeiss, Oberkochen, Germany).

Human Kidney Biopsy Samples

Human kidney biopsy samples were obtained from diagnostic renal biopsies performed at Chiba University Hospital. This study was conducted under informed consent and was approved by the Ethics Committee on Human Research of the Chiba University Hospital (Reference No. 1178).

Measurement of Urine Albumin, Urine Creatinine, and Plasma Creatinine

Urine albumin was measured using a Mouse Albumin ELISA KIT (Fujifilm—Wako Shibayagi, Gunma, Japan) on the basis of the manufacturer's protocol. Urine and plasma creatinine were measured using LabAssay Creatinine (Wako, Osaka, Japan) on the basis of the manufacturer's protocol.

Histologic Analysis

Kidneys were removed, cut longitudinally into half, and fixed with 4% paraformaldehyde (PFA) in PBS at 4°C overnight. After dehydration, the kidneys were embedded in paraffin. Paraffin blocks were sectioned in 2.5-µm thicknesses and stained with periodic acid–Schiff for histologic evaluation.

Western Blotting

Glomerular, human embryonic kidney 293 (HEK293), and COS-7 cellular lysates were prepared in CHAPS buffer and supplemented with protease inhibitor (Complete Mini) and phosphatase inhibitor (PhosSTOP), as previously described.5 Proteins were eluted with ×4 Laemmli buffer/2-mercaptoethanol and then analyzed using western blotting. Antibodies used in this study are listed in Supplemental Table 1.

Cell Culture and Transient Transfection

Culturing conditionally immortalized murine podocytes was performed as previously explained.16 Transient transfection to HEK293 cells was performed as previously described.5 To induce apoptosis, podocytes were treated with 0.075 µg/ml ADR. Ivermectin was administered at concentrations of 0.05, 0.1, 0.25, 0.5, 1, and 10 µM to investigate the importin-⍺ inhibition on dendrin nuclear translocation.

Plasmid Construct

A full-length cDNA clone of rat dendrin and rat dendrin lacking NLS1 domain (amino acids 59–77) were cloned inframe into pFLAGCMV-6a (Sigma-Aldrich, St. Louis, MO) vectors. To generate enhanced green fluorescent protein plasmids human dendrin (wild type), an open reading frame sequence of human dendrin was cloned into an enhanced green fluorescent protein plasmids-C1 vector (Clontech, Mountain View, CA). The NLS1 with all alanine mutation in three clusters of basic amino acids (NLS1 all-ala) and NLS2 all-ala mutants were constructed as follows: A mutational double-strand DNA fragment, including the NLS1 domain (amino acids 34∼84), was synthesized (Integrated DNA Technologies, Skokie, IL) and the other domain of plasmid was amplified by inverse PCR. These fragments were fused by using In-Fusion cloning system (Takara, Shiga, Japan). Other mutational plasmids were constructed as follows: mismatched unidirectional PCR primers, in which overlapping terminal regions were synthesized, respectively (Sigma-aldrich, St. Louis, MO), and all regions were amplified by inverse PCR using all-ala mutant plasmid as a template and the PCR product was directly transformed into Escherichia coli (E. coli) DH5α (Toyobo, Osaka, Japan). All constructs were verified using Sanger sequencing.

Establishment of Dendrin and dendrinΔNLS1 OE Podocyte

A piggyBac transposon system was used to establish the OE of dendrin (dendrin-OE) and dendrin lacking NLS1 (dendrinΔNLS1-OE) in cultured murine podocytes.17 The green fluorescent protein (GFP) sequence was removed from the pPB-EF1α-EiP-A piggyBac vector, and Flag-tagged dendrin-OE and dendrinΔNLS1-OE were cloned to the vector (pPB-EF1α-Flag-dendrin-iP-A and pPB-EF1α-Flag-dendrinΔNLS1-iP-A). A control podocyte, pPB-EF1α-Control-iP-A, was constructed using inverse PCR. These piggyBac vectors and pHL-EF1a-hcPBase (piggyBac transposed expression vector) were cotransfected through lipofection into undifferentiated cultured podocytes. After 48 hours, puromycin (1.25 µg/ml) was applied to the culture for 4 days. After the selection, single cells were isolated from the remaining puromycin-resistant colonies. Dendrin expression was confirmed using western blotting and immunofluorescence staining after the expansion of each single-cell clone.

Pulldown Assays

Glutathione S-transferase (GST) pull-down assay with GST-dendrin was performed as previously reported.11 Complete Mini and PhosSTOP supplemented lysis buffer were used to prepare glomerular lysate. GST-fusion N-terminus of dendrin protein was expressed in E. coli BL21 and immobilized on GST-agarose beads (GE Healthcare). The beads were washed in assay buffer (1% Triton-X 100 in PBS), and glomerular lysates were added to the beads. Bound proteins were eluted with ×4 Laemmli buffer/2-mercaptoethanol. Elutes and glomerular lysates (serving as input) were analyzed using western blotting.

Wound Healing Assay

Wound healing assay was performed as previously described.18 Each coverslip was then scratched with a sterile 200 µl pipette tip, washed with PBS, and placed into a fresh medium. The cells were viewed with light microscope, and the surface area was measured as a baseline. After 24 hours, cells were fixed with 4% PFA, images were captured, and the surface area was measured. The average scratched surface area of each well on 24 hours was compared with the baseline from the respective wells and presented as a percentage.

Detachment Assay

The detachment assay was performed as previously described with a slight modification.19 Differentiated podocytes were trypsinized and resuspended in media at 3.5 × 105 cells/ml. Then, 1 ml of cell suspension was added to each well of 12-well plate. The cells were incubated for 24 hours to allow the attachment. Then, 0.4 ml of 0.05% trypsin-EDTA (Nacalai Tesque, Kyoto, Japan) was added to each well for 2 minutes, and 0.4 ml fetal bovine serum was added to attenuate the trypsinization. The well was washed three times with PBS to remove detached cells. The cells were stained using 4′,6-diamidino-2-phenylindole (DAPI) and viewed in Keyence BZ-X810 microscope (Keyence Corporation, Osaka, Japan). The average number of attached cells was compared with the control or dendrin-OE podocytes.

Flowcytometry Apoptosis and Cell Size Assay

Apoptotic podocytes were detected by staining using allophycocyanin (APC)–labeled annexin V and 7-aminoactinomycin D (7-AAD) (BioLegend, San Diego, CA).20 The cells were collected 24 hours after treatment with ADR. The cells were washed, suspended, and stained with APC-annexin V and 7-AAD with concentration as recommended by the manufacturer. At least 10,000 cells per sample were analyzed on fluorescence-activated cell sorting Aria III (BD) using FCS Express 7. Early apoptotic cells were identified as annexin V (+) 7-AAD (−) cells, late apoptotic cells as annexin V (+) 7-AAD (+) cells, and viable cells as annexin V (−) 7-AAD (−) cells.

Forward scatter height was used as a surrogate to measure cell size. The viable cell population of control podocytes was used as reference cells. The relative cell size of each subpopulation was calculated as follows:

x;$ of FSC−H of specific populationx$ of FSC−H of reference cells

The Δ cell size in each cell group was calculated as follows:

x$ of FSC−H of viable cells−x$ of FSC−H of early apoptotic cellsx$ of FSC−H of reference cells

Human Transcriptomic Data Analysis

To examine the expression of importins, MAGI2, and dendrin in human kidney, we extracted expression data from the NEPHROSEQ databases (www.nephroseq.org, University of Michigan, Ann Arbor, MI). The expression data were analyzed from Higgins Normal Tissue Panel, Hodgin FSGS Glomeruli, and Nakagawa CKD Kidney datasets.21–23

Statistical Analysis

Data are presented as mean±SEM. The unpaired t test was used to compare two sets of data, and one-way ANOVA was used to compare multiple groups of data, unless indicated in the figure. Significant results were considered when P < 0.05.

Results

Dendrin Deficiency Attenuates Nephropathy in ADR and MAGI2 podKO Mice

To investigate the role of dendrin in nephropathy, we injected WT and dendrin KO mice with ADR. Dendrin KO injected with ADR (dendrin KO+ADR) mice have milder body weight decrease compared with WT+ADR mice (Figure 1A). Dendrin deficiency reduced albuminuria but not serum creatinine in ADR-injected mice (Figure 1, B and C). The loss of dendrin reduced the percentage of glomerulosclerosis (Figure 1, D and E) and preserved the number of podocytes per glomeruli (Figure 1F, Supplemental Figure 1, A and B) in ADR-injected mice.

Figure 1.

Figure 1

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Dendrin deficiency attenuates nephropathy in adriamycin (ADR)-injected mice and prolonged the lifespan of MAGI2 podKO. (A) The percentage of body weight change in wild type (WT) and dendrin KO mice treated with ADR. (B and C) ualbumin/creatinine ratio and plasma creatinine of dendrin KO or WT mice injected with ADR 10 mg/kg body wt r.o. (D) Representative PAS staining image of the kidney, sacrificed on 28 days after ADR injection. (E) Quantification of sclerotic glomeruli per total glomeruli. (F) The average number of WT-1–positive cells per glomeruli in WT untreated, WT+ADR, and dendrin KO+ADR mice (20 glomeruli counted per mice), analyzed using confocal microscopy. (G) Kaplan–Meier survival curves of MAGI2 podKO/dendrin KO (n=10), MAGI2 podKO (n=14), and dendrin KO mice (n=6). Survival of MAGI2 podKO versus MAGI2 podKO/dendrin KO (median=16.5 versus 21.5, P = 0.0010). (H and I) uACR and plasma creatinine of MAGI2 podKO and MAGI2 podKO/dendrin KO. (J and K) Representative image and the quantification of PAS staining in 8-week-old MAGI2 podKO versus MAGI2 podKO/dendrin KO mice's kidney. (L) The average number of WT-1–positive cells per glomeruli in MAGI2 podKO versus MAGI2 podKO/dendrin KO. Scale bar, 40 µm. Data are presented as means±SEM (unless otherwise indicated) and analyzed with the unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001. PAS, periodic acid–Schiff. Figure 1 can be viewed in color online at www.jasn.org.

Previously, MAGI2 podKO mice showed nuclear translocation of dendrin, proteinuria, and shorter lifespan.24,25 To investigate the role of dendrin in MAGI2 deficiency, we generated MAGI2 podKO/dendrin KO (Supplemental Figure 1C). Dendrin KO mice live beyond the observation period. Dendrin deficiency prolonged the lifespan of MAGI2 podKO mice (Figure 1G). MAGI2 podKO/dendrin KO has lower albuminuria than the MAGI2 podKO group (Figure 1H). However, there was no significant difference in the plasma creatinine level (Figure 1I). MAGI2 podKO/dendrin KO mice also have lower percentage of glomerulosclerosis (Figure 1, J and K) and higher number of podocytes per glomeruli than the MAGI2 podKO group (Figure 1L). These data suggested that dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis.

Nuclear Translocation of Dendrin Promoted Podocyte Apoptosis

Previously, we reported that ADR and TGF-β enhance dendrin nuclear translocation.9,11 However, whether dendrin nuclear translocation was a consequence or cause of podocyte injury is unknown. To elucidate the effect of dendrin nuclear translocation, we generated dendrin-OE and dendrinΔNLS1-OE podocyte using piggyBac transposon system (Figure 2A). Podocytes carrying empty vectors are used as control. Dendrin expression in dendrin-OE and dendrinΔNLS1-OE podocytes was confirmed using western blot (Figure 2, B and C). The three types of OE podocytes expressed different patterns of dendrin. Dendrin-OE predominantly expressed dendrin in the nuclei, while dendrinΔNLS1-OE podocytes mainly expressed dendrin in the cytoplasm (Figure 2, D–F).

Figure 2.

Figure 2

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NLS1 sequence of dendrin induced nuclear localization of dendrin promoting podocytes apoptosis. (A) Construct of plasmid for generation of control, dendrin-OE, and dendrinΔNLS1-OE podocytes. (B and C) Western blot and its quantification measuring dendrin and GAPDH expression. (D) Representative confocal microscopy image of dendrin immunofluorescence stain in control, dendrin-OE, and dendrinΔNLS1-OE podocytes. (E) Histogram quantification of fluorescence intensity along the dotted line in B. (F) Quantification of dendrin overlaps with 4′,6-diamidino-2-phenylindole (DAPI) in dendrin-OE (n=30) and dendrinΔNLS1-OE (n=30) podocytes. (G and H) Representative confocal microscopy of vinculin, paxillin, and ACTN4 staining in differentiated control, dendrin-OE, and dendrinΔNLS1-OE podocytes. (I and J) Quantification of paxillin-positive area and paxillin-ACTN4 colocalization. (K) Representative flowcytometry analysis detecting 7-AAD and APC-annexin V in untreated and ADR-treated control, dendrin-OE, and dendrinΔNLS1-OE podocytes. (L and M) The percentage of viable and apoptotic cells in untreated and ADR-treated podocytes. Scale bar, 10 µm. Data are presented as means±SEM. and analyzed with the unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001. Figure 2 can be viewed in color online at www.jasn.org.

The subcellular localization of dendrin affects focal adhesion (FA). We checked the distribution of FA proteins paxillin and vinculin as well as actin-binding protein α-actinin-4 (ACTN4) (Figure 2, G and H). Thick paxillin and vinculin staining represented mature FA. Control podocytes showed mature FA in the cell body and the edge of the cells. However, dendrin-OE podocytes lack mature FA in the cell body. Quantitatively, the percentage of FA area per cell, marked by paxillin positive area of the cells, is decreased in dendrin-OE compared with control and dendrinΔNLS1-OE podocytes (Figure 2I). The colocalization of paxillin and ACTN4 in dendrin-OE was also less than control and dendrinΔNLS1-OE podocytes (Figure 2J). Together, nuclear-localized dendrin reduced the abundance and altered the distribution of FA.

The lack of FA and podocyte migration contributes to podocyte detachment, which could lead to detachment-induced apoptosis.26 To investigate the effect of dendrin localization on podocyte survival, we performed apoptosis assays in OE podocytes (Figure 2K). Nuclear dendrin enhanced ADR-induced apoptosis and reduced podocyte viability (Figure 2, I and J). Under no treatment, dendrin-OE podocytes showed higher apoptosis and lowered viable cell percentages compared with both control and dendrin ΔNLS1-OE podocytes (Figure 2, L and M, and Supplemental Figure 2, A–D). Intrinsic apoptotic signaling was enhanced in dendrin-OE podocytes, indicated by the increased cleaved caspase-3 expression after ADR treatment (Supplemental Figure 2F). These findings indicated that nuclear dendrin promoted apoptosis in podocytes with or without ADR treatment.

Two Clusters of Basic Amino Acids within NLS1 Are Required for Dendrin Nuclear Translocation

The NLS1 sequence of dendrin carries three clusters of basic amino acids. Thus, dendrin's NLS1 may act as classical or nonclassical NLS. To investigate the importance of basic amino acid residues in NLS1, we constructed mutations in lysine and arginine to alanine in three clusters within NLS1 and transfected the construct to COS-7 and HEK293 cells (Figure 3A). All alanine mutation in three clusters of basic amino acids of within the NLS1 sequence (NLS1 all-ala) caused the exclusion of dendrin from the nuclei of HEK293 cells (Figure 3B). In the mutation study, mutants 5 and 8 showed partial nuclear translocation of dendrin. Only mutant 7 showed complete nuclear localization of dendrin (Figure 3, B–D). These findings suggested that the first and third sections of basic amino acid clusters in the NLS1 sequence are essential for dendrin nuclear translocation.

Figure 3.

Figure 3

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Two clusters of basic amino acid in NLS1 are crucial for nuclear localization of dendrin. (A) Construct of plasmid showing amino acid sequence of WT and mutant NLS1. Blue is WT clusters of basic amino acid; red is mutant clusters of basic amino acids. (B) Representative confocal microscopy image of GFP-dendrin and DAPI immunofluorescence staining in HEK293 cells transfected with human dendrin carrying WT or mutant NLS1 sequence. (C) Fluorescence intensity quantification along the dashed line in B. Green represents dendrin; blue represents DAPI. (D) Quantification of dendrin signals overlap with DAPI (n=10 each group). Data are presented as means±SEM and analyzed with the unpaired t test. #P < 0.0001. Figure 3 can be viewed in color online at www.jasn.org.

Dendrin Interact with Importin-α through NLS1

Confirming that dendrin has a classical bipartite NLS sequence, we hypothesize that nuclear transport adaptor importin-α may mediate the nuclear translocation of dendrin. Pulldown assay using glomerular lysate and GST-dendrin as bait showed that human dendrin interacts with importin-α1, importin-α3, importin-α4, and importin-α7 (Figure 4A). Importin-α3 is specifically expressed in mouse glomeruli among the other investigated importin-α subfamilies (Figure 4B). Importin-α1, importin-α3, importin-α4, and importin-α7 are endogenously expressed in HEK293 cells, cultured podocytes, and glomeruli (Figure 4C). The analysis of mRNA expression from NEPHROSEQ database revealed that importin-α3 is highly expressed in the human glomeruli (Supplemental Figure 3, E and F).21 Furthermore, importin-α3 expression was increased in the glomeruli of individuals with subnephrotic FSGS and in the kidney of individuals with CKD (Supplemental Figure 3, G and H).22,23 Because the expression of importin-α3 is particularly high in the glomeruli and differentiated podocytes, we decided to focus on importin-α3.

Figure 4.

Figure 4

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Importin-⍺ interacts with dendrin and expressed specifically in the glomeruli. (A) GST pull-down assay using GST-N-terminus-dendrin in glomerular lysate showing importin-⍺1, importin-⍺3, importin-⍺4, and importin-⍺7 interact with human dendrin. (B) Western blot analysis of endogenous importin-⍺1, importin-⍺3, importin-⍺4, and importin-⍺7 expression in HEK293 cells, whole kidney, and isolated glomeruli of WT mice. (C) Importin-⍺1, importin-⍺3, importin-⍺4, and importin-⍺7 are endogenously expressed in HEK293 cells, undifferentiated and differentiated cultured podocytes. (D–G) Confocal microscopy analysis of endogenous importin-⍺1 (D), importin-⍺3 (E), importin-⍺4 (F), and importin-⍺7 (G) in HEK293 cells transfected with GFP-dendrin and GFP-dendrinΔNLS1. Scale bar, 10 µm. (H and I) Representative confocal microscopy analysis of dendrin and importin-⍺3 immunofluorescence stains in dendrin-OE and dendrinΔNLS1-OE podocytes. The histograms represent the fluorescence intensity along the dashed line. Figure 4 can be viewed in color online at www.jasn.org.

To know whether importin-α has a spatial correlation with dendrin, we transfect HEK293 cells with full-length GFP-dendrin and GFP-dendrinΔNLS1. Importin-α1, importin-α3, importin-α4, and importin-α7 are colocalized with GFP-dendrin in the nuclei, but not with GFP-dendrin ΔNLS1 (Figure 4, D–G). We also confirmed that importin-α3 colocalized with dendrin in dendrin-OE podocytes (Figure 4, H and I). Together, these data suggested that importin-α has a spatial correlation with dendrin.

Importin-α Inhibition Prevents Dendrin Nuclear Localization and Attenuates Podocytes Apoptosis

Ivermectin was reported to dissociate the heterodimer formation of importin-α/β in baby hamster kidney cell lines.27,28 Thus, we treated dendrin-OE podocytes with ivermectin and evaluated the localization of importin-α3 and dendrin. Treatment with 0.05, 0.1, and 0.25 µM ivermectin for 24 hours partially shifted the distribution of dendrin from nuclei to the cytoplasm of dendrin-OE podocytes. Ivermectin 1 and 10 µM treatment entirely shifted dendrin and importin-α3 from nuclei to the cytoplasm (Figure 5, A–C). These data suggested that the inhibition of importin-α/β prevents nuclear localization of dendrin.

Figure 5.

Figure 5

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Inhibition of importin-⍺ prevents nuclear localization of dendrin and reduce apoptosis in podocytes. (A) Representative confocal microscopy images of dendrin and importin-⍺3 immunofluorescence stains in dendrin-OE treated with DMSO or ivermectin. (B) Histogram quantification of fluorescence intensity along the dotted line in A. (C) Quantification of dendrin overlaps with DAPI. (D and E) Representative confocal microscopy of vinculin, paxillin, and ACTN4 staining in dendrin-OE podocytes treated with DMSO or ivermectin. (F and G) Quantification of paxillin-positive area in the cell. (H) Representative flowcytometry analysis detecting 7-AAD and APC-annexin V in dendrin-OE treated with DMSO or ivermectin. (I and J) The percentage of total apoptotic and viable dendrin-OE podocytes in DMSO (n=3) and ivermectin 0.1 µM (n=3), 0.25 µM (n=3), and 1 µM (n=3) groups. Scale bar, 25 µm. Data are presented as means±SEM. and analyzed with the unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001. Figure 5 can be viewed in color online at www.jasn.org.

Next, we evaluated the FA and apoptosis after ivermectin treatment. Inhibition of dendrin nuclear localization rescued the abundance of FA in podocytes and the distribution of FA to cell bodies (Figure 5, D–G). The percentage of FA area per cell is increased after the treatment of ivermectin. We performed an apoptosis assay in ivermectin-treated dendrin-OE podocytes (Figure 5H). Dendrin-OE podocytes treated with 0.25 and 1 µM ivermectin have lower apoptosis and higher cell viability than the control (Figure 5, I and J). Together, these findings suggested that the inhibition of importin-α attenuated podocyte apoptosis.

The early hallmark of apoptosis is apoptotic cell volume decrease (AVD) before cytochrome-c release, caspase-3 activation, and DNA laddering.29 We performed a cell size assay to evaluate the AVD in control, dendrin-OE, and dendrinΔNLS1-OE podocytes treated with ADR. The early apoptotic cell size is significantly reduced compared with the viable cells, regardless of the podocyte group (Supplemental Figure 4, A–C). However, the degree of cell shrinkage among the groups is different. Dendrin-OE podocytes have a significantly higher degree of cell shrinkage during early apoptosis than other groups (Supplemental Figure 4, D and E). These findings suggested that nuclear localization of dendrin promoted AVD, which may enhance apoptosis.

Next, we treated dendrin-OE podocytes with ivermectin. The average cell size of early apoptotic and viable cells in the ivermectin-treated dendrin-OE group is significantly larger than 0.025% DMSO-treated dendrin-OE podocytes (Supplemental Figure 5, A–C). However, during early apoptosis, DMSO-treated dendrin-OE has a significantly higher degree of AVD than ivermectin-treated podocytes (Supplemental Figure 5, D and E). Together, these data indicated that the inhibition of dendrin nuclear translocation attenuated the AVD.

Nuclear-Localized Dendrin Promotes c-Jun N-terminal kinase Phosphorylation and Cell Detachment

c-Jun N-terminal kinase (JNK) signaling regulates cellular attachment and motility.30,31 Therefore, we examined the phosphorylation of JNK in control, dendrin-OE, and dendrinΔNLS1-OE podocytes. The ADR treatment induced JNK phosphorylation in dendrin-OE podocytes (Figure 6, A and B). Under ADR stress, dendrin-OE podocytes showed higher JNK phosphorylation than control and dendrinΔNLS1-OE podocytes (Figure 6, C and D). The inhibition of importin-α by ivermectin decreased the JNK phosphorylation in ADR-treated dendrin-OE podocytes (Figure 6, E and F).

Figure 6.

Figure 6

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Nuclear-localized dendrin promotes JNK phosphorylation. (A and B) A western blot and its quantification measuring pJNK and JNK expression in dendrin-OE podocytes treated with ADR. (C and D) A western blot and its quantification comparing pJNK and JNK expression in ADR-treated control, dendrin-OE, and dendrinΔNLS1-OE podocytes. (E and F) A western blot and its quantification comparing pJNK and JNK expression in dendrin-OE podocytes treated with ADR+DMSO and ADR+ivermectin. (G) The area of wound scrapes in control, dendrin-OE, and dendrinΔNLS1-OE podocytes at 0 and 24 hours. (H) Quantification of wound scrapes the area of groups in G. (I) The area of wound scrapes in dendrin-OE podocytes treated with DMSO or ivermectin at 0 and 24 hours. (J) Quantification of wound scrapes the area of groups in I. (K and L) A representative DAPI staining image and a quantification of attached podocytes after 2 minutes of trypsinization. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001. Figure 6 can be viewed in color online at www.jasn.org.

We also examined the cell motility and detachment of podocytes. The migration analysis revealed the hypermotility of dendrin-OE compared with control and dendrin ΔNLS1-OE podocytes (Figure 6, G and H). The treatment using importin-α inhibitor reduced the motility of dendrin-OE podocytes (Figure 6, I and J). To examine the cellular detachment, we briefly exposed control, dendrin-OE, and dendrin ΔNLS1-OE podocytes to trypsin and measured the cell attachment. Dendrin-OE podocytes have lower attachment than control and dendrin ΔNLS1-OE podocytes (Figure 6, K and L). The treatment using importin-α inhibitor rescued the cell attachment in dendrin-OE podocytes. Together, the OE of full-length dendrin promotes JNK phosphorylation, cell motility, and cell detachment.

Inhibition of Importin-α Prevents Dendrin Nuclear Localization and Attenuates Nephropathy in ADR-Injected Mice and MAGI2 podKO Mice

To investigate the effect of the inhibition of dendrin nuclear translocation in vivo, we treated ADR mice and MAGI2 podKO mice with ivermectin. There is no difference in body weight changes between ADR+DMSO and ADR+ivermectin groups (Figure 7A). However, the urine ACR (uACR) in the ADR+ivermectin group is significantly lower than the ADR+DMSO group (Figure 7B). The level of plasma creatinine in both groups was not significantly different (Figure 7C). Ivermectin also significantly reduced glomerulosclerosis and preserved the number of podocytes per glomeruli in the ADR+ivermectin group compared with the ADR+DMSO group (Figure 7, D–F, Supplemental Figure 6, A and B).

Figure 7.

Figure 7

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Inhibition of importin-⍺ attenuates dendrin nuclear localization and nephrosis in ADR-treated mice and MAGI2 podKO mice. (A) The percentage of body weight change in ADR-injected (10 mg/kg body wt r.o.) mice treated with 1.5% DMSO in saline i.p. (n=7) or ivermectin 2.5 mg/kg body wt per day i.p. (n=7). (B and C) uACR and plasma creatinine of ADR+DMSO and ADR+ivermectin. (D) Representative PAS staining of the kidney, sacrificed on day 28 after ADR injection. (E) The average number of sclerotic glomeruli of ADR+DMSO (n=7) and ADR+ivermectin mice (n=7). (F) The average number of WT-1 positive cells per glomeruli in ADR+DMSO (n=5) and ADR+ivermectin mice (n=5), analyzed using confocal microscopy. (G) The percentage of body weight change in MAGI2 podKO+DMSO (n=5) and MAGI2 podKO+ivermectin mice (n=5) starting from 6 weeks of age. (H and I) uACR and plasma creatinine of MAGI2 podKO+DMSO and MAGI2 podKO+ivermectin mice. (J) Representative PAS staining of MAGI2 podKO+DMSO and MAGI2 podKO+ivermectin mice. (K) Quantification of sclerotic glomeruli per total glomeruli. (L) The average number of WT-1 positive cells per glomeruli in MAGI2 podKO+DMSO and MAGI2 podKO+ivermectin mice. (M and N) Representative image of dendrin/importin-⍺3 immunofluorescence staining and the quantification of nuclear dendrin in the kidney of 14 days ADR+DMSO and ADR+ivermectin mice. (O and P) Representative image of dendrin/importin-⍺3 immunofluorescence staining and the quantification of nuclear dendrin in the kidney of MAGI2 podKO+DMSO and MAGI2 podKO+ivermectin mice on age 8 weeks (day 14 of treatment). Scale bar, 40 µm. Data are presented as means±SEM and analyzed with the unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001. Figure 7 can be viewed in color online at www.jasn.org.

We treated MAGI2 podKO mice with DMSO (MAGI2 podKO+DMSO) or ivermectin (MAGI2 podKO+ivermectin) for 14 days starting from age 6 weeks. The MAGI2 podKO+DMSO and MAGI2 podKO+ivermectin mice exhibited body weight decrease without any significant difference between the groups (Figure 7G). The uACR of MAGI2 podKO+DMSO and MAGI2 podKO+ivermectin was higher than the control groups at age 6, 7, and 8 weeks (Figure 7H). After 2 weeks of ivermectin treatment, the uACR of MAGI2 podKO+ivermectin groups was significantly lower than the MAGI2 podKO+DMSO group. There is no difference in plasma creatinine between WT, MAGI2 podKO+DMSO, and MAGI2 podKO+ivermectin groups 14 days after treatment (Figure 7I). The MAGI2 podKO+ivermectin group showed less glomerulosclerosis and more WT-1–positive cells per glomeruli than the MAGI2 podKO+DMSO group (Figure 7, K and L, Supplemental Figure 6, C and D). These data suggested that ivermectin treatment reduced albuminuria, glomerulosclerosis, and podocyte loss in MAGI2 podKO mice.

Observing that ivermectin injection attenuated albuminuria in both ADR-induced nephropathy and MAGI2 podKO mice, we wanted to know whether ivermectin injection inhibits importin-α3 and dendrin nuclear translocation. Some of dendrin signal colocalized with importin-α3 and DAPI after 14 days of ADR injection (Figure 7M). In ADR+ivermectin groups, we observed that importin-α3 localized in the cytoplasm. Ivermectin reduced the number of nuclear dendrin per glomeruli in ADR-injected mice (Figure 7N). In the MAGI2 podKO+DMSO group, some dendrin colocalized with importin-α3 in the nuclei (Figure 7O). The number of nuclear dendrin per glomeruli in MAGI2 podKO+ivermectin is less than in the MAGI2 podKO+DMSO group (Figure 7P). These findings suggested that importin-α inhibition reduced nuclear localization of dendrin in the glomeruli.

Importin-α3 Colocalized with Nuclear Dendrin in Human Glomerular Diseases

To investigate the importance of importin-α3 in human glomerular diseases, we assess the colocalization of dendrin and importin-α3 in human kidney biopsy. In the glomeruli of patients with minimal change disease (MCD), importin-α3 and dendrin were expressed as a capillary pattern (Figure 8A). In FSGS and IgAN, dendrin translocated to the nucleus (Figure 8B) and colocalized with importin-α3 (Figure 8C). Although we did not find any statistically significant difference, we observed an increasing trend of dendrin nuclear translocation in the LN group. However, in both FSGS and IgAN, the intensity of importin-α3 staining was not different from MCD (Figure 8D). This result shows that individuals with glomerular diseases demonstrated nuclear translocation of both dendrin and importin-α3.

Figure 8.

Figure 8

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Importin-⍺3 expression is associated with dendrin in human glomerular diseases. (A) Representative glomerular immunofluorescence images of dendrin (red) and importin-⍺3 (green) in human glomerular diseases. Scale bar, 72 and 14 µm. (B–D) Quantification of nuclear-localized dendrin, colocalization of nuclear-translocated dendrin and importin-⍺3, and importin-⍺3 immunofluorescence intensity in human glomerular diseases. (E) Schematic diagram of interaction of bipartite NLS sequence of dendrin with importin-⍺ which leads to nuclear translocation of dendrin and podocytes loss. Data are presented as means±SEM and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; #P < 0.0001. Figure 8 can be viewed in color online at www.jasn.org.

Discussion

This study investigated the role of dendrin and its subcellular localization in podocyte injury. A previous study demonstrated that dendrin ablation prolongs the lifespan of CD2AP KO mice.32 This study found that dendrin deficiency also extends the lifespan of MAGI2 podKO mice. Furthermore, dendrin ablation attenuates proteinuria, glomerulosclerosis, and podocyte loss in ADR nephropathy and MAGI2 podKO mice. There is a possibility that dendrin ablation exercised its protective effect through cells other than podocytes since we use general dendrin KO mice. However, since dendrin is specifically expressed in the podocyte, we suggested that ablation of dendrin in podocyte is responsible for the protective effect.

Despite the finding that dendrin deficiency attenuated nephropathy, aiming to reduce dendrin expression may not be a straightforward approach to attenuate nephropathy. Human mRNA expression data showed that dendrin was positively correlated with eGFR in patients with CKD.22 On the other hand, nuclear translocation of dendrin was consistently observed in various kidney diseases, such as IgAN, LN, membranous nephropathy, and FSGS.9,10 Thus, the subcellular localization rather than the expression of dendrin is potentially affecting podocyte survival.

We hypothesized that mainly nuclear-localized dendrin promotes cell detachment–induced cell death or anoikis. This study provides at least two findings suggesting that the loss of cell attachment preceded the apoptosis in dendrin-OE podocytes. First, nuclear-localized dendrin promoted the FA alteration and cell detachment without ADR stimulation, when the apoptotic marker cleaved caspase-3 was still undetected. Second, dendrin promotes several characteristics, such as JNK activation and AVD, which are known to be upstream from caspases activation during the apoptosis. Phosphorylation of JNK was previously reported to regulate BH3-only proteins leading to anoikis.33,34 In addition, AVD also precedes caspase-3 activation and DNA laddering during apoptosis.29,35,36 Therefore, our data suggested that the loss of cell adhesion precede the cell death in dendrin-OE podocytes from the standpoint that cleaved caspase-3 marks the early event in apoptosis.

From a mechanism perspective, bipartite NLS and importin-α are essential for dendrin nuclear translocation. Previous studies reported that proteins with classical bipartite NLS require binding to armadillo repeats of importin-α, which subsequently tether them to importin-β.37–40 There is evidence that classical NLS could recognize several subfamilies of importin-α.38 In our studies, importin-α3 is relatively abundant in differentiated podocytes and glomeruli compared with other subfamilies. Thus, importin-α3 is possibly the importin-α subfamily playing an important role in dendrin nuclear translocation.

The finding of classical bipartite NLS in dendrin prompted us to use importin-α/β inhibitor to prevent dendrin nuclear translocation. Our study clarified that ivermectin treatment could inhibit the nuclear translocation of dendrin in vitro and in vivo. Previously, ivermectin is identified as a specific inhibitor of importin-α/β–mediated nuclear transport.27,28,41 The previous studies reported conflicting findings about the action of ivermectin in nuclear transport.27,28,42 Our study suggested the later mechanism for dendrin nuclear translocation.

Although this study showed the role of dendrin in a pathologic condition, the function of dendrin in physiologic podocytes is still unclear. In physiologic glomeruli, dendrin is localized in the cytoplasm of human and murine podocytes.9,10 In this study, cytoplasmic dendrin prevents cell detachment by stabilizing the FA, indicating its role in maintaining cellular attachment and preventing podocyte loss. Therefore, it would be interesting to study dendrin deficiency in models featuring podocyte loss and impaired attachment, such as ACTN4-KO mice or diabetic nephropathy.

Hypothetically, any condition that activates TGF-β signaling, such as diabetes, could induce the nuclear localization of dendrin. Previously, we reported that TGF-β stimulation promotes dendrin nuclear translocation.11 Since dendrin is expressed in the kidney and the brain synapse, it would be interesting to see whether nuclear-localized dendrin also has some role in the cerebral and neuronal complications of diabetes. A previous study reported that dendrin interacts with synaptic scaffold protein KIBRA through its PY motifs.43 In the kidney, KIBRA deficiency disrupts directional cell migration and promotes podocyte injury by inhibiting the function of YAP.12,44 In the brain, KIBRA modulates synaptic transmission, spatial learning, and memory.43 Therefore, investigating the implication of dendrin-KIBRA in the diabetic milieu would be of interest.

In summary, we showed that the nuclear translocation of dendrin enhances podocyte loss by promoting a cell detachment–induced apoptosis or anoikis (Figure 8E). The interaction of classical bipartite NLS1 with importin-⍺ mediates dendrin nuclear translocation. The inhibition of dendrin nuclear translocation could attenuate proteinuria, podocyte loss, and glomerulosclerosis in vivo. Thus, targeting the dendrin nuclear translocation might be a potential strategy to treat kidney diseases in which dendrin nuclear translocation is observed.

Supplementary Material

jasn-34-1222-s001.pdf (2.3MB, pdf)
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Acknowledgments

We thank Prof. Lawrence B. Holzman (University of Pennsylvania) for the Neph2-cre mice and Prof. Karl Tryggvason (Karolinska Institute) for the dendrin−/− mice. We thank Ms. Chihiro Makino and Ms. Mayu Miyaki for their excellent technical assistance. We thank Dr. Motoko Kimura, Dr. Kiyoshi Hirahara, and Prof. Toshinori Nakayama (Department of Immunology, Graduate School of Medicine, Chiba University) for their technical support with confocal microscopy. We thank the LGS program of Chiba University Graduate School of Medicine for the generous support to MAE to carry out this PhD project.

Disclosures

M. Aizawa reports Honoraria: AbbVie GK, AstraZeneca K.K., Bayer Yakuhin, Ltd., Kowa Company, Ltd., Kyowa Kirin Co., Ltd., Novartis Pharma K.K., Otsuka Pharmaceutical, and Terumo Corporation. Outside the contents of the study, K. Asanuma have received research funding from Mitsubishi Tanabe Pharmaceutical Corporation. K. Asanuma also reports Honoraria: AstraZeneca, Daiichi Sankyo, and Kyouwa Kirin. M. Kikyo reports Employer: Mistubishi Tanabe Pharma Corporation. S.-i. Makino reports Research Funding: Bayer Pharma. J. Patrakka reports Research Funding: AstraZeneca and Guard Therapeutics. N. Shirata reports Employer: Mitsubishi Tanabe Pharma Corporation. All remaining authors have nothing to disclose.

Funding

This work was supported in part by a Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Scientific Research, Grant/Award Nos. 17K19653 and 18H02823 (K.A.).

Author Contributions

Conceptualization: Katsuhiko Asanuma, Maulana A. Empitu, Mitsuhiro Kikyo, Naritoshi Shirata, Hiroyuki Yamada.

Data curation: Katsuhiko Asanuma, Maulana A. Empitu, Mitsuhiro Kikyo, Naritoshi Shirata.

Formal analysis: Maulana A. Empitu, Mitsuhiro Kikyo, Naritoshi Shirata.

Funding acquisition: Katsuhiko Asanuma.

Investigation: Maulana A. Empitu, Ika N. Kadariswantiningsih, Mitsuhiro Kikyo, Naritoshi Shirata.

Methodology: Katsuhiko Asanuma, Maulana A. Empitu, Ika N. Kadariswantiningsih, Mitsuhiro Kikyo, Shin-ichi Makino, Naritoshi Shirata, Hiroyuki Yamada.

Resources: Masashi Aizawa, Katsuhiko Asanuma, Ika N. Kadariswantiningsih, Shin-ichi Makino, Katsuhiko Nishimori, Jaakko Patrakka, Hiroyuki Yamada.

Supervision: Katsuhiko Asanuma.

Validation: Katsuhiko Asanuma, Maulana A. Empitu.

Visualization: Maulana A. Empitu.

Writing – original draft: Maulana A. Empitu.

Writing – review & editing: Katsuhiko Asanuma, Maulana A. Empitu, Ika N. Kadariswantiningsih.

Data Sharing Statement

All data used in this study is available in this article.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/JSN/E429.

Supplemental Figure 1. Dendrin deficiency reduced podocyte loss in ADR-treated and MAGI2 podKO mice.

Supplemental Figure 2. Apoptosis quantification in control, dendrin-OE, and dendrinΔNLS1-OE podocytes.

Supplemental Figure 3. Dendrin, MAGI-2, and importin-α proteins mRNA expressions in human tissue.

Supplemental Figure 4. Cell size assay in control, dendrin-OE, and dendrinΔNLS1-OE podocytes treated with ADR.

Supplemental Figure 5. Cell size assay in dendrin-OE podocytes treated with ivermectin.

Supplemental Figure 6. Importin-α inhibitor reduced pococyte loss in ADR-treated mice and MAGI2 podKO mice.

Supplemental Table 1. List of antibodies used in this study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data used in this study is available in this article.

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