Arp2/3-dependent regulation of ciliogenesis governs adaptive distal tubular epithelial cell states in kidney disease

Abstract

Proteinuric kidney disease substantially affects renal tubules through incompletely understood mechanisms. We identify elongation of primary cilia in distal renal tubules in the context of glomerular nephropathy. In renal biopsies and mouse models, tubular injury correlates with ciliary elongation, tubule dilation, and disruption of the cortical actin cytoskeleton. In vitro studies implicate biophysical cues of the glomerular filtrate and subsequent dysregulation of the actin cytoskeleton as contributing factors, confirmed by conditional deletion of N-WASP and Arp2/3 in vivo and in vitro. Electron and fluorescence microscopy revealed enlarged ciliary pockets, basal body mislocalization, and intracellular cilia formation in Arp3 knockout conditions. Transcriptome analysis identifies the essential role of cilia in maintaining adaptive tubular cell states, while persistent activation leads to disease progression through extracellular matrix remodeling, exemplified by Tenascin-C. Our findings establish cilia as central mediators of tubular adaptation to injury and identify the Arp2/3-dependent actin cytoskeleton as a critical regulator, providing essential insights into the pathogenesis of chronic kidney disease.

Fig. 1.. Proteinuric glomerular disease is associated with cilia elongation in distal renal tubules.

(A and B) Immunofluorescence (IF) analysis of distal tubules in human kidney biopsies with diagnoses of FSGS, IgAN, or healthy controls (Ctrl.). Cilia were stained with acetyl–α-tubulin, the thick ascending limb (TAL) of the nephron with NKCC2, all tubule compartments with wheat germ agglutinin (WGA) lectin and nuclei with Hoechst. Maximum intensity projections of z-stacks were generated to show the entire cilium. White arrows indicate normal, yellow arrows indicate elongated, and white arrowheads indicate shortened cilia. (B) Mean ciliary length per patient (FSGS, N = 7; IgAN, N = 6; control N = 7, dots indicate individual patients analyzed; error bars indicate means and S.E.M.; *P < 0.05) and single cilia measurements (violin plot; Ctrl., N = 1367; FSGS, N = 2052; IgAN, N = 1540). (C) IF analysis of murine models of glomerular disease. White arrows indicate normal, and yellow arrows indicate elongated cilia. (D) The mean cilia length in distal tubules (TAL and collecting duct tubules) was quantified (dots indicate individual animals analyzed—Nphs2 WT, Nphs2 mutant, Parva WT, Parva KO, Pdss2 mutant, NOH, and NOH control N = 5 each; Pdss2 WT N = 4; Col4A3 WT and KO N = 6 each; error bars indicate means and S.E.M.; n.s., not significant; **P < 0.01, ***P < 0.001, and ****P < 0.0001). (E) Correlation analysis between lumen diameter and mean ciliary length per tubule in disease models with elongated cilia. (F and G) Quantification of dilated tubules and proteinaceous casts in distal (medullary) tubules per area in the indicated disease models (dots indicate individual animals analyzed). (H) Correlation analysis between proteinaceous cast formation and dilation of distal tubules. Red dots indicate animals with elongated cilia, and blue dots with normal cilia length. (I) Representative IF of a distal tubule showing elongated cilia (white arrow) and intraluminal proteinaceous cast formation [white asterisk; visualized via autofluorescence (AF)].

Fig. 2.. Combined biological and physical properties of the proteinuric glomerular filtrate affect ciliary elongation via the actin-cytoskeleton.

(A and B) Analysis of cilia length in ciliated mIMCD3 cells exposed to albumin (N = 3), human serum (N = 4), basement membrane extracellular matrix (ECM) [100 μg/ml (low) or 1 mg/ml (high); N = 3 each], increased medium viscosity of 150 or 1500 cP (methylcellulose; N = 4 each), 1500 cP and high ECM (N = 3), laminar flow of 0.2 or 2 dyn/cm² (N = 3 each) or negative control medium (N = 11). Scatter plots show mean cilia length per replicate (statistical comparisons to Ctrl. samples is shown; dots indicate independent experiments per condition; error bars indicate means and S.E.M.; n.s., not significant; ***P < 0.001, ****P < 0.0001). Violin plots show individual cilia measurements per condition (statistical analysis of selected comparisons is shown; n.s., not significant; ****P < 0.0001). (B) Representative images of indicated treatment conditions. Cells were stained for acetyl–α-tubulin, filamentous actin (F-actin) with phalloidin, and nuclei with Hoechst. White arrows indicate short and red arrows elongated cilia. (C) N-WASP, WAVE2, and CTTN regulate Arp2/3 complex–dependent polymerization of branched actin filaments at the cell cortex, suppressing cilia elongation. (D) Representative images of mIMCD3 cells cultured in control or 1500-cP high-viscosity medium for 24 hours. Cells were stained for CTTN and F-actin to visualize the cortical (branched) actin cytoskeleton (white asterisks indicate the leading edge, and white arrows indicate filopodia). (E to G) CTTN, N-WASP, WAVE2, and ARP3 stainings in the collecting duct (CD) of proteinuric Parva KO animals demonstrate a reduction in apical localization (arrowheads indicate the apical cell cortex). CDs were stained with Dolichos biflorus agglutinin (DBA) lectin, cilia with acetyl–α-tubulin, and nuclei with Hoechst (maximum intensity projections are shown).

Fig. 3.. Specific deletion of N-WASP (Wasl) in distal tubules results in elongation of primary cilia.

(A) A conditional Wasl (N-WASP) KO for the distal tubule compartment was generated by combining the Wasl^fl/fl allele with the Cdh16-Cre transgene. (B) IF confirms the loss of N-WASP expression in distal renal tubules of Wasl^fl/fl*Cdh16-Cre mice. Cre recombinase–positive tubular cells were labeled by enhanced green fluorescent protein (EGFP), applying the mT/mG reporter allele [tubules were stained with WGA and nuclei (blue) with Hoechst; orange arrow indicates interstitial cells without Cre expression]. (C) Quantification of blood urea nitrogen (BUN) in Wasl WT and Wasl^fl/fl*Cdh16-Cre (KO) animals at 5 months of age (N = 5 animals per genotype; dots indicate individual animals analyzed; error bars indicate means and S.E.M.; n.s., not significant). (D to G) Representative images of PAS or IF-stained kidneys from Wasl WT and Wasl KO animals at 5 months of age. Black boxes indicate selected regions for magnified images. KO animals show occasional dilatation of CD tubules (red asterisks) and reduced cell density (red arrowheads). The staining of CD tubules by DBA lectin shows increased proportion of DBA-negative cells (orange arrows) in CDs of KO animals (AF, autofluorescence). Quantifications show no formation of tubular cysts (cystic index), interstitial fibrosis, and tubular atrophy (IF-TA score) but a reduced fraction of DBA-positive CD cells in KO kidneys (N = 5 animals per genotype; n.s., not significant; ***P < 0.001). (H) IF staining of WT and Wasl^fl/fl*Cdh16-Cre KO mice for cilia (acetyl–α-tubulin) and distal tubule compartments using NKCC2 (TAL) and DBA lectin (CD). Maximum intensity projections are shown. Orange arrows indicate representative cilia. (I and J) Quantification of mean cilia length in indicated tubule compartments (N = 5 animals per genotype at 5 months of age; dots indicate individual animals analyzed; error bars indicate means and S.E.M.; **P < 0.01, ***P < 0.001).

Fig. 4.. Specific deletion of ARP3 (Actr3) in distal tubules results in dilation of tubules accompanied by progressive kidney disease.

(A) Conditional Actr3 (ARP3) KO mice were generated by combining the Actr3^fl/fl allele with the Cdh16-Cre transgene. (B) IF staining confirms the loss of ARP3 expression in distal renal tubules of Actr3^fl/fl*Cdh16-Cre mice. Cre-positive tubular cells were labeled by EGFP, applying the mT/mG reporter allele [tubules were stained with WGA and nuclei (blue) with Hoechst]. The white arrow indicates the presence of ARP3-positive interstitial cells in Actr3^fl/fl*Cdh16-Cre mice. (C) Kidneys of Actr3 KO mice exhibited a pale and smaller appearance compared to WT. (D) Quantification of BUN in Actr3 WT and KO animals at 2 and 3 weeks (w) of age (N = 5 animals per time point and genotype; dots indicate individual animals analyzed; error bars indicate means and S.E.M.; **P < 0.01). (E) Representative images of PAS or multiplex IF (mIF)–stained kidneys from Actr3 WT and KO animals. Black boxes indicate selected regions for magnified images. KO animals show progressive dilatation of medullary tubules (red asterisks) and interstitial fibrosis (red arrowheads). SMA and COL1A1 indicate a marked expansion of mesenchymal cells and deposition of ECM in the interstitium of KO animals. 7-plex mIF analysis shows infiltrating immune cells in Actr3 KO kidneys (F4/80⁺ macrophages (white arrows); CD3⁺ (arrowheads), CD4⁺, CD8⁺ and FOXP3⁺, T cell populations; LAMC1, ECM and basement membranes). (F to K) Quantifications of kidney staining [N = 5 animals per time point and genotype [(F) to (I)] or N = 4 for mIF [(J) and (K)]; dots indicate individual animals analyzed; error bars indicate means and S.E.M.; n.s., not significant; *P < 0.05, **P < 0.01]. Graphs show cystic index (F), IF-TA score (G), and inflammation (immune cell infiltrates) (I), SMA within the medulla (H), F4/80⁺ macrophages per area (J), and CD3⁺ T cells per area (K).

Fig. 5.. Loss of ARP3 results in elongation of primary cilia in kidney distal tubule cells in vitro and in vivo.

(A) IF staining of WT and Actr3^fl/fl*Cdh16-Cre KO mice for cilia (acetyl–α-tubulin) in distal tubule compartments using NKCC2 (TAL), NCC (DCT), DBA lectin (CD), and Hoechst (nuclei). Maximum intensity projections are shown. Orange arrows indicate representative cilia. (B to D) Quantification of mean cilia length (N = 4 animals per genotype at 2 weeks of age; dots indicate individual animals; error bars indicate means and S.E.M.; *P < 0.05, ***P < 0.001). (E) Violin plot of single cilia measurements (Actr3 WT N = 1722, Actr3 KO N = 1525, Wasl WT N = 1243, Wasl KO N = 1445; ****P < 0.0001). (F) Validation of mIMCD3 WT control (Ctrl.-1 and -2) and Actr3 KO (KO-1 and -2) cell lines. Western blot for ARP3 (ACTR3) and ARP2 (ACTR2) confirmed loss of ARP3 and indicated the degradation of the Arp2/3 complex. (G and H) Staining for CTTN and F-actin (phalloidin) demonstrates the loss of CTTN-positive leading edges and lamellipodia in Actr3 KO cells. White boxes indicate regions for magnified images. White asterisks indicate CTTN at the cell cortex in WT, and white arrows loss in KO cells. The percentage of CTTN-positive cell cortex circumference was quantified (N = 25 cells per genotype; dots indicate individual cells analyzed; error bars indicate means and S.E.M.; ****P < 0.0001). (I) Representative cells stained for cilia (acetyl–α-tubulin), basal bodies (γ-tubulin), and nuclei (Hoechst) demonstrate the elongation of cilia in Actr3 KO cells. White arrows indicate basal bodies. (J and K) Quantification of ciliated cells and cilia length in control and Actr3 KO cells (N = 3 independent experiments; dots indicate results per genotype and experiment; error bars indicate means and S.E.M.; **P < 0.01, ***P < 0.001). (L) Violin plot of single cilia measurements per genotype (Ctrl.-1&2 N = 374 and KO-1&2 N = 559 cilia).

Fig. 6.. Loss of Ift88 and inhibition of dynein function reduces cilia formation and length in Actr3 KO cells.

(A) Western blot quantification of the soluble and insoluble α-tubulin fractions in Actr3 cells [N = 2 independent experiments; Actr3 Ctrl.-1&2 (total N = 4) and KO-1&2 (total N = 4) cell lines were pooled for statistical analysis, dots indicate measurements per experiment and genotype; error bars indicate means and S.E.M.; n.s., not significant]. (B to E) Quantification of mean cilia length in 100 nM or 1 μM cytochalasin D, 10 μM Y-27632, FCS, or FCS readdition for 90 min treated Ctrl.-1 and Actr3 KO-1 cells (N = 4 or 3 independent experiments; dots indicate mean per genotype and experiment; error bars indicate means and S.E.M.; n.s., not significant, *P < 0.05, **P < 0.01). (F to H) Quantification of IFT81-positive ciliary bulges and percentage of IFT81-positive ciliary area in CD tubules of WT and Actr3 KO animals. Representative images of IFT81, acetyl–α-tubulin, DBA lectin, and Hoechst (nucleus) staining (maximum intensity projections are shown). Orange arrows indicate IFT81-positive regions of the ciliary shaft (N = 4 animals per genotype at 2 weeks of age; dots indicate individual animals analyzed; error bars indicate means and S.E.M.; n.s., not significant, **P < 0.01). (I to K) Analysis of cilia in Actr3 KO-1 Ift88 KO (Actr3 Ift88 double KO – “dKO”), Actr3 KO-1 IFT88 WT (KO-1), and 0.5 μM ciliobrevin D (KO-1 CB)–mIMCD3 cells. Representative images show acetyl–α-tubulin, F-actin (Phalloidin), and Hoechst (nucleus, Nucl.) in the indicated cell lines. White boxes indicate regions for magnified images. Graphs show quantification of cilia length and ciliated cells in indicated cell lines (N = 3 independent experiments; dots indicate mean per genotype and experiment; error bars indicate means and S.E.M.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 7.. IC and EC localization of the primary cilium is regulated by ARP3 and the actin cytoskeleton.

(A) SEM of Ctrl.-1 and Actr3 KO-1 mIMCD3 cells demonstrate ciliary shaft elongation (arrowheads) and the emergence of ciliary pockets (arrow) in KO cells. (B) CLEM reveals the presence of partially and completely IC localized cilia in Actr3 KO cells [cilia (acetyl–α-tubulin), basal bodies (γ-tubulin), and nuclei (Hoechst)]. (C) Analysis of IC (magenta) and EC (green) ciliary localization using the sspH-Smo-GFP reporter system. Representative images illustrate the partial and complete IC localization of cilia in Ctrl., Actr3 KO, dKO, and 1 μM cytochalasin D (Cyto. D)–treated cells. (D to F) Quantification of cilia localization, fraction of EC to total cilia shaft length, and EC shaft length in Actr3 or WT cells treated with DMSO (WT ctrl.) or 1 μM cytochalasin D. Stacked bar graphs indicate percentages of completely EC, PIC, or completely IC localized cilia (N = 3 independent experiments; dots indicate mean values per genotype and experiment; Actr3 Ctrl.-1&-2 were pooled (total N = 4); error bars indicate means and S.E.M.; **P < 0.01, ****P < 0.0001). (G) Analysis of interdependencies between cilia length and IC localization of the axoneme in Actr3 KO-1&-2 cells. Scatter dot plot demonstrates the relationship between total cilia length and RICAL of cilia with PIC localization. Graphs show linear interpolation analysis of cilia measurements and simulated datasets of models with positive (A, green), negative (B, orange), or no correlation (C, blue) between these parameters (N = 125 KO-1&-2 cilia analyzed; error bars indicate 95% confidence intervals). (H) Volcano plot of cilia with total IC or (partial) EC localization (non-IC) in Actr3 KO-1&-2 cells (N = 113 IC and 499 non-IC cilia; n.s., not significant).

Fig. 8.. ARP3-dependent regulation of cilia reprograms the transcriptome of kidney tubule cells.

(A) Transcriptome analysis of kidney medullas from Actr3 KO and WT mice at 2 weeks of age, nonstarved Ctrl.-1/-2 and Actr3 KO-1/-2 and starved Ctrl.-1, KO-1, Actr3 KO-1 Ift88 KO (dKO) and ciliobrevin D–treated KO-1 (CB) cells. Datasets of Pkd1, Pkd2, Ift140, and Dync2h1 KO mIMCD3 cells were reanalyzed (experiments were color-coded). (B) Differential gene expression analysis (DGEA) of nonstarved (green) and starved (blue) Actr3 KO versus Ctrl. cell lines [red dots, adjusted P value <0.05 with absolute log₂ fold change (FC) >1]. (C-1) Intersection analysis with reference datasets (Pkd1, Pkd2, Ift140, and Dync2h1). Bar graphs show significantly regulated genes in KO versus Ctrl. datasets (adjusted P value <0.05) and in the indicated number of reference datasets. (C-2) Box plots indicate absolute log₂ FCs of genes for the comparison of Actr3 KO versus Ctrl. cells or mean absolute log₂ FCs across all datasets. (D) DGEA of starved dKO or CB-treated KO-1 versus control KO-1 cells. (E) Normalized log₂ FC of indicated experiments to respective controls. (F) Venn diagram of significantly and matching regulated genes (adjusted P value <0.05 per dataset, inverse regulation by dKO and CB). (G) GO-term analysis of genes with matching regulation in all datasets. (H) Heatmap of significant regulated cilia genes. (I) DGEA of Actr3 KO and WT mice. (J) Intersection analysis of genes (adjusted P value <0.05) in nonstarved (green) or starved KO cells (blue), and Actr3 KO mice (brown). Graphs show matching and significantly regulated genes per intersection. TFs and co-TFs filtered by FC and inverse regulation in dKO and CB cells. (K) Heatmap of identified TFs (n.s., not significant). (L and M) IF analysis of LHX1 and CITED2 in DBA lectin or NKCC2 positive tubules (arrowheads indicate increased LHX1 and arrows loss of CITED2).

Fig. 9.. Cilia elongation in distal renal tubules is associated with an adaptive epithelial cell state.

(A) GSEA for adaptive, cycling, or degenerative tubule cell states. Bubble plots indicate normalized enrichment scores (NES) (Actr3 KO versus WT mice (brown), nonstarved (green) and starved (blue) Actr3 KO versus Ctrl. cells, dKO (Actr3 KO-1 Ift88 KO), and ciliobrevin D (CB)–treated KO-1 versus KO-1). (B and C) Analysis of the adaptive marker ITGB6 (cilia stained by acetyl–α-tubulin; tubules with elongated (arrows) and nonelongated cilia (arrowheads), casts (asterisks); N = 3 WT and N = 5 Nphs2^R231Q/A286V animals; dots indicate mean per animal; dashed lines indicate the same animal; ***P < 0.001). (D) Silver staining of distal tubule basement membranes (TBM) in CKD caused by FSGS and in Actr3 KO mice (arrows indicate thickened TBMs). (E) GSEA of matrisome gene sets (NES for the indicated conditions; n.s., not significant). (F) Intersection analysis of matrisome genes with matching regulation in indicated datasets (adjusted P value <0.05; coregulation in Actr3 KO and inverse regulation by dKO and CB). (G to J) Analysis of ECM dynamics in Actr3 cells cultured on Matrigel substrates (mean fluorescence intensity, MFI; N = 30 cells per condition; n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (I) Tnc expression in indicated datasets. (J) Representative Tenascin-C (TNC) and phalloidin (F-Actin)–stained cells. (K and L) Analysis of DBA-positive tubules show TNC negative, low, or high expressing cells (arrows indicate cilia and asterisks interstitial cells; N = 3 WT and N = 5 Nphs2^R231Q/A286V animals; dots indicate mean per animal; dashed lines indicate the same animal; *P < 0.05, ***P < 0.001, ****P < 0.0001). (M to Q) Analysis of cell size and adherent cells cultured on basement membrane (BM) Matrigel or TNC-coated surfaces under no-flow and laminar-flow conditions (N = 3 independent experiments; dots indicate mean per experiment; error bars indicate means and S.E.M.; n.s., not significant, **P < 0.01).

Fig. 10.. Graphic summary of the proposed role of cilia elongation in distal renal tubules.

(A) Proteinuric glomerular diseases cause cilia elongation and thickening of the TBM in the distal tubules of the nephron. (B) Proteinuric glomerular filtrate influences the N-WASP/WAVE – Arp2/3 complex–dependent cortical branched actin cytoskeleton, inducing cilia elongation and a shift in the mode of ciliogenesis. Subsequent ciliary signaling alters transcriptional programs to induce an adaptive epithelial cell state in response to tubular injury. This includes altered expression of TFs such as LHX1, matrisome genes such as TNC and markers of an adaptive cell state such as ITGB6. However, prolonged activation of these programs induces features of a degenerative cell state, including pathological remodeling of the TBM and activation of dedifferentiating signaling cascades, e.g. via TNC-ITGB6.