(A) Levels of Tgfb1, Ctsl, and control gene in glomeruli of TGF-β1 Tg and WT animals. Phenotype categorization is as described previously (23). (B) H&E-stained sections of the renal cortex, showing increased CatL staining in TGF-β1 Tg mice. Kidneys of WT and TGF-β1 Tg mice were stained for CatL using anti–cytosolic CatL antibody, and nuclei were stained using DAPI. (C) CatL staining levels in sections from B. ***P < 0.001. (D) H&E-stained sections showing dendrin localization in the nucleus of the podocytes in TGF-β1 Tg animals (arrows). Kidneys of WT and TGF-β1 mice were stained for dendrin using anti-dendrin antibody. (E) Dendrin staining levels in sections from D. ***P < 0.001. (F and G) H&E-stained sections showing increased CatL staining in glomerulus of 3-week-old Cd2ap^–/– mice (arrows). Kidneys of WT and Cd2ap^–/– mice were stained for CatL using anti–cytosolic CatL antibody, and nuclei were stained using DAPI. (H) Dendrin localization in glomerulus of WT and Cd2ap^–/– mice at different stages of development. WT1 was used to stain podocyte nuclei (red). In the young glomerulus, dendrin (green) was localized at the membrane (ribbon staining pattern), without any colocalization with WT1. At 4 weeks, dendrin staining was still localized to the plasma membrane in WT mice, but was predominantly found in the nucleus costaining with WT1 in Cd2ap^–/– mice. Scale bars: 20 μm (B, D, F, and G); 30 μm (H).

(A) Ctsl mRNA levels, determined by RT-PCR, in high-Tgfb1 Cd2ap^–/– podocytes infected with different shRNA constructs (C2, C5, C6) downregulating endogenous CatL. Con, high-Tgfb1 Cd2ap^–/– podocytes not infected with lentiviruses; Scr, cells infected with lentiviruses expressing a scrambled oligo. (B) CatL levels in high-Tgfb1 Cd2ap^–/– podocytes infected with lentiviruses expressing different shRNA constructs to downregulate CatL at relative volumes as indicated. (C) Time course of CatL activity in high-Tgfb1 Cd2ap^–/– podocytes and in high-Tgfb1 Cd2ap^–/– podocytes infected with lentiviruses to downregulate CatL in the absence and presence of CA074 at neutral pH. (D) Time course of CatL activity in the presence of CA074 in high-Tgfb1 Cd2ap^–/– podocytes, high-Tgfb1 Cd2ap^–/– podocytes infected with lentiviruses to downregulate CatL, and WT podocytes at neutral pH. (E and F) Protein levels in WT cells, high-Tgfb1 Cd2ap^–/– podocytes, and high-Tgfb1 Cd2ap^–/– podocytes treated with E64 or infected with lentiviruses to downregulate CatL (shRNA-C6). Dyn, dynamin; Synpo, synaptopodin. GAPDH was used as a loading control. (G) Quantitation of protein levels from Western blots in E and F.

(A) Ddn mRNA levels, determined by RT-PCR, in high-Tgfb1 Cd2ap^–/– podocytes infected with different shRNA constructs (D2–D4) downregulating endogenous dendrin. Con, uninfected high-Tgfb1 Cd2ap^–/– podocytes; Scr, high-Tgfb1 Cd2ap^–/– podocytes infected with lentiviruses expressing a scrambled oligo. (B) Dendrin levels in high-Tgfb1 Cd2ap^–/– podocytes infected with lentiviruses expressing the 2 most efficient shRNA constructs, D3 and D4. (C) Organization of the actin cytoskeleton and FAs in high-Tgfb1 Cd2ap^–/– podocytes with dendrin downregulation. FAs and F-actin were visualized with anti-paxillin and rhodamine-phalloidin, respectively. (D) Number of FAs within WT and high-Tgfb1 Cd2ap^–/– podocytes with dendrin downregulation. Data (mean ± standard deviation) represent measurements of >50 cells in C. ***P < 0.001. (E) Ctsl mRNA levels, determined by RT-PCR, in high-Tgfb1 Cd2ap^–/– podocytes with dendrin downregulation. (F) Protein levels of CatL, dynamin, synaptopodin, RhoA, and GAPDH in high-Tgfb1 Cd2ap^–/– podocytes and cells infected with lentiviruses. (G) Time course of CatL activity in high-Tgfb1 Cd2ap^–/– podocytes, high-Tgfb1 Cd2ap^–/– podocytes infected with lentiviruses, and low-Tgfb1 Cd2ap^–/– podocytes at neutral pH. (H) Loss of CD2AP rendered podocytes hypersensitive to proapoptotic signals, as shown by specific enrichment of mono- and oligonucleosomes released into the cytoplasm of WT and high-Tgfb1 Cd2ap^–/– podocytes treated with different apoptosis inducers. Stauro, staurosporine; Actino D, actinomycin D; Angio II, angiotensin II. (I) TGF-β1–induced apoptosis was reversed by downregulation of CatL or dendrin in high-Tgfb1 Cd2ap^–/– podocytes. Scale bars: 20 μm.

(A) Immunoblot of cleaved CD2AP fragments tagged with N-terminal GFP. At pH 7.0, CD2AP cleaved into a stable 55-kDa fragment (squares), as detected with anti-GFP antibody. We detect the same fragment with the N-CD2AP antiserum. This antiserum also detected a weak band corresponding to a 44-kDa fragment (triangle). (B) Match of cleavage fragments with predicted CatL cleavage site QPLGS. (C) Deletion of the CatL cleavage site LSAAE protected CD2AP from limited proteolysis into p32 (circle). (D) Match of p32 with predicted CatL cleavage site LSAAE. (E) CatL cleaved CD2AP-FLAG, yielding p32 (circle), detected by anti–C-CD2AP. (F) WT Ctsl cleaved CD2AP in HEK 293 cells. Cytosolic CatL (CatL M1) was sufficient to cleave CD2AP, yielding p32 (circle). These cleavage reactions were prevented by incubation of the cells with E64. (G) Coimmunoprecipitation of nephrin, synaptopodin, and dendrin from HEK 293 cells transfected with full-length CD2AP, N-terminal CD2AP, and p32. (H) Structural domains of CD2AP, together with major CatL cleavage sites, predicted sizes of resulting fragments from CatL digestion, and recognition sites of the antibodies used. SH3, src homology domain 3; PR, proline-rich motif; CC, coiled-coil domain. (I) Immunofluorescent staining of kidney biopsies from patients with MCD and FSGS. N-terminal CD2AP was reduced only in progressive disease (i.e., FSGS). Scale bar: 30 μm.

(A) Actin cytoskeleton and FA organization in fully differentiated low- and high-Tgfb1 Cd2ap^–/– cells. Note the loss of well-defined stress fibers and dramatic increase in number of transverse arcs in high-Tgfb1 Cd2ap^–/– cells. FAs and F-actin were visualized with anti-paxillin antibodies and rhodamine-phalloidin, respectively. (B) Low-Tgfb1 Cd2ap^–/– podocytes could be transformed into high-Tgfb1 Cd2ap^–/– podocytes by high TGF-β1 levels. WT and low-Tgfb1 Cd2ap^–/– passage cells were treated with 5 ng/ml TGF-β1 in the media for 24 hours. Actin cytoskeleton was monitored by staining cells with rhodamine-phalloidin, and dendrin localization was monitored using anti-dendrin antibody (green). (C–E) mRNA levels for Tgfb1 (C) and Ctsl (D and E), determined by RT-PCR in podocytes. When indicated, cells were treated with 5 ng/ml TGF-β1 for 24 hours or with 50 μM LPS for 24 hours. Podocytes expressing Actn4 mutant are also shown in E. (F) Subcellular fractionation of low- and high-Tgfb1 Cd2ap^–/– podocytes in isotonic sucrose. Total proteins from soluble (S) and particulate (P) fractions were probed for CatL and for the lysosomal protein markers Lamp-2 and mannosidase (Manno), then analyzed by Western blotting. GAPDH was used as a loading control. Strong cytosolic CatL induction (asterisk) was observed in both soluble and pellet fractions of high-Tgfb1 Cd2ap^–/– podocytes. Scale bars: 20 μm.

(A) Organization of the actin cytoskeleton and FAs in podocytes in which CatL was downregulated. FAs and F-actin were visualized with anti-paxillin and rhodamine-phalloidin, respectively. (B) Quantitation representing measurements of >50 cells shown in A. Data are mean ± standard deviation. ***P < 0.001. Scale bars: 20 μm.

(A and B) Nuclear dendrin induced transcription from the CatL promoter, but not from the CatB promoter. HEK 293 cells were cotransfected with pSEAP reporter vector containing the promoter of interest, dendrin, and Metridia luciferase to normalize for transfection efficiency. Mutant dendrin lacking its nuclear localization signal (dNLS) was ineffective. Promoter activity and response to dendrin depended on a region between bp –1,215 and bp –339, as revealed by partial deletion promoter constructs. ***P < 0.001. (C) Strategy used to identify the dendrin binding site within the CatL promoter. The promoter fragment between bp –1,215 and –339 was divided into 4 parts. EMSA with these unlabeled DNA fragments, visualized by SYBR green, revealed dendrin binding to fragment F (data not shown), which was then further divided into overlapping fragments to fine-map the dendrin binding site. (D) EMSA demonstrating specific dendrin binding to one of the biotin-labeled 60-bp oligonucleotides that were completely abolished by a 200-fold excess of unlabeled oligonucleotide. (E) The 60-bp oligonucleotide that exhibited dendrin binding was divided into 3 overlapping 24-bp oligonucleotides to further narrow the dendrin binding site. Mutation of 3 nucleotides in the 5′ region, but not in the central or 3′ region, of the 24-bp oligonucleotide 4-1 completely abolished dendrin binding, as demonstrated by the inability of a 200-fold excess of unlabeled mutated oligonucleotide to compete with the labeled WT oligonucleotide.