Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy

Abstract

Podocyte injury leading to albuminuria is a characteristic feature of diabetic nephropathy (DN). Hyperglycemia and advanced glycation end products (AGEs) are major determinants of DN. However, the underlying mechanisms of podocyte injury remain poorly understood. The cytosolic protein TNFAIP2/M-Sec is required for tunneling nanotubes (TNTs) formation, which are membrane channels that transiently connect cells, allowing organelle transfer. Podocytes express TNFAIP2 and form TNTs, but the potential relevance of the TNFAIP2-TNT system in DN is unknown. We studied TNFAIP2 expression in both human and experimental DN and the renal effect of tnfaip2 deletion in streptozotocin-induced DN. Moreover, we explored the role of the TNFAIP2-TNT system in podocytes exposed to diabetes-related insults. TNFAIP2 was overexpressed by podocytes in both human and experimental DN and exposre of podocytes to high glucose and AGEs induced the TNFAIP2-TNT system. In diabetic mice, tnfaip2 deletion exacerbated albuminuria, renal function loss, podocyte injury, and mesangial expansion. Moreover, blockade of the autophagic flux due to lysosomal dysfunction was observed in diabetes-injured podocytes both in vitro and in vivo and exacerbated by tnfaip2 deletion. TNTs allowed autophagosome and lysosome exchange between podocytes, thereby ameliorating AGE-induced lysosomal dysfunction and apoptosis. This protective effect was abolished by tnfaip2 deletion, TNT inhibition, and donor cell lysosome damage. By contrast, Tnfaip2 overexpression enhanced TNT-mediated transfer and prevented AGE-induced autophagy and lysosome dysfunction and apoptosis. In conclusion, TNFAIP2 plays an important protective role in podocytes in the context of DN by allowing TNT-mediated autophagosome and lysosome exchange and may represent a novel druggable target.Abbreviations: AGEs: advanced glycation end products; AKT1: AKT serine/threonine kinase 1; AO: acridine orange; ALs: autolysosomes; APs: autophagosomes; BM: bone marrow; BSA: bovine serum albumin; CTSD: cathepsin D; DIC: differential interference contrast; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; HG: high glucose; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PI3K: phosphoinositide 3-kinase; STZ: streptozotocin; TNF: tumor necrosis factor; TNFAIP2: tumor necrosis factor, alpha-induced protein 2; TNTs: tunneling nanotubes; WT: wild type.

Keywords: Advanced glycation end products; albuminuria; autophagosomes; experimental diabetes; hyperglycemia; lysosomes; nephrin; podocytes; renal function loss; slit diaphragm.

Figure 1.

TNFAIP2 expression in both human and experimental diabetic nephropathy. (A) Glomerular TNFAIP2 protein expression was assessed by immunohistochemistry in renal cortex sections from control subjects (n = 8) and patients with either incipient (DN-micro, n = 3) or overt diabetic nephropathy (DN-macro, n = 12) (original magnification 200X, scale bar: 100 µm). Focal segmental glomerulosclerosis (FSGS) sections were used as a positive control. The percent area of positive staining is shown in the graph (***p < 0.001 DN vs. control). (B) Double immunofluorescence for TNFAIP2 and the podocyte marker SYNPO (synaptopodin) was carried out on renal sections from DN patients. The merged image shows colocalization (original magnification 200X, scale bar: 100 µm). (C) Representative immunohistochemistry images of glomerular TNFAIP2 protein expression in renal cortex sections from non-diabetic (ND) and diabetic mice (DM) after 12 weeks of diabetes (magnification 400X, scale bar: 50 µm). (D) The graph shows the percent area of glomerular TNFAIP2-positive staining (n = 5 mice per group; ***p < 0.001 DM vs. ND). (E) Tnfaip2 mRNA levels were measured in the glomeruli isolated from ND and DM mice by real time-PCR and results corrected for the expression of the housekeeping gene Hprt (n = 5 mice per group; ***p < 0.001 ND vs. DM). (F) Staining for NPHS2/Podocin and TNFAIP2 on serial renal cortical sections obtained from DM mice showed a podocyte distribution (magnification 400X, scale bar: 50 µm). (G) Representative immunoblotting of TNFAIP2 in cultured podocytes exposed to high glucose concentrations (HG) for 48 h with and without the PI3K inhibitor, LY294002 (LY). TUBB/Tubulin was used as a loading control. (H) Results of densitometry analysis are shown in the graph (n = 3; ***p < 0.001 HG vs. others). (I and J) Immunoblottings showing TNFAIP2 expression in podocytes exposed to AGEs, mechanical stretch (10% elongation) or vehicle (V). Results of densitometry analyses are shown in the graphs (p = ns).

Figure 2.

The TNFAIP2-TNT system in cultured podocytes exposed to diabetes-related insults. (A) Podocytes pre-exposed to high glucose concentrations (HG) for 48 h were stained with wheat germ agglutinins (WGA) Alexa Fluor 488 to reveal TNTs. A representative image showing a TNT-like channel, interconnecting two podocytes, is shown (magnification 630X, scale bar: 50 µm). (B) Serial Z-stack images, acquired with a step-size of 0.25-µm proved that TNTs did not adhere to the substrate. In panel C, colors represent the Z-depth (depth coding; red: bottom, blue: top). (D) F-actin was labeled with Cell-Light Actin-RFP to show that TNTs have an actin backbone (magnification 630X, scale bar: 50 µm). (E and F) Podocytes were exposed to normal (NG) or high glucose (HG) (E) and vehicle or AGEs (F) with and without inhibitors of AKT1 (AKT1-VIII inhibitor (AKT1i); 1 µM), PI3K (LY-294002 (LY); 50 µM), and TP53 (p-nitro-pifithrin (TP53i); 5 µM), stained with WGA Alexa Fluor 488, and analyzed by fluorescent live cell microscopy. The graphs show the percentage of podocytes connected by one or more TNTs (n = 3; ***p < 0.001 HG and HG+p53i vs. others; **p < 0.01 AGEs and AGEs+p53i vs. others). (G) Murine podocytes were transfected with either Tnfaip2 shRNA (tnfaip2^−/−) or a mock plasmid (Tnfaip2^+/+). Knockdown efficiency was assessed by immunoblotting and TUBB/tubulin used as internal control. (h and i) tnfaip2^−/− and Tnfaip2^+/+ podocytes were exposed to (H) vehicle or AGEs and (I) NG or HG for 48 h, stained with WGA Alexa Fluor 488, and analyzed by fluorescent live cell microscopy to reveal TNTs. The graphs show the percentage of podocytes connected by one or more TNTs (***p < 0.001 Tnfaip2^+/+ HG and Tnfaip2^+/+AGEs vs. others).

Figure 3.

Effect of tnfaip2 deletion on diabetes-induced glomerular structural abnormalities, markers of fibrosis, and inflammation. Renal cortex samples from diabetic (DM) and non-diabetic (ND) both WT (ND-WT, DM-WT) and tnfaip2 KO (ND-KO, DM-KO) mice were studied 12 weeks after diabetes onset. (A) Representative periodic acid Schiff (PAS) staining images (magnification 400X, scale bar: 50 µm) and (B) quantification of glomerulosclerosis are shown (n = 6 mice per group; ***p < 0.001 DM-WT vs. ND-WT; DM-KO vs. DM-WT). (C) Electron microscopy images showing a more prominent mesangial expansion in the glomeruli from DM-KO mice compared to DM-WT mice (magnification 3200X). (D) Immunofluorescence images of glomerular FN1 (fibronectin 1) are shown (magnification 400X, scale bar: 50 µm). (E) Quantification of the percent area of glomerular FN1-positive staining (n = 6 mice per group; ***p < 0.001 DM-WT vs. ND-WT; DM-KO vs. DM-WT). (F) Tgfb1 mRNA levels were measured by real-time PCR in total renal cortex and corrected for the expression of the housekeeping gene Hprt (n = 5 mice per group; *p < 0.05 DM-WT vs. ND-WT; DM-KO vs. DM-WT). (G) Glomerular macrophage accrual was evaluated by counting the number of LGALS3/MAC2-positive cells per glomerulus (n = 6 mice per group; ***p < 0.001 DM-WT vs. ND-WT; DM-KO vs. DM-WT). mRNA levels of Ly6c2 (H), Ccl2 (I), Ccr2 (J), and Tnf (K) were measured in the renal cortex by real time-PCR and the results corrected for the expression of the housekeeping gene Hprt (n = 5 mice per group; *p < 0.05 DM-WT vs. ND-WT; **p < 0.01 DM-KO vs. DM-WT). (L) Schematic illustration of the protocol used for generating diabetic (DM) tnfaip2 KO chimeric mice. Recipient tnfaip2 KO mice were lethally irradiated, and then reconstituted with bone marrow (BM) from either WT (DM-KO-c^WT) or KO (DM-KO-c^KO) mice. (M) PCR genotyping of peripheral blood cells from chimeric animals. M: marker; NC: no template control. (N) ALB (albumin) excretion rate (AER) was measured after 12 weeks of diabetes in transplanted animals. (*p < 0.05 DM-WT-c^WT vs. ND-WT-c^WT; DM-KO-c^WT and DM-KO-c^KO vs. DM-WT-c^WT. (O) Representative images of PAS staining of renal cortex sections from transplanted animals (magnification 400X, scale bar: 50 µm).

Figure 4.

Effect of tnfaip2 deletion on diabetes-induced podocyte abnormalities. Renal cortex samples from diabetic (DM) and non-diabetic (ND) WT (ND-WT, DM-WT) and tnfaip2 KO (ND-KO, DM-KO) mice were studied 12 weeks after diabetes onset. (A) Representative immunofluorescence images of NPHS2/Podocin and NPHS1/Nephrin (magnification 400X, scale bar: 50 µm). (B) The graph shows the percent area of NPHS1/Nephrin and NPHS2/Podocin positive staining (n = 6 mice per group; ***p < 0.001 DM-WT vs. ND-WT; *p < 0.05 DM-KO vs. DM-WT). (C) Nphs1 and Nphs2 mRNA levels were measured by real-time PCR in total renal cortex and the results corrected for the expression of Wt1 (n = 5 mice per group; ***p < 0.001 DM-WT vs. ND-WT; *p < 0.05 DM-KO vs. DM-WT). (D) Electron microscopy images showing glomeruli from ND-WT, DM-WT, and DM-KO mice. The extent of foot processes effacement was greater in DM-KO than in DM-WT animals (magnification 3200X). (E) Apoptosis was assessed by TUNEL assay (green) and nuclei counterstained with DAPI (magnification 400X, scale bar: 50 µm). The percentage of apoptotic cells per glomerular area is shown in the graph (n = 6 mice per group; ***p < 0.001 DM-KO vs. others). (F) Representative immunostaining for CDKN1C/P57 (podocyte marker) (magnification 400X, scale bar: 50 µm). The number of CDKN1C/P57-positive cells per glomerular area is reported in the graph (n = 6 per group; ***p < 0.001 DM-KO vs. others). (G) Primary podocytes obtained from both WT and tnfaip2 KO mice were exposed to either vehicle or AGEs for 48 h and apoptosis assessed by TUNEL assay (apoptotic cells in pink). Nuclei were counterstained with DAPI (magnification 100X, scale bar: 200 µm). The percentage of apoptotic cells is shown in the graph (***p < 0.001 WT-AGEs vs. vehicle; KO-AGEs vs. WT-AGEs). (H) Primary podocytes obtained from both WT and tnfaip2 KO mice were exposed to either vehicle or AGEs for 48 h in the presence/absence of latrunculin-B (LatB, 100 nM) and apoptosis assessed by ANXA5/Annexin V staining (green). Nuclei counterstained with DAPI (magnification 100X, scale bar: 200 µm). The percentage of apoptotic cells is shown in the graph (***p < 0.001 AGEs vs. vehicle; AGEs-LatB. vs. AGEs).

Figure 5.

Effect of tnfaip2 deletion on diabetes-induced autophagy abnormalities. Markers of autophagy were studied in the glomeruli of diabetic and non-diabetic WT (ND-WT, DM-WT) and tnfaip2 KO (ND-KO, DM-KO) mice and in primary podocytes from ND-WT and ND-KO animals exposed to normal glucose (NG), high glucose (HG), vehicle, or AGEs for 48 h. (A) Immunoblotting showing MAP1LC3/LC3-II expression in isolated glomeruli (TUBB/Tubulin loading control). The graph shows the results of densitometry analysis (n = 5 per group; *p < 0.05 DM-WT vs. ND-WT; ***p < 0.001 DM-KO vs. DM-WT). (B) Representative images of glomerular SQSTM1/p62 immunostaining (magnification 400X, scale bar: 50 µm). (C) The graph shows the number of SQSTM1/p62-positive cells per glomerular area (n = 6 per group; ***p < 0.001 DM-WT vs. ND-WT; DM-KO vs. DM-WT). (D and E) Immunoblotting showing MAP1LC3/LC3-II expression (TUBB/Tubulin loading control) in cultured podocytes exposed to (D) vehicle or AGEs and (E) NG or HG. Results of densitometry analysis are shown in the graph (n = 3; *p < 0.05 WT-AGE vs. vehicle; ***p < 0.001 KO-AGE vs. WT-AGE; **p < 0.01 WT-HG vs. WT-NG and KO-HG). (F and G) Co-staining (yellow dots) for SQSTM1/p62 (green) and MAP1LC3/LC3-II (red) in cultured podocytes exposed to (F) vehicle or AGEs and (G) NG or HG. Nuclei were counterstained with DAPI (magnification X630, bar: 50 µm). (H and I) Quantification of the percent SQSTM1/p62⁺ and MAP1LC3/LC3⁺ area is shown in the graphs (n = 3; ***p < 0.001 WT-AGE/WT-HG vs. vehicle/NG; KO-AGE/KO-HG vs. WT-AGE/WT-HG). mRNA levels of (J) Becn1, (K) Atg5, (L) Atg7 were measured by real time-PCR in the renal cortex of experimental animals and the results corrected for the expression of the housekeeping gene Hprt (n = 5 per group; p = ns). (M and N) Becn1 mRNA levels were assessed by real time-PCR in cultured podocytes exposed to (M) vehicle or AGEs, (N) NG or HG. Results were corrected for the expression of the housekeeping gene Rn18s (n = 3; p = ns).

Figure 6.

Effect of tnfaip2 deletion and/or diabetes on both autophagic flux and autolysosome (AL) formation. (A) Autophagy flux was monitored by tandem fluorescent-tagged LC3 in primary WT and tnfaip2 KO podocytes exposed to either vehicle or AGEs for 48 h in the presence or absence of latrunculin B (LatB). Nuclei were counterstained with DAPI. Yellow puncta autophagosomes (APs) and free red puncta (acidic AL) are shown in the RFP+GFP-LC3 panels (magnification X630, bar: 50 µm). (B) Quantification of the number of yellow and free red puncta per cell is shown in the graph (n = 3; ***p < 0.001 WT-AGE vs. vehicle; **p < 0.01 KO-AGE and LatB-AGE vs. WT-AGE). (C) The relative GFP:RFP ratio was assessed by ImageJ and results are shown in the graph (**p < 0.01 WT-AGE vs. vehicle; *p < 0.05 KO-AGE and LatB-AGE vs. WT-AGE). (D) Fusion of APs with lysosomes was assessed by double immunofluorescence for MAPLC3/LC3 (red) and LAMP1 (green). Merged images showed co-localization (magnification X630, bar: 50 µm). (E) The graph shows the percentage of the yellow positive area per cell (***p < 0.001 WT-AGE vs. WT-vehicle; KO-AGE vs. WT-AGE).

Figure 7.

Effect of tnfaip2 deletion and re-expression on diabetes-induced changes on lysosomes, autophagosomes (APs), and apoptosis. Lysosome both integrity and function was assessed in primary murine WT and tnfaip2 KO podocytes exposed to either vehicle or AGEs for 48 h. The effect of tnfaip2 re-expression on lysosomal integrity and function, AP accumulation, and apoptosis was tested by transfecting KO podocytes with an adenovirus expressing either Tnfaip2 (KO^Adv+) or a control mock vector (KO^Adv-). (A) Representative immunofluorescence images of podocytes stained with Acridine Orange (AO) (magnification X100, bar: 200 µm). Quantification of green to red AO fluorescence is reported in the graph below (n = 3; ***p < 0.001 WT-AGE and KO^Adv+-AGE vs. WT-vehicle; KO-AGE and KO^Adv–AGE vs. WT-AGE). (B) Colocalization of CTSD (red) with LAMP1⁺ lysosomes (green). Nuclei were stained with DAPI (magnification X630, bar: 50 µm). (C) Tnfaip2 mRNA levels were measured by real-time PCR in WT, KO^Adv+ and KO^Adv- podocytes and the results corrected for the expression of Rn18s (n = 3; ***p < 0.001 KO^Adv- vs. others). (D) Effect of Tnfaip2 re-expression on AP accumulation as shown by costaining (yellow dots) for SQSTM1/p62 (green) and MAP1LC3/LC3 (red). Nuclei were counterstained with DAPI (magnification X630, bar: 50 µm). (E) Quantification of CTSD activity expressed as relative fluorescence units (RFU) per number of cells (**p < 0.01 WT-AGE and KO^Adv+-AGE vs. WT-vehicle; KO-AGE and KO^Adv–AGE vs. WT-AGE). (F) Effect of Tnfaip2 re-expression on podocyte apoptosis assessed by TUNEL assay (apoptotic cells in pink) (magnification 100X, scale bar: 200 µm). (G) The percentage of apoptotic cells is shown in the graph (**p < 0.01 WT-AGE and KO^Adv+-AGE vs. WT-vehicle; KO-AGE and KO^Adv–AGE vs. WT-AGE).

Figure 8.

TNFAIP2-TNT-mediated lysosome and autophagosome (AP) exchange in AGE-treated podocytes. (A) Podocytes stained with CellTracker Blue were exposed to either vehicle or AGEs for 48 h, and then cocultured with donor podocytes carrying LAMP1-GFP-labeled lysosomes. The images show green lysosomes within the TNT and in the cytosol (*) of a recipient podocyte (magnification X630, bar: 50 µm, DIC images showing the TNT). (B and C) Lysosome transfer (GFP and Blue dually labeled cells) was quantified by flow cytometry in recipient podocytes exposed to either vehicle or AGEs and co-cultured with donor WT and tnfaip2 KO podocytes. WT donor podocytes were treated with latrunculin-B (WT-LatB 100 nM, 1 h) to prevent TNT formation. KO and WT donor podocytes were transfected with an adenovirus expressing either tnfaip2 (KO^Adv+, WT^Adv+) or a mock vector (n = 3; **p < 0.01 WT-AGE and KO^Adv+-AGE vs. WT-Vehicle; KO-AGE, WT^LatB-AGE vs. WT-AGE; p < 0.05 WT^Adv+-AGE vs. WT-AGE). (D) Recipient podocytes were stained with CellTracker Blue and exposed to high AGE concentration (200 µg/ml, 48 h; H-AGE). Donor podocytes with LAMP1-GFP-labeled lysosomes were exposed to lower AGE concentrations (50 µg/ml, 48 h; L-AGE). In coculture, green lysosomes were seen in the cytosol (*) of recipient podocytes (magnification X630, bar: 50 µm, DIC images showing the TNT). (E) Donor (GFP-labeled lysosomes) and recipient (Recip, CellTracker Blue) podocytes were pre-exposed to either H-AGE (200 µg/ml) or L-AGE (50 µg/ml) concentrations and then co-cultured. Lysosome transfer (GFP and Blue dually labeled cells) from donor towards recipient (Recip) podocytes was quantified by flow cytometry (***p < 0.001 Donor-L-AGE to Recip-H-AGE vs. others). (F) Donor podocytes exposed to AGEs for 48 h were transfected with tandem fluorescent-tagged LC3 to label APs (yellow punta), then co-cultured with healthy recipient podocytes. The image shows yellow puncta within the TNT and in the cytosol (*) of a recipient podocyte (magnification X630, bar: 50 µm, DIC images showing the TNT).

Figure 9.

Effect of TNFAIP2-TNT-mediated lysosome exchange on autophagosome (AP) accumulation, lysosome dysfunction, and apoptosis in a co-culture system. Podocytes were exposed to either vehicle or AGEs for 48 h. The effect of the TNFAIP2-TNT system on autophagy, lysosome dysfunction, and apoptosis was assessed by co-culturing recipient AGE-treated podocytes with donor (D) cells transfected with an adenovirus expressing Tnfaip2 (WT^Adv+), mock vector (WT^Adv-) or pre-exposed to leupeptin (Leup, 200 µg/ml WT^Leup). (A) Immunostaining for SQSTM1/p62 (pink). Blue recipient podocyte (CellTracker Blue) and green donor podocytes (CellTracker Green) (magnification 630X, scale bar: 50 µm) and quantification of SQSTM1/p62⁺ positive area per cell (n = 3; ***p < 0.001 AGE and AGE+Donor-WT^Leup vs. vehicle; **p < 0.01 AGE-Donor-WT^Adv- vs. AGE andAGE-Donor-WT^Adv+). (B) Acridine Orange (AO red and green) staining. Blue donor podocytes (CellTracker Blue) (magnification 630X, scale bar: 50 µm) and quantification of green to red AO fluorescence (n = 3; ***p < 0.001 AGE and AGE+Donor-WT^Leup vs. vehicle; **p < 0.01 AGE-Donor-WT^Adv- vs. AGE and AGE-Donor-WT^Adv+). (C) The graph shows the percentage of apoptotic podocytes as assessed by ANXA5/Annexin V staining (n = 3; ***p < 0.001 AGE and AGE+Donor-WT^Leup vs. vehicle; **p < 0.01 AGE-Donor-WT^Adv- vs. AGE and AGE-Donor-WT^Adv+).

Figure 10.

Effect of Tnfaip2 over-expression in monoculture of AGE-treated podocytes. WT podocytes and podocytes transfected with an adenovirus expressing Tnfaip2 (WT^Adv+) or a mock vector (WT^Adv-) were exposed to either vehicle or AGEs for 48 h. (A) Double immunostaining (yellow dots-autophagosomes) for MAP1LC3/LC3⁺ and SQSTM1/p62⁺ (magnification X630, bar: 50 µm, nuclei: DAPI) and quantification of the percent SQSTM1/p62⁺ and MAP1LC3/LC3⁺ positive area (n = 3; ***p < 0.001 AGE-WT^Adv- vs. others). (B) Acridine Orange (AO) staining (magnification 100X, scale bar: 200 µm) and quantification of green and red AO fluorescence (n = 3; ***p < 0.001 AGE-WT^Adv- vs. others). (C) CTSD activity expressed as relative fluorescence units (RFU) per number of cells (n = 3; ***p < 0.001 AGE-WT^Adv- vs. others). (D) Images of apoptotic podocytes (pink, TUNEL assay) (magnification 100X, scale bar: 200 µm, nuclei: DAPI) and quantification of the percentage of TUNEL positive cells (n = 3; ***p < 0.001 AGE-WT^Adv- vs. others). (E) Tnfaip2 mRNA levels were measured by real-time PCR in WT^Adv+ and WT^Adv- podocytes and the results corrected for the expression of Rn18s (n = 3; ***p < 0.001 WT^Adv+ vs. WT^Adv+).