Glucose-derived AGEs induce Notch signaling in podocytes in vitro
Glucose-derived AGEs were prepared as described in the ‘Research design and methods’ section by incubating D-glucose and BSA. We have noticed browning in D-glucose-BSA preparation after 2 weeks, and the extent of AGE formation was demonstrated by measuring non-tryptophan AGE fluorescence (figure 1A). We assessed the degree of glycation of BSA during incubation with D-glucose by measuring its free amino groups. There was about a 50% reduction in free amino groups in D-glucose-BSA preparation (figure 1B). High-molecular-weight aggregates were observed in D-glucose-BSA preparation (figure 1C). Free amino groups of BSA can react with the carbonyl group of glucose and form AGEs via the Maillard reaction. Therefore, we assessed the formation of AGEs in the D-glucose-BSA preparation by immunoblotting with the anti-AGE antibody. The data suggest AGEs were selectively noticed in D-glucose-BSA preparation (figure 1D). Since we confirmed the formation of glucose-derived AGEs in D-glucose-BSA preparation, hereafter they are referred to as AGEs. Then, we investigated the effect of AGEs on Notch signaling components in human podocytes. We found that AGEs induced RAGE and NICD1 expression in a dose-dependent manner (figure 1E), whereas BSA naïve to AGE modification failed to elicit NICD1 expression (figure 1F). We have also observed that AGEs induced Notch ligand (JAG1), NICD1, and Notch downstream transcription factor HES1 in podocytes (figure 1G). Since γ-secretase cleaves Notch1 to release NICD1, we measured γ-secretase activity in cells treated with AGEs. We observed a dose-dependent increase in γ-secretase activity with AGE treatment (figure 1H). All these data suggest that Notch signaling is activated in human podocytes exposed to AGEs.
Figure 1AGEs induce Notch signaling in podocytes. (A) Non-tryptophan AGE fluorescence was measured to demonstrate the formation of AGEs in D-glucose+BSA preparations at Ex: 370 nm and Em: 400–500 nm. (B) Quantification of free amines in BSA and D-glucose+BSA preparations of various concentrations (50–200 μg/mL of BSA) by TNBS assay. (C) BSA and D-glucose+BSA preparations were subjected to SDS-PAGE and stained with Coomassie blue. The arrowhead indicates high-molecular-weight aggregates in the stacking region of the gel. (D) Immunoblots showing the presence of AGEs in D-glucose+BSA preparations. M indicates the standard protein marker (D,E). (E) Immunoblots showing the expression of RAGE, NICD1, and β-actin in HPC cells treated with AGEs (25–200 µg/mL) for 48 hours. (F) Immunoblots showing the expression of RAGE, NICD1, and β-actin in HPC treated with BSA alone (25–200 µg/mL) for 48 hours. (G) Immunoblots showing the expression of RAGE, JAG1, NICD1, HES1, and β-actin in HPC treated with AGEs (100 µg/mL) for indicated time intervals (24–72 hours). (E–G) The fold expression was presented after normalizing with β-actin. (H) γ-secretase activity in HPC treated with (50–200 µg/mL) or without AGEs for 48 hours (n=6). *P<0.05, ****P<0.0001. Data are presented as mean±SD (n=3). AGEs, advanced glycation end-products; a.u, arbitrary unit; BSA, bovine serum albumin; CTL, control; Em, emission; Ex, excitation; HES1, hairy and enhancer of split homolog1; HPC, human podocyte; JAG1, jagged1; NICD1, Notch intracellular domain; RAGE, receptor for AGEs; SDS-PAGE, sodium dodecyl sulfate poly-acrylamide gel electrophoresis; TNBS, trinitrobenzene sulfonic acid.
Both RAGE and γ-secretase are required for AGE-activated Notch signaling
To confirm the role of γ-secretase in AGE-induced Notch1 activation, we next treated the human podocytes with AGEs in the absence or presence of a well-established γ-secretase inhibitor, DAPT. As expected, DAPT treatment to the AGE-exposed human podocytes decreased the γ-secretase activity (online supplementary figure S1A). Further, we noticed reduced γ-secretase activity in podocytes that were treated simultaneously with AGEs and FPS-ZM1, a RAGE inhibitor (online supplementary figure S1A). The ability of AGEs to induce NOTCH1, JAG1, or HES1 is ameliorated in the presence of FPS-ZM1 as measured by qRT-PCR (figure 2A–C) and western blotting (figure 2D). Furthermore, DAPT also prevented HES1 expression in AGE-treated human podocytes as measured by qRT-PCR (figure 2C) and immunoblotting (figure 2D). Interestingly, NICD1 levels were decreased following treatment with DAPT (figure 2D). As anticipated, DAPT treatment does not affect the expression of both NOTCH1 (figure 2A) and its ligand JAG1 (figure 2B,D). Next, we studied the essential role of RAGE and NOTCH1 in AGE-induced Notch1 signaling by siRNA-mediated knocking down of RAGE and NOTCH1 expression, respectively. Knockdown of RAGE expression resulted in blunting of AGE-induced expression of NOTCH1, NICD1, JAG1, and HES1 (figure 2E), while knockdown of NOTCH1 expression resulted in blunting of AGE-induced expression of NICD1, JAG1, and HES1 expression (figure 2E). Furthermore, AGE-induced γ-secretase activity is also ameliorated by the knocking down of RAGE and NOTCH1 expression, respectively (online supplementary figure S1B).
Figure 2AGE-activated Notch signaling promotes EMT in podocytes. (A–C) qRT-PCR analysis showing the expression of (A) NOTCH1, (B) JAG1, and (C) HES1 in HPC treated with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1. β-actin was used as an internal control. **P<0.01, ***P<0.0001. (D) Immunoblots showing the expression of NICD1, JAG1, HES1, and β-actin in HPC treated with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1 (48 hours). The fold change values were presented after normalizing with β-actin. (E) HPC cells transfected with specific siRNA targeting RAGE and Notch1 or scramble RNA (Scr) were subjected to immunoblotting for RAGE, NOTCH, JAG1, and NICD1. (F) Expression of E-CAD, N-CAD, and vimentin in HPC (CTL, AGEs, AGEs+DAPT, and AGEs+FPS-ZM1) was analyzed by qRT-PCR. The expression of β-actin was used as an internal control. ****P<0.0001. (G) Immunoblotting analysis of expression of E-CAD, N-CAD and vimentin in HPC (CTL, AGEs, AGEs+DAPT, and AGEs+FPS-ZM1). (E,G) The fold change values were presented after normalizing with β-actin expression. (H) Phalloidin staining of podocytes showing F-actin arrangement. The white arrows indicate filopodia formation. Scale bar=20 µm. (I) Quantification of the average number of filopodia formation observed from the phalloidin staining (n=16). ****P<0.0001. Data are presented as mean±SD (n=3). AGEs, advanced glycation end-products; CTL, control; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; E-CAD, E-Cadherin; EMT, epithelial to mesenchymal transition; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; HES1, hairy and enhancer of split gene1; HPC, human podocyte; JAG1, jagged1; N-CAD, N-Cadherin; NICD1, Notch intracellular domain; qRT-PCR, quantitative reverse transcription-PCR; RAGE, receptor for AGEs; siRNA, small interfering RNA.
Activation of Notch1 signaling was shown to enhance the migratory properties of podocytes via EMT.10 Furthermore, in our earlier study, podocytes exposed to N(ε)-carboxymethyl lysine were shown to possess enhanced migration properties and undergo phenotypic switch via EMT.17 Therefore, we assessed the migratory property of podocytes treated with AGEs in the presence and absence of DAPT and FPS-ZM1. Both DAPT and FPS-ZM1 attenuated AGE-induced podocyte motility (online supplementary figure S1C). As we could see the enhanced migration of podocytes treated with AGEs, we assessed the expression of epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin and vimentin). Loss of E-cadherin and increased N-cadherin, that is, cadherin switch, are hallmark features of EMT. Exposure of podocytes to AGEs manifested in cadherin switch and increased expression of vimentin (figure 2F,G), whereas DAPT and FPS-ZM1 prevented AGE-induced cadherin switch and vimentin expression (figure 2F,G). EMT is accompanied by dramatic changes in cytoskeleton remodeling.26 Therefore, to assess the cytoskeletal abnormalities induced by AGEs, we stained podocytes with phalloidin, which specifically stains F-actin. DAPT and FPS-ZM1 prevented AGE-induced F-actin reorganization in human podocytes (figure 2H). We measured an average number of filopodia per cell and found that DAPT and FPS-ZM1 prevented AGE-induced filopodia formation in human podocytes (figure 2I). Motonishi et al27 showed that SIRT1 regulates the integrity of the podocyte actin cytoskeleton and prevents glomerular injury. Therefore, we assessed SIRT1 expression in AGE-treated podocytes and found that AGEs marginally induced SIRT1 expression (online supplementary file 1). All these data suggest that podocytes undergo EMT on treatment with AGEs, and Notch activation is required for EMT of AGE-treated podocytes in vitro.
AGE-induced Notch1 signaling is abrogated by RAGE inhibitor in vivo
We investigated for co-localization of NICD1 and HES1 in the nucleus in response to AGE treatment. As expected there is a predominant accumulation of both NICD1 and HES1 in the nucleus of podocytes as demonstrated by immunofluorescence (figure 3A) and immunoblotting of nuclear fraction from podocytes exposed to AGEs (figure 3B). A large body of evidence reported the accumulation of AGEs in glomeruli from patients with DN or experimental animals.26 Therefore we investigated the expression of RAGE and components of Notch signaling in mice administered with AGEs for 4 weeks. We have observed enhanced RAGE expression in glomerular sections from AGE-administered mice (figure 3C). Furthermore, elevated glomerular expression of NICD1, JAG1, and HES1 was observed in AGE-injected mice (figure 3D,E), whereas coadministration of DAPT ameliorated AGE-induced NICD1 and HES1 expression (figure 3D,E). Indeed simultaneous administration of RAGE inhibitor (FPS-ZM1) attenuated the AGE-induced expression of NICD1, JAG1, and HES1 (figure 3D,E). Furthermore, both DAPT and RAGE inhibitor blunted the AGE-induced cadherin switch and vimentin expression in glomerular lysates (figure 3F). Together the data suggest AGEs activate Notch signaling and induce EMT in glomerular podocytes.
Figure 3AGEs induce Notch signaling in mice glomeruli and inhibition of γ-secretase and RAGE ameliorates EMT in mice kidney. (A) Immunofluorescence for the nuclear co-localization study of NICD1 (Cy3, red) and HES1 (Cy5, far-red) in HPC treated with or without (CTL) AGEs, AGEs+DAPT and AGEs+FPS-ZM1. Magnification ×630. Scale bar=20 µm. (B) Immunoblots for NICD1, HES1 and histone H2B, the nuclear extract of HPC, treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1. The fold change values were presented with the expression of the respective genes after normalizing with H2B. (C) Immunostaining for RAGE expression in mice glomeruli from with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6, each group). Magnification ×630. Scale bar=20 µm. (D) Double immunostaining with anti-NICD1 (Alexa Fluor 555) and anti-WT1 (Cy3, red) in glomerular sections from with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 treatment (n=6, each group). Magnification ×630. Scale bar=20 µm. (E) Immunoblotting analysis for NICD1, JAG1, HES1, and β-actin in mice glomerular lysates from with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 treated mice (n=6, each group). The fold change values were presented with the expression of the respective genes after normalizing with β-actin. (F) Immunoblotting analysis for E-CAD, N-CAD, vimentin, and β-actin in glomerular lysates from with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1 treated mice. The fold change values were presented with the expression of the respective genes after normalizing with β-actin. Data are presented as mean±SD (n=3). AGEs, advanced glycation end-products; CTL, control; DAPI, 4′,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; E-CAD, E-Cadherin; EMT, epithelial to mesenchymal transition; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; HES1, hairy and enhancer of split gene1; HPC, human podocyte; JAG1, jagged1; N-CAD, N-Cadherin; NICD1, Notch intracellular domain; RAGE, receptor for AGEs.
Notch1 signaling is required for AGE-induced glomerular fibrosis
DN is presented with progressive renal fibrosis.28 29 AGEs were shown to induce fibrosis and contribute to the pathology of DN.30 Therefore, we examined the paraffin-embedded sections of AGE-treated mice for fibrosis. We noticed renal fibrosis in the mice administered with AGEs, as analyzed by PAS and MT staining (figure 4A). Glomerular damage score was found elevated in AGE-treated mice (figure 4B). It is noteworthy that both DAPT and RAGE inhibitor had prevented AGE-induced fibrosis and glomerular damage (figure 4A,B). Next, we measured the expression of fibrotic markers in AGE-treated mice and mice treated with DAPT and RAGE inhibitor. Indeed, DAPT and FPS-ZM1 inhibited the AGE-induced fibrotic markers: collagen IV, α-SMA, and fibronectin (figure 4C–F).
Figure 4AGE-induced Notch activation leads to fibrosis and podocyte foot process effacement. (A) Representative images of PAS and MT staining in mice glomeruli from with or without AGEs, AGEs+DAPT, and AGEs+FPS-ZM1 treatment. Magnification ×400. Scale bar=50 µm. (B) The glomerular damage score was quantified as described in the ‘Research design and methods’ section. (C–F) Immunohistochemical staining for Col IV, α-SMA, and fibronectin in glomerular sections from mice treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Magnification ×400. Scale bar=50 µm. The intensity of glomerular expression of Col IV, α-SMA, and fibronectin was quantified using ImageJ (NIH). ****P<0.0001. (G) Representative TEM images of podocytes from mice treated with or without (CTL), AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Scale bar=1 µm. (H) Representative images of immunohistochemical staining for WT1 (podocytes) in the glomerulus from mice treated with or without (CTL) AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Magnification ×630. The arrows indicate detached podocytes. Scale bar=20 µm. (I) The percentage of glomerulus with detached podocytes was quantified with the help of ImageJ (NIH). ****P<0.0001. Data are presented as mean±SD. AGEs, advanced glycation end-products; Col IV, collagen IV; CTL, Control; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; MT, Masson’s trichrome; NIH, National Institutes of Health; PAS, periodic acid-Schiff; α-SMA, alpha-smooth muscle actin; TEM, transmission of electron microscopy.
Glomerular fibrosis is presented with thickening of GBM that may manifest in the podocyte foot process effacement.31 Since we observed elevated expression of fibrotic markers in AGE-injected mice, we next assessed the morphology of GBM and podocytes in these mice. Although TEM analysis of AGE-administered mice shows thickening of GBM and podocyte foot process effacement, co-treatment with DAPT or FPS-ZM1 abrogated this adverse effect of AGEs on glomerular architecture (figure 4G). The thickening of GBM might lead to the dehiscence of podocytes.32 Suztak et al have reported that the depletion of podocytes occurs at the onset of DN.33 Next, we assessed whether podocyte depletion occurred in AGE-administered mice. Quantification of WT1, a podocyte-specific marker, revealed the presence of detached podocyte in many glomeruli, whereas DAPT and RAGE inhibitor prevented AGE-dependent podocyte depletion (figure 4H,I). Earlier, studies have shown that podocytes undergo apoptosis in response to noxious stimuli.34 Therefore, we assessed podocyte apoptosis in AGE-treated human podocytes (in vitro) and mice (in vivo). Glomerular sections from mice treated with AGEs showed increased TUNEL positive cells (online supplementary figure S2A), elevated cleaved caspase 3, and Bax expression (online supplementary figure S2B,C). Quantification of condensed nuclei from AGE-treated human podocyte cells revealed a 40%±15% presence of podocyte apoptotic bodies (online supplementary figure S2D). Together the data confirm that AGE-induced Notch signaling leads to glomerular fibrosis and podocyte depletion.
Inhibiting Notch1 activation abrogates AGE-induced proteinuria
As we have stated, podocytes are instrumental in regulating glomerular permselectivity and either podocyte injury or loss elicits proteinuria. Since AGE treatment showed podocyte foot process effacement (figure 4G), we measured the permselectivity of podocytes in vitro by albumin influx assay. Exposure of podocyte monolayer to AGEs resulted in increased permeability to albumin, whereas DAPT or FPS-ZM1 prevented AGE-induced albumin leakage across podocyte monolayer (figure 5A). The expression of predominant slit-diaphragm proteins (podocin and nephrin) decreased in the glomerulus of AGE-treated mice (figure 5B,C). Since damage to the slit-diaphragm contributes to proteinuria, we measured the renal function parameters and found that the administration of AGEs to mice resulted in increased urinary albumin to creatinine ratio (UACR) and a decline in GFR (figure 5D and online supplementary figure S3A). Mice treated with AGEs had increased the amount of protein in the urine (online supplementary figure S3B), whereas co-treatment of mice with DAPT or FPS-ZM1 rescued the expression of podocin and nephrin and blocked AGE-induced proteinuria (figure 5A–D and online supplementary figure S3A,B). Together the data suggest that AGEs impair the podocyte function and induce proteinuria through RAGE and Notch1 signaling.
Figure 5Blockade of Notch and RAGE protects mice from proteinuria, and elevated levels of AGEs correlate with Notch activation in people with DN. (A) AGEs alter podocyte permeability in vitro. Albumin permeability across the podocyte monolayer was determined after 48 hours of exposure to AGEs (n=3). ****P<0.0001. (B) Immunoblotting study for podocin and nephrin expression in glomerular lysates and (C) by immunohistochemical staining for podocin in the glomerulus from mice treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). Magnification ×630. Scale bar=20 µm. The fold change values were presented with the expression of the respective genes after normalizing with β-actin. (D) UACR was estimated in mice treated with or without AGEs, AGEs+DAPT and AGEs+FPS-ZM1 (n=6). ****P<0.0001. (E) Immunoblotting analysis for AGEs in urine samples of DN (n=5) and non-diabetic group (n=3). The arrowhead indicates the positive staining for AGEs in the urine samples of a patient with DN. (F) Immunohistochemical staining of glomerular serial sections from patients with DN (n=16) and non-diabetic groups (n=10) for AGEs (DyLight488, green), RAGE (Cy3, red), NICD1 (Alexa Fluor 555), and HES1 (Cy5, far-red). Magnification ×630. Scale bar=20 µm. (G) A proposed model depicting the adverse effect of AGEs on podocytes. Activation of Notch signaling via AGE–RAGE interaction induces the thickening of GBM, EMT, and dehiscence of podocytes, which eventually result in impaired glomerular permselectivity and proteinuria. AGEs, advanced glycation end-products; CTL, control; DAPI, 4′,6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester; DN, diabetic nephropathy; EMT, epithelial to mesenchymal transition; FPS-ZM1, N-Benzyl-4-chloro-N-cyclohexylbenzamide; GBM, glomerular basement membrane; HES1, hairy and enhancer of split gene1; NICD1, Notch intracellular domain; RAGE, receptor for AGEs; SD, slit-diaphragm; UACR, urinary albumin to creatinine ratio.
Activated Notch signaling in subjects with DN
AGEs are elevated in plasma and accumulate in several tissues including the kidney in patients with diabetes. AGEs are considered a new therapeutic target in chronic kidney disease.35 Elevated HbA1c and glycated albumin levels in subjects with DN were associated with decreased renal function as evidenced by increased UACR (online supplementary table S1) and increased urinary protein levels compared with control subjects (online supplementary figure S3C). Non-tryptophan AGE fluorescence also revealed the presence of AGEs in the serum from subjects with DN (online supplementary figure S3D). Indeed, AGEs were observed in urine samples from subjects diagnosed with DN (figure 5E). Notch signaling is inactive in the adult kidney, whereas activated Notch signaling is reported in glomerular diseases particularly in podocytes.15 Therefore, to confirm our data that AGEs activate Notch1 signaling in humans, we analyzed glomerular NICD1 expression in people with DN. Immunohistochemical analysis of kidney from people with DN revealed increased expression of AGE, RAGE, NICD1, and HES1 compared with non-diabetics (figure 5). Moreover, Nephroseq analysis suggests that there is a strong correlation in the expression of RAGE, Notch1, Hes1, and EMT marker (vimentin) in non-diabetics versus DN mouse kidney data sets (online supplementary figure S3E). Together the data suggest that activated Notch signaling in subjects with DN is concomitant with EMT of glomerular podocytes.