NR1 was purchased from Sigma Chemical Co. (St. Louis, MO, USA), and the purity of NR1 was >98%. The autophagy inhibitor, 3-methyladenine (3-MA), monodansylcadaverine (MDC), and 2′,7′-dichlorofluorescein diacetate (DCF) were also purchased from Sigma Chemical Co. Small interfering RNAs (siRNAs) against human Beclin 1 and scramble siRNA were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Hoechst 33342, the Click-iT TUNEL Alexa Fluor 488 Imaging Assay kit for microscopy and high-content screening (HCS), SYBR-Green PCR Master Mix, NuPage Novex 10% Bis-Tris gel, NuPage transfer buffer, TRIzol, the High Capacity cDNA Reverse Transcription kit, the Dead Cell Apoptosis kit with Annexin V Alexa Fluor 488 and propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Polyvinylidene fluoride membranes were purchased from Pall Life Sciences (Port Washington, NY, USA). Immobilon Western Chemiluminescent HRP Substrate was purchased from Millipore Corp. (Billerica, MA, USA). The CellTiter-Glo luminescent cell viability assay kit and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay kit were purchased from Promega Corp. (Madison, WI, USA)

The following antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA): nephrin (sc-377246), desmin (sc-65983), pro-caspase-3 (sc-7148), pro-caspase-9 (sc-56073), cleaved caspase-9 (sc-22182), Bax (sc-493), Bid (sc-135847), Bcl-2 (sc-492), Bcl-xL (sc-8392), poly(ADP-ribose) polymerase (PARP; sc-7150) and β-actin (sc-47778). Besides, rat anti-mouse podocalyxin (PCX) antibody (MAB1556) was purchased from R&D Systems (Minneapolis, MN, USA). In addition, antibodies against podocin (ab50339), CD2AP (ab84829), active caspase-3 (ab49822), Beclin (ab62557), PI3K p85 (ab189403), phosphorylated (p-)Beclin 1 (S234; ab183335), mTOR (ab2732), p-mTOR (S2448; ab109268), p-mTOR (S2481; ab137133), p-PI3K p85 (Y607; ab182651) were from Abcam (Cambridge, UK). Antibodies to light chain 3 (LC3; #12741), Akt (pan; #2920), cleaved caspase-9 (#9509), p-Akt (T308; #9275), p-Akt (S473, #4060), p-p70S6K (Thr389; #9206) and p-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1; T37/46; #2855) were all purchased from Cell Signaling Technology, Inc. Goat anti-rabbit IgG antibody conjugated with HRP (#11-035-003), goat anti-mouse IgG antibody conjugated with HRP (#115-035-003), and goat anti-rabbit IgG antibody conjugated with Cy3 (#111-165-144) were purchased from Jackson ImmunoResearch Laboratories, West Grove, PA, USA.

Conditionally immortalized human podocytes were kindly provided by Dr Guisen Li (Division of Nephrology, from the Sichuan Provincial People's Hospital) and were cultured as previously described (24). In brief, in order to maintain an undifferentiated proliferative state, podocytes were cultured at 33°C with RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies). Before use, when the cells had reached 70–80% confluence, they were cultured at 37°C to reach differentiation. The podocytes were divided into the following groups: i) the low glucose (LG) group: the cells were incubated in RPMI-1640 containing 5 mM D-glucose; ii) the high glucose (HG) group: the cells were incubated in RPMI-1640 containing 30 mM D-glucose; iii) the NR1-treated (NR1) group: the cells were incubated in RPMI-1640 containing 30 mM D-glucose and then treated with 20 µM NR1 for 24 h.

To ensure that NR1 can abolish the growth inhibitory effects of high glucose on podocytes, podocyte viability was measured within 24 h of exposure to a series of NR1 concentrations (0, 1, 5, 10, 20, 50 and 100 µM). The number of viable cells was assessed by CellTiter-Glo luminescent cell viability assay kit according to the manufacturer's instructions in triplicate. Cell morphology was also observed under a light microscope (Leica Microsystems GmbH, Wetzlar, Germany).

The podocytes were grown to 80% confluence on 6-well plates under growth-restricted conditions and treated with low glucose, high glucose, or a combination of both high glucose and 20 µM NR1 for 24 h. Hoechst 33342 was added to achieve a final concentration of 10 µM and the cells were analyzed after 5 min of Hoechst staining. Apoptotic cells with condensed chromatin were manually counted for a minimum of 20 high-powered fields per experimental condition by two blinded technicians. The number of apoptotic nuclei was expressed as a percentage of the total number of apoptotic nuclei.

Following the experimental treatments, the differentiated human podocytes were dispersed from 6-well culture plates, collected in PBS, washed with FACS buffer, and stained using the Dead Cell Apoptosis kit with Annexin V-PI following the manufacturer's instructions. Cells (10⁴) were acquired using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA). The number of apoptotic cells was analyzed using CellQuest software 3.3 (Becton-Dickinson) and was expressed as a percentage of the number of total cells.

Podocyte apoptosis was analyzed by TUNEL assay according to the manufacturer's recommendations. Following fixation in 4% buffered paraformaldehyde (PFA), the cells were incubated with terminal deoxynucleotidyl transferase using FITC-labeled nucleotides. DAPI served as a nuclear counterstain (blue). TUNEL-positive cells (green) in 15–20 randomly selected fields were manually counted out of the total number of cells.

Podocyte apoptosis was also assessed by measuring the activity of caspase-3 using the caspase-3 activity kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. In brief, the cells were lysed, and the supernatant was collected, quantified and incubated with the caspase-3-specific color substrate Ac-DEVD-pNA. Caspase-3 activity was determination by measuring optical density at OD400 nm.

The podocytes were grown in triplicate on type I collagen-coated glass coverslips, fixed with 4% PFA, and permeabilized with Triton X-100. Following the removal of the remaining Triton X-100, the cells were incubated with anti-active caspase-3 antibodies overnight, followed by Cy3-conjugated secondary antibody. For nuclear staining, the cells were stained with DAPI. For the negative contro, PBS was used instead of the primary antibody. The total cell population and the number of cleaved caspase-3-positive cells were manually counted.

Following incubation with 20 µM NR1 for the indicated periods of time, the podocytes were cultured with 0.05 mM MDC at 37°C for 60 min. The fluorescence intensity of the cells was analyzed by flow cytometry. The cells were respectively transfected with Beclin 1 siRNA and scramble siRNA at 33 nM using DharmaFect (#T-2004; Dharmacon, Lafayette, CO, USA) according to the manufacturer's instructions (25). The transfected cells were used for subsequent experiments 24 h later.

The cells were lysed and the protein lysates were centrifuged at 12,000 rpm for 30 min. Supernatants were collected and loaded on a SDS-PAGE gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for electrophoresis. Following electrophoresis, the proteins were transferred onto PVDF membranes in transfer buffer. The membranes were blocked with 5% non-fat dry milk in TBS for 1 h. The blots were incubated overnight at 4°C with primary antibodies at the dilutions recommended by the manufacturer, followed by incubating with horseradish peroxidase (HRP)-conjugated secondary antibody, and then visualized by Immobilon Western Chemiluminescent HRP substrate (26).

RNA was extracted using the RNeasy micro kit (Qiagen, Hilden, Germany) and reverse transcribed [1 µg total RNA, 1 µl random primer (50 µmol/l; Applied Biosystems, Foster City, CA, USA), 1X reverse transcriptase buffer, and 10 units reverse transcriptase (Promega Corp.) in a total volume of 20 µl]. The RNA and primer were heated to 72°C and slowly cooled before reverse transcription at 42°C for 1 h. The room temperature reaction was then diluted to 100 µl with RNase-free water (Applied Biosystems) as previously described (27). For quantitative PCR (qPCR), 2.5% of the total room temperature reaction was used as input for PCR using SYBR-Green Master Mix (Applied Biosystems) and templates were subjected to 36 rounds of PCR (94°C for 30 sec; 56°C for 30 sec; 72°C for 1 min) on an ABI 7900 Real-Time PCR system. As previously described (28–30), the following primers were used: podocin forward, 5′-GGCTGTGGAGGCTGAAGC-3′ and reverse 5′-CTCAGAAGCAGCCTTTTCCG-3′; nephrin forward, 5′-CGGTACAGGATCTGGCTGTT-3′ and reverse 5′-CTCTCTCCACCTCGTCATACA-3′; podocalyxin forward, 5′-CTACGGACTCATCTAACAAA-3′ and reverse, 5′-AGATAACC GATGACGGTA-3′; desmin forward, 5′-CAGAATTGAATCTCTCAACG-3′ and reverse, 5′-CTTCAGAAATGTTCTTAGCC-3′; CD2AP forward, 5′-AACAGATACCGAAGGTAAAA-3′ and reverse, 5′-ATCAAATGGATTATCTCCCC-3′ and GAPDH forward, 5′-TGGTCACCAGGGCTGCTT-3′ and reverse, 5′-AGCTTCCCGTTCTCAGCCTT-3′. The 2^−ΔΔCt method was used to calculate the relative amount of the target RNAs, as previously described (31).

Data are expressed as the means ± SEM from at least 3 independent experiments. Statistical significance was determined by one-way ANOVA with Bonferroni correction for thye comparison of 3 groups or the Student's t-test for the comparison of 2 groups.

Accumulating evidence has indicated that podocyte apoptosis is an early step in the progression of DN (15–17). We thus hypothesized that NR1 may exert protective effects on podocytes by inhibiting apoptosis. Cell viability was measured to determine the dose response of podocytes to NR1 (Fig. 1A). NR1 at the concentration of 20 µM was proven to be effective in protecting the podocytes, as evidenced by a significant increase in cell viability compared to the group exposed to high glucose. Subsequent time-course experiments using 20 µM NR1 revealed that NR1 treatment increased cell viability significantly after 8 h and resulted in a 22.7% increase at 24 h (Fig. 1B). NR1 restored cell viability in the HG-treated podocytes, indicating its protective effects on podocytes. To induce significant changes in cell viability, NRI treatment at 20 µM for 24 h of the HG-treated podocytes was used in all subsequent experiments.

To determine whether NR1 protects podocytes from apoptosis, cell morphology was assessed under a light miscroscope (Fig. 1C). The podocytes in the HG group exhibited marked apoptotic morphological changes, including membrane blebbing, cell volume reduction and rounding. These morphological changes were attenuated in the podocytes in the NR1 group (Fig. 1C). Moreover, the percentage apoptosis was measured by Hoechst 33342 staining (Fig. 2A and B). As shown in Fig. 2A, considerably greater DNA fragmentation in the nuclei was observed in the podocytes in the HG group than in those in the LG group, and normal, round and homogeneously stained nuclei were observed in the NR1-treated podocytes. Statistical analysis also indicated that exposure to high glucose resulted in increased apoptosis events that were attenuated by NR1 (Fig. 2B). There was no statistically significant difference in the percentage apoptosis between the cells in the LG group and those in the NR1 group. In order to validate the results obtained by Hoechst 33342 staining, Annexin V-PI staining and FACS analysis were performed as a second independent method to measure apoptosis (Fig. 3). The FACS data revealed similar results as the Hoechst staining. In addition, TUNEL staining was performed as a third independent method to measure apoptosis (Fig. 4A), and the numbers of TUNEL-positive cell percentages were manually calculated (Fig. 4B). Consistent with the other two methods, the TUNEL staining results also demonstrated that high glucose induced the apoptosis of podocytes and that this effect was reversed by NR1. Taken together, these data convincingly demonstrated that NR1 inhibits podocyte apoptosis in vitro.

It has been previously demonstrated that the slit diaphragm bridges adjacent foot processes and functions as the ultimate molecular size filter (32). Given the clinical significance of the slit diaphragm proteins, we further analyzed the expression levels of podocin, nephrin and CD2AP. As shown in Fig. 5A, the expression levels of these proteins were significantly decreased under high glucose conditions and NR1 reversed this effect. Consistent with the results of western blot analysis, NR1 also restored the mRNA levels of podocin, nephrin and CD2AP in the HG-treated podocytes (Fig. 5B). As PCX inhibits cell-matrix interactions (33), and desmin is a marker of podocyte injury (34,35), the protein expression of PCX and desmin was assessed by western blot analysis. Exposure to high glucose enhanced the expression levels of PCX and desmin in podocytes, while NR1 inhibited these effects (Fig. 5).

To further determine how NR1 inhibits podocyte apoptosis, podocytes in the LG, HG and NR1 groups were stained with cleaved caspase-3 antibody. The results revealed that high glucose contributed to podocyte apoptosis by increasing cleaved caspase-3 expression, while NR1 significantly inhibited the expression of cleaved caspase-3 (Fig. 6A). Furthermore, caspase-3 activity assay (Fig. 6B) revealed that high glucose enhanced caspase-3 activity in the podocytes, while NR1 significantly attenuated this effect. Western blot analysis of proteins involved in the apoptotic pathway (Fig. 7) revealed decreased expression levels of pro-caspase-3 and -9, and increased expression levels of cleaved caspase-3 and -9 in the HG group podocytes, indicating the activation of caspase-9 and caspase-3. However the cleavage of caspase-3 and -9 was partially inhibited by NR1. In addition, the upregulation of the levels of pro-apoptotic Bax and Bid proteins, as well as the downregulation of the anti-apoptotic Bcl-2 and Bcl-xL proteins were observed in the podocytes in the HG group; however, NR1 downregulated the expression levels of Bax and Bid, and upregulated the expression levels of Bcl-2 and Bcl-xL in the HG-treated podocytes. Further experiments of the PARP cleavage product indicated that the exposure of the podocytes to HG increased the cleavage of PARP, and treatment with NR1 resulted in a substantial decrease in the cleavage of PARP. Taken together, our data demonstrate that NR1 significantly attenuates HG-induced podocyte apoptosis via the caspase-dependent pathway.

Increasing evidence indicates that podocytes have a higher level of constitutive autophagy than other intrinsic renal cells do, and we thus investigated the expression levels of autophagy-related proteins. Western blot ananlysis (Fig. 8A) revealed that HG increased the expression of LC3-II, and decreased the expression of LC3-I and Beclin 1 in podocytes. However, NR1 significantly reversed these effects on these proteins in the HG-treated cells to levels comparable to those of the LG group. To further investigate the role of autophagy in NR1 treatment, Beclin 1 was knocked down by siRNA (Fig. 8B). By measuring the MDC fluorescence intensity in the presence of Beclin 1 siRNA, we found that the silencing of Beclin 1 resulted in reduced autophagy in the NR1-treated podocytes (Fig. 8C). To further evaluate whether NR1 induces autophagy, the chemical autphagy inhibitor, 3-MA, was added to the medium of podocytes in each group. The MDC fluorescence intensity of the podocytes treated with 3-MA also revealed that 3-MA inhibited NR1-induced autophagy, while 3-MA had no effect on the HG-treated podocytes (Fig. 8D). These observations suggested that NR1 increased the basal autophagy that was inhibited by HG.

In order to further explore the mechanisms through which NR1 inhibits apoptosis and increases autophagy, we investigated whether the PI3K/Akt/mTOR signaling pathway is involved in these mechanisms. As shown in Fig. 9, the phosphorylation levels of PI3K (p85), Akt, mTOR and p70S6K and 4E-BP1, which are downstream genes of mTOR, were markedly decreased in the podocytes in the HG group. However, NR1 increased the phosphorylation levels of these proteins, indicating that the activation of PI3K/Akt/mTOR pathway may be involved in NR1 induced podocyte protection. These data suggested that the NR1-induced protective effects on podocytes were dependent on the phosphorylation and activation of the PI3K/Akt/mTOR pathway.