Palmitate-mediated lipotoxicity is known to induce mesangial cell apoptosis [8,9]. As expected, palmitate treatment increased caspase-3 cleavage in cultured mesangial cells (Figure 1A). Moreover, palmitate treatment activated PERK signaling (PERK phosphorylation, eIF2α phosphorylation, and CHOP expression) and ATF6 signaling pathways (ATF6 cleavage) but did not activate the IRE1 signaling pathway (Figure 1B). To confirm whether ER stress signaling activation is linked to mesangial cell apoptosis, mesangial cells were treated with thapsigargin, which triggers ER stress by inhibiting sarcoplasmic/ER Ca²⁺-ATPase (SERCA), leading to Ca²⁺ depletion. Thapsigargin treatment increased caspase-3 cleavage, suggesting that ER stress signaling is involved in mesangial cell apoptosis (Figure 1C). To further confirm, we employed the PERK inhibitor GSK2606414, which blocks PERK phosphorylation, and the S1P inhibitor AEBSF, which blocks ATF6 cleavage. As shown in Figure 1D, palmitate-induced caspase-3 cleavage was attenuated by GSK2606414 and AEBSF treatment. In addition, palmitate-induced caspase 3/7 activity and the increased number of apoptotic cells were diminished by treatment with the two inhibitors (Figure 1E,F). These results suggest that lipotoxicity induces mesangial cell apoptosis via PERK and ATF6 signaling pathways.

Type I PRMTs, which induce asymmetric dimethyl arginine generation, are closely related to the progression of diabetic nephropathy [18]. Mesangial cells in the glomerulus are crucial for the maintenance of a normal glomerular filtration rate. Nevertheless, the expression of type I PRMTs in mesangial cells under pathologic conditions has not been explored. Palmitate treatment increased PRMT1 expression, whereas the expression of PRMT3 and PRMT4, which are also type I PRMTs, was not altered (Figure 2A). Immunofluorescence studies also revealed that PRMT1 expression is increased by palmitate treatment and that its expression is observed predominantly in the nuclei of mesangial cells (Figure 2B). Moreover, PRMT1 activity, determined by western blotting using an antibody for ASYM24, which recognizes asymmetrically dimethylated arginine, was elevated by palmitate treatment (Figure 2C).

To determine whether palmitate-induced ER stress and apoptosis is due to elevated PRMT1 expression, endogenous PRMT1 was silenced by prmt1 siRNA transfection without the alteration of other type I PRMTs. (Figure 3A). The knockdown of PRMT1 attenuated palmitate-induced ER stress signaling, except for IRE1 phosphorylation (Figure 3B). Moreover, palmitate-induced mesangial cell apoptosis was diminished by prmt1 siRNA transfection (Figure 3C–E). These results suggest that PRMT1 mediates palmitate-induced ER stress activation and mesangial cell apoptosis.

To confirm in vivo, we investigated the glomeruli of high fat diet (HFD) mice since HFD is known to contribute to intrarenal-lipotoxicity [8,19,20]. We used PRMT1 haploinsufficient (prmt1^+/−) mice to evaluate the roles of PRMT1 in vivo as PRMT1 knockout mice are embryonically lethal [21]. The number of apoptotic cells, as determined by a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, in the glomerulus was significantly elevated in mice fed HFD for 12 weeks (Figure 4A). However, HFD-induced glomerular apoptosis was significantly attenuated in prmt1^+/− mice compared with wild-type (WT; prmt1^+/+) mice (Figure 4A), implying that PRMT1 could have a significant function in glomerular apoptosis.

Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s nutrient mixture F-12, and fetal bovine serum (FBS) were purchased from Life Technologies (Gibco BRL, Grand Island, NY, USA). Palmitate and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The palmitate was prepared as described previously [30]. Thapsigargin and GSK2606414 were purchased from Millipore (Billerica, MA, USA). p-IRE antibody (ab48187), ATF6 antibody (ab11909), PRMT1 antibody (ab3768), and PRMT4 antibody (ab110024) were purchased from Abcam (Cambridge, UK). caspase-3 antibody (#9662), p-eIF2α antibody (#9721), and CHOP antibody (#2895) were purchased from Cell Signaling Technology (Beverly, MA, USA). ASYM24 antibody (#07-414) was obtained from Millipore. β-actin antibody (sc-1616) and p-PERK antibody (sc-32577-R) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All reagents were of the highest purity commercially available.

The rat mesangial cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in DMEM/Ham’s F-12 (1:1) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO₂ in air. Stock cultures of cells were subcultured once a week (split ratio 1:8). Cells were grown to confluence in 60 mm dishes in DMEM/Ham’s F-12 with 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer, 10% FBS, 5.5 mM glucose, 0.35% additional sodium bicarbonate, 2.5 mM l-glutamine, and 1% penicillin/streptomycin at 37 °C. The media was changed every other day. Passaged cells were plated to yield near-confluent cultures at the end of the experiments.

Cell pellets were lysed in Mammalian Protein Extraction Reagent (M-PER) (Thermo Fisher Scientific, Waltham, MA, USA). Protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail I + II (Sigma-Aldrich) were added, and then the proteins were extracted according to the manufacturer’s instructions. Western blotting was performed as described previously [17].

Caspase-3/7 activity was measured with the Caspase-Glo 3/7 Assay Kit (Promega, Madison, WI, USA). Mesangial cells were seeded in a white-walled 96-well plate and then treated with palmitate. After 24 h, the cells were processed according to the manufacture’s instruction. GloMax Microplate Reader (Promega) was used for luminescence analysis.

Annexin V/Propidium iodide staining was performed with a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Billerica, MA, USA) according to modified manufacturer’s instructions. Mesangial cells cultured in 6-well plates were washed with phosphate-buffered saline (PBS) and then filled with 1 mL of Accutase (Innovative Cell Technologies, San Diego, CA, USA). After two minutes, the plates were gently tapped to detach the cells. The resuspended cells were immediately washed twice with cold PBS and then resuspended in a pre-chilled binding buffer, which contained fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). After a seven minute incubation on ice in the dark, apoptotic cells were analyzed by flow cytometry (Accuri C6, BD Biosciences). FITC and PI double negative cells (LL; lower-left) were regarded as healthy cells. FITC-positive and PI-negative cells (LR; lower-right) were considered as early apoptotic cells, and FITC and PI double positive cells (UR; upper-right) were late apoptotic cells. FITC-negative and PI positive cells (UL; upper-left) were regarded as necrotic. Apoptotic cells were determined by the sum of early apoptotic cells and late apoptotic cells (LR + UR; red frame).

The cells were washed twice in PBS and fixed for 10 min with 4% paraformaldehyde in PBS. After three washes in PBS, fixed cells were permeabilized with 0.2% Triton X-100. 1% bovine serum albumin (BSA) solution was used for blocking. The cells were incubated with PRMT1 antibody (dilution ratio—1:100) for 15 h at 4 °C. After three washes in PBS, the cells were incubated with anti-rabbit FITC secondary antibody (Sigma-Aldrich). Then the cells were mounted on slides and the nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) present in the ProLong Gold Antifade Mounting Medium (Invitrogen, Carlsbad, CA, USA). Immunofluorescence imaging was performed as described previously [17].

siRNA for PRMT1 (Santa Cruz Biotechnology; sc-41069) and scrambled siRNA (Quiagen, Hilden, Germany) were used for silencing the endogenous PRMT1 expression. 40 nM of each siRNA was transfected into mesangial cells using Lipofectamin™ RNAiMAX reagent (Invitrogen), following the reverse transfection method, as instructed by the manufacturers.

Animal experiments were performed in accordance with the “Guide for Animal Experiments” (Edited by the Korean Academy of Medical Sciences) and approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU121119-3; 19 November 2012). PRMT1 haploinsufficiency mice were kindly provided by Seung-Hoi Koo (Department of Life Science, Korea University, Seoul, Korea). Twelve male C57BL/6J mice were separated into two groups and age matched; six PRMT1 haploinsufficiency mice were one group. The normal diet group was challenged with 10% kcal fat containing feed (Research Diets, NJ, USA; D12450B) and the high fat diet group was challenged with 60% kcal fat containing feed (Research Diets, NJ, USA; D12492) for 12 weeks. After 12 weeks, all the mice were sacrificed and the kidneys were extracted. To make a tissue slide, fixed kidneys in 10% neutral buffered formalin were embedded in paraffin and cut at a 5 μm thickness using a microtome. The kidney sections were deparaffinized with Histochoice, rehydrated with serial diluted ethanol, and washed with distilled water. Staining was performed using the DeadEnd™ Fluorometric TUNEL System (Promega) according to the manufacturer’s instructions. The stained tissues were mounted on slides, and the nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) present in the ProLong Gold Antifade Mounting Medium (Invitrogen) and observed using a fluorescence microscope (Eclipse Ni-U, Intensilight C-HGFI, Nikon, Japan).

The results were expressed as the mean ± SEM. Data are representative of three or four independent experiments. For two group comparisons, a student t test was used, and for multiple comparisons, one-way ANOVA by SPSS (SPSS Inc., Chicago, IL, USA), followed by the Tukey post hoc test, was used. A value of p < 0.05 was considered significant.