Regulation of podocyte survival and endoplasmic reticulum stress by fatty acids

Abstract

Apoptosis of podocytes is considered critical in the pathogenesis of diabetic nephropathy (DN). Free fatty acids (FFAs) are critically involved in the pathogenesis of diabetes mellitus type 2, in particular the regulation of pancreatic β cell survival. The objectives of this study were to elucidate the role of palmitic acid, palmitoleic, and oleic acid in the regulation of podocyte cell death and endoplasmic reticulum (ER) stress. We show that palmitic acid increases podocyte cell death, both apoptosis and necrosis of podocytes, in a dose and time-dependent fashion. Palmitic acid induces podocyte ER stress, leading to an unfolded protein response as reflected by the induction of the ER chaperone immunoglobulin heavy chain binding protein (BiP) and proapoptotic C/EBP homologous protein (CHOP) transcription factor. Of note, the monounsaturated palmitoleic and oleic acid can attenuate the palmitic acid-induced upregulation of CHOP, thereby preventing cell death. Similarly, gene silencing of CHOP protects against palmitic acid-induced podocyte apoptosis. Our results offer a rationale for interventional studies aimed at testing whether dietary shifting of the FFA balance toward unsaturated FFAs can delay the progression of DN.

Fig. 1.

Palmitic acid induces apoptosis and necrosis of podocytes in a dose-dependent manner. A: representative flow cytometry results for podocytes exposed to increasing concentrations of palmitic acid (125–500 μM) or BSA (at a concentration equivalent to cells treated with 500 μM palmitic acid complexed to BSA) for 38 h. The abscissa and ordinate represent the fluorescence intensity of annexin V Alexa 647 and propidium iodide (PI), respectively. B: quantitative analysis of palmitic acid-induced podocyte cell death. Bar graph represents the mean percentages ± SD of annexin V-positive/PI-negative (early apoptotic) and annexin V-positive/PI-positive (late apoptotic/necrotic) podocytes (n = 3). *P < 0.05, **P < 0.01.

Fig. 2.

Palmitic acid induces time-dependent podocyte apoptosis. A: palmitic acid-induced podocyte cell death was determined by annexin V/PI staining followed by flow cytometry. B: quantitative analysis. Bar graph represents the mean fold-increase ± SD in annexin V-positive/PI-negative (apoptotic) cells incubated with palmitic acid or BSA for indicated time points (n = 3). *P = 0.05, **P < 0.05, ***P < 0.01. C: bar graph representing the mean fold-increase ± SD in annexin V-positive/PI-positive (necrotic) cells incubated with palmitic acid or BSA for indicated time points (n = 3). **P < 0.05, ***P < 0.01. D: bar graphs representing intact cells recovered in cell pellets (= total cells in culture dishes minus floating cellular debris in supernatant after centrifugation at 550 g), which were used for flow experiments shown in A–C. Palmitic acid significantly decreased the number of recovered cells after 34 and 48 h (**P < 0.05), reflecting the increase in necrotic cellular debris that could not be recovered in cell pellets.

Fig. 3.

Palmitic acid activates caspase 3 in podocytes. Western blot analysis of active, cleaved caspase 3 protein steady-state levels in podocytes exposed to staurosporine (stauro), fatty acid-free BSA (BSA), or palmitic acid (palm) is shown. A: 0.25 μM staurosporine (serving as a positive control) and palmitic acid caused a strong activation of caspase 3 after 16 h. No signal was seen after 1-h exposure to palmitic acid or after treatment with BSA (negative control). β-Actin served as a loading control. Representative results of 3 independent experiments are shown. B: dose-dependent activation of caspase 3 by palmitic acid. For control condition (BSA), the BSA concentration equivalent to cells exposed to 500 μM palmitic acid complexed to BSA was used. β-Actin served as a loading control. Representative results of 2 independent experiments are shown.

Fig. 4.

Palmitic acid induced the endoplasmic reticulum (ER) chaperone immunoglobulin heavy chain binding protein (BiP) and C/EBP homologous protein (CHOP) in podocytes. A: palmitic acid-induced upregulation of BiP (top) and quantitative analysis of BiP levels normalized to β-actin (bottom). The expression level of the control condition (BSA) was set to 100%; n = 4. *P < 0.01. B: palmitic acid induced upregulation of CHOP (top) and quantitative analysis of CHOP levels normalized to β-actin (bottom). The expression level of the control condition (BSA) was set to 100%; n = 4. *P < 0.01. C: time- and dose-dependent upregulation of CHOP protein levels by palmitic acid. For the control condition (BSA), the BSA concentration was equivalent to cells exposed to 500 μM palmitic acid complexed to BSA. β-Actin served as a loading control. Representative results of 3 independent experiments are shown. D: RT-PCR of X-box binding protein-1 (XBP-1) mRNA in podocytes treated with 500 μM palmitic acid for 12 h. Tunicamycin (Tn; 5 ng/ml) was used as a positive control. In the control condition (BSA), a strong band for unspliced (u) XBP-1 is visible, whereas sXBP-1 and hXBP-1 are strongly increased after treatment with palmitic acid or tunicamycin. Representative results of 3 independent experiments. E. BiP and CHOP expression after exposure for 24 h as follows. Lane 1, control medium containing 5 mM glucose and fatty acid free BSA (BSA); lane 2, control medium (BSA) supplemented with additional 17 mM mannitol (M); lane 3, control medium (BSA) supplemented with additional 17 mM glucose [high glucose (HG)]; lane 4, BSA complexed with 500 μM palmitic acid (palm); lane 5, palmitic acid (palm) and HG; lane 6, 5 ng/ml TGF-β with HG; lane 7, 5 ng/ml TGF-β; lane 8, 0.25 μM staurosporine (stauro). Upregulation of CHOP is only seen with palmitic acid, whereas BiP is induced by palmitic acid and TGF-β (faint band also for HG and M). β-Actin served as a loading control. Representative results of 4 independent experiments are shown.

Fig. 5.

Palmitoleic or oleic acid prevent palmitic acid-induced podocyte death and attenuate CHOP induction. A: palmitoleic acid blocks palmitic acid-induced podocyte cell death (n = 3, *P < 0.05, **P < 0.01, compared with palmitic acid). B: palmitoleic acid blocks palmitic acid-induced upregulation of BiP and CHOP protein steady-state levels. β-Actin served as a loading control. Representative result of 2 independent experiments is shown. C: oleic acid blocks palmitic acid-induced podocyte cell death (n = 3, *P < 0.01 compared with palmitic acid). D: oleic acid blocks palmitic acid-induced upregulation of BiP and CHOP protein steady-state levels. β-Actin served as a loading control. Representative result of 2 independent experiments is shown.

Fig. 6.

Gene silencing of CHOP protects against palmitic acid-induced podocyte cell death. A: gene-silencing of CHOP suppresses the tunicamycin-induced upregulation of BiP and CHOP protein levels. β-Actin served as a loading control. Representative result of 3 independent experiments are shown. B: quantitative analysis showing a significant reduction of tunicamycin-induced upregulation of CHOP and BiP in cells infected with CHOP short-hairpin (sh) RNA (*P < 0.01, **P < 0.001). C: gene silencing of CHOP suppresses the palmitic acid-induced upregulation of CHOP and BiP protein levels. Representative result of 3 independent experiments is shown. D–F: gene silencing of CHOP protects against palmitic acid-induced apoptosis (D) and overall increase in cell death (F).