Diabetes Metab J.  2020 Aug;44(4):581-591. 10.4093/dmj.2019.0063.

Inhibition of Ceramide Accumulation in Podocytes by Myriocin Prevents Diabetic Nephropathy

• Woo CY
• Baek JY
• Kim AR
• Hong CH
• Yoon JE
• Kim HS
• Yoo HJ
• Park TS
• Kc R
• Lee KU
• Koh EH

• KMID: 2513017
• DOI: http://doi.org/10.4093/dmj.2019.0063

Abstract

Ceramides are associated with metabolic complications including diabetic nephropathy in patients with diabetes. Recent studies have reported that podocytes play a pivotal role in the progression of diabetic nephropathy. Also, mitochondrial dysfunction is known to be an early event in podocyte injury. Thus, we tested the hypothesis that ceramide accumulation in podocytes induces mitochondrial damage through reactive oxygen species (ROS) production in patients with diabetic nephropathy.

We used Otsuka Long Evans Tokushima Fatty (OLETF) rats and high-fat diet (HFD)-fed mice. We fed the animals either a control- or a myriocin-containing diet to evaluate the effects of the ceramide. Also, we assessed the effects of ceramide on intracellular ROS generation and on podocyte autophagy in cultured podocytes.

OLETF rats and HFD-fed mice showed albuminuria, histologic features of diabetic nephropathy, and podocyte injury, whereas myriocin treatment effectively treated these abnormalities. Cultured podocytes exposed to agents predicted to be risk factors (high glucose, high free fatty acid, and angiotensin II in combination [GFA]) showed an increase in ceramide accumulation and ROS generation in podocyte mitochondria. Pretreatment with myriocin reversed GFA-induced mitochondrial ROS generation and prevented cell death. Myriocin-pretreated cells were protected from GFA-induced disruption of mitochondrial integrity.

We showed that mitochondrial ceramide accumulation may result in podocyte damage through ROS production. Therefore, this signaling pathway could become a pharmacological target to abate the development of diabetic kidney disease.

Keyword

Diabetic nephropathies; Ceramides; Mitochondria; Podocytes; Reactive oxygen species; Thermozymocidin

Figure

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  Fig. 1 Myriocin prevented albuminuria and podocyte injury in Otsuka Long Evans Tokushima Fatty (OLETF) rats. OLETF rats at 18 weeks of age were treated with myriocin for 4 weeks. (A) Twenty-four hours urinary albumin excretion. Data are presented as mean±standard error of the mean (n=7). (B) Kidney cortex ceramide concentration determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) (n=5). (C) Representative images of immunostaining for periodic acid-Schiff (PAS) and desmin, a marker of podocyte injury and epithelial-mesenchymal transition. Scale bars, 50 µm (n=3). Con, control. aP<0.050.01 vs. Long Evans Tokushima Otsuka (LETO) rats, bP<0.050.05 vs. untreated OLETF rats.

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  Fig. 2 Treatment with myriocin prevented albuminuria and podocyte injury in high-fat diet (HFD)-fed mice. (A) Twenty-four hours urinary albumin excretion (n=7). Data are presented as mean±standard error of the mean. (B) Kidney cortex ceramide concentration determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) (n=5). (C) Representative images of immunostaining for periodic acid-Schiff (PAS) and desmin. Scale bars, 50 µm (n=3). (D) The panels show representative images of the immunofluorescence analysis of glomeruli from control, HFD diet, and HFD-fed treated myriocin (HFD+Myr) mice. Myriocin restored the nephrin expression in HFD-fed mice. Green fluorescence denotes nephrin in the glomeruli, and blue fluorescence (DAPI) is indicative of the presence of a nucleus (×300). Con, control. aP<0.050.01 vs. control diet mice, bP<0.050.05 vs. untreated HFD-fed mice.

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  Fig. 3 Glucose, free fatty acid, and angiotensin II (GFA) treatment induced podocyte apoptosis. Myriocin pretreatment prevented GFA-induced ceramide accumulation and alleviated the deleterious effects of GFA. (A) Apoptosis was determined at 4 hours using a Cell Death enzyme-linked immunosorbent assay (ELISA) kit (n=5). (B) Representative immunoblots for cleaved caspase-3 and caspase-9 (n=5). (C) Ceramide concentration determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) (n=5). (D) Cell viability measured by the cell counting kit 8 (CCK-8) method (n=5). Data are presented as mean±standard error of the mean. Con, control; OD, optical density. aP<0.050.05 vs. untreated cells, bP<0.050.01 vs. GFA-treated cells.

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  Fig. 4 Myriocin decreased mitochondrial reactive oxygen species production through decreased ceramide accumulation and prevented glucose, free fatty acid, and angiotensin II (GFA)-induced mitochondrial morphological alterations. (A) Immunofluorescence corresponding to MitoSOX (red), ceramide (green), and nucleus (blue). Scale bars, 50 µm (n=3). (B) Transmission Electron Microscope (TEM) of podocyte mitochondria. Scale bars, 1 µm. We pretreated podocytes for 24 hours with or without 1 µM myriocin in serum-free medium and then incubated them with GFA for 4 hours (n=4).

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