Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Daniel L. Galvan, Jianyin Long, Nathanael Green, Benny H. Chang, Jamie S. Lin, Paul Schumacker, Luan D. Truong, Paul Overbeek, Farhad R. Danesh
Daniel L. Galvan, Jianyin Long, Nathanael Green, Benny H. Chang, Jamie S. Lin, Paul Schumacker, Luan D. Truong, Paul Overbeek, Farhad R. Danesh
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Research Article Metabolism Nephrology

Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice

Abstract

Phosphorylation of dynamin-related protein 1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here, we established the role of Drp1 serine 600 (Drp1S600) phosphorylation in mitochondrial fission in vivo and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in the progression of diabetic nephropathy (DN). We generated a knockin mouse in which S600 was mutated to alanine (Drp1S600A). We found that diabetic Drp1S600A mice exhibited improved biochemical and histological features of DN along with reduced mitochondrial fission and diminished mitochondrial ROS in vivo. Importantly, we observed that the effect of Drp1S600 phosphorylation on mitochondrial fission in the diabetic milieu was stimulus dependent but not cell type dependent. Mechanistically, we show that mitochondrial fission in high-glucose conditions occurs through concomitant binding of phosphorylated Drp1S600 with mitochondrial fission factor (MFF) and actin-related protein 3 (Arp3), ultimately leading to accumulation of F-actin and Drp1 on the mitochondria. Taken together, these findings establish the idea that a single phosphorylation site in Drp1 can regulate mitochondrial fission and progression of DN in vivo and highlight the stimulus-specific consequences of Drp1S600 phosphorylation in mitochondrial dynamics.

Authors

Daniel L. Galvan, Jianyin Long, Nathanael Green, Benny H. Chang, Jamie S. Lin, Paul Schumacker, Luan D. Truong, Paul Overbeek, Farhad R. Danesh

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Figure 2

Drp1S600A mutation protects against progression of DN.

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Drp1S600A mutation protects against progression of DN. (A) Breeding sch...
(A) Breeding scheme to obtain diabetic Drp1S600A-mutant mice and representative images of diabetic mice of each genotype. (B) Immunostaining of glomeruli from diabetic Drp1, WT, and homozygous mutant mice. Glomeruli were stained with an antibody against the p-Drp1 (S637) site (green). Podocytes were identified with an antibody against podocin (red), and nuclei were stained with DAPI (blue). Images were acquired by confocal microscopy (original magnification, ×60). (C) ACR analysis of nondiabetic db/m mice,Drp1 WT mice, and mice of the 3 diabetic genotypes at 12, 16, and 20 weeks demonstrating a significant reduction in albuminuria in 16- and 20-week-old diabetic Drp1S600A heterozygous and homozygous mutant mice compared with diabetic db/db mice. (D) Body weight and (E) blood glucose levels at different time points for the groups described in C. (F) Representative PAS-stained images (scale bar: 50 μm), TEM images (scale bar: 500 nm), SEM images (scale bar: 10 μm), and SEM inset images (scale bar: 1 μm) of kidney glomeruli from mutant allele mice. Images are from a sampling of 3 to 5 animals. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple comparisons test. Results are presented as the mean ± standard error of the mean (n = 3–8/group).