Early diabetic kidney disease (DKD) is marked by dramatic metabolic reprogramming due to nutrient excess, mitochondrial dysfunction, and increased renal energy requirements from hyperfiltration. We hypothesized that changes in metabolism in DKD may be regulated by Sirtuin 5 (SIRT5), a deacylase that removes post-translational modifications derived from acyl-coenzyme A and has been demonstrated to regulate numerous metabolic pathways. We found decreased malonylation in kidney cortex (~80% proximal tubules) of type 2 diabetic BKS db/db mice, associated with increased SIRT5 expression. Proteomics analysis of malonylated peptides found that proteins with significantly decreased malonylated lysines in the db/db cortex were enriched in non-mitochondrial metabolic pathways: glycolysis and peroxisomal fatty acid oxidation (FAO). To confirm relevance of these findings in human disease, we analyzed diabetic kidney transcriptomic data from a cohort of Southwestern American Indians which revealed tubulointerstitial specific increase in Sirt5 expression. Overexpression of SIRT5 in cultured human proximal tubules demonstrated increased aerobic glycolysis with reduced mitochondrial metabolism, and conversely with decreased SIRT5 expression, there was reduced glycolysis and increased mitochondrial metabolism. These findings suggest that SIRT5 may lead to differential nutrient partitioning and utilization in DKD. Our findings highlight a previously unrecognized role for SIRT5 in metabolic reprogramming in DKD.

Peptides were dissolved in 25 µL 0.1% FA and 10 µL of the sample was injected for analysis. Peptides were separated on reverse phase Thermo Scientific Acclaim PepMap 100: 75 µm x 2 cm (C18, 3 µm bead, 100 Å pore size) trap column and Thermo Scientific Acclaim PepMap C18, 2 µm particle size, RSLC 75 µm x 25 cm column. Data were acquired with Orbitrap QExactive coupled to Thermo Scientific Easy nLC-1000 UHPLC. Samples were run using a 90 min gradient from 5% “B” to 35% “B”. Buffer “A” was 0.1% FA in water and buffer “B” was 0.1% FA in acetonitrile. All data were acquired in positive mode with a lock mass of 445.12002 using data dependent acquisition with a 15 sec dynamic exclusion. MS1 data were acquired in profile mode at a resolution of 70,000 with a maximum time of 75 msec and a range of 350 to 1500 m/z. MS2 spectra were acquired in profile mode at a resolution of 15,000 and maximum integration time of 75 msec. The isolation window was 1.6 m/z and the collision energy was 20.

9.5 mg of peptides from 5 db/+ and 5 db/db mice kidney cortex were affinity enriched for malonylated peptides by incubation with anti-malonyllysine antibody conjugated to Dynabeads in IAP buffer (50 mM MOPS–NaOH, pH 7.2, 10 mM Na2HPO4 50 mM NaCl) with 0.1% NP-40 at 4 °C for 24 hrs. Pre-selection, 250 ρg of heavy-labeled Malonyl-lysine (Kmal) peptide standards were added to each sample. The Kmal heavy-labeled peptide standard was synthesized by the University of Michigan peptide core. The synthesized sequence was as follows: H2N-TV*DGPSG(K/Malonyl)LWR-OH (V*: Valine 13C5, 15N). Beads were washed twice with IAP + 0.1% 0.1% NP-40 buffer, twice with IAP and once with water. Samples were eluted with 0.1% TFA in water three times. Samples were desalted using desalting spin column as per manufacturer’s directions. Peptides were dried in a speed-vac for 1 hr and samples were stored at -80 °C until analysis.