Altered retinoic acid metabolism in diabetic mouse kidney identified by O isotopic labeling and 2D mass spectrometry

Abstract

Background: Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes.

Methodology/principal findings: Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, (18)O- and (16)O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change >or=1.5 and p<or=0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARbeta/delta mRNA.

Conclusions/significance: Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy.

Figure 1. Retinoic acid is a key signaling hub in the highest ranked network generated by IPA.

Protein-protein associations are indicated by edges containing single lines, whereas proteins that act upon another protein (controlling their expression) are indicated by arrows. Nodes are represented by shapes and colors: Shapes indicate function: enzymes (diamond), transcription regulators (oval), nuclear receptors (rectangle), cytokines (square), transporter (trapezoid), and “other” (circles). The red color indicates those proteins that are significantly increased in abundance in the diabetic kidney while green indicates those that are significantly decreased. The intensity of the color represents the degree of change. Abbreviations of proteins identified as significantly changed from the mass spectrometry dataset (indicated in green and red) are: GGT1, gamma-glutamyltransferase 1; ACOX1, acyl-Coenzyme A oxidase 1; CYP4B1, cytochrome P450, family 4, subfamily B, polypeptide 1; ADH1C, alcohol dehydrogenase 1C; CANX, calnexin; PAH, phenylalanine hydroxylase; EEF, elongation factor; GSTA5, glutathione S-transferase alpha 5.

Figure 2. Real time-PCR and western blot of RALDH1 (ALDH1A1) and real time-PCR of ADH.

Real time-PCR was analyzed using the ΔCT method and results are scaled to the mean control values ±SE (n = 7). Western blots were performed in duplicate and normalized to β-actin (n = 7) and results are represented as mean±SE. A. RNA was extracted from 50 mg of kidney cortical tissue and cDNA synthesis performed according to the manufacturer's instructions. Relative quantification of RALDH1 and ADH mRNA was performed using a MyiQ Single-Color Real-Time PCR Detection System. Control (n = 4) and diabetic (n = 7) results are normalized to the expression level of cyclophilin within each animal, and then plotted as fold change compared to the average control value. B. RALDH1 Western blots. Total tissue extracts were prepared from renal cortex of 3-month diabetic and age-matched control mouse kidneys. An equal amount of protein (50 µg/lane) was used for all animals, and representative control (lanes 1–4) and db/db (lanes 5–8) Western blots are shown. C. Densitometric quantification of RALDH1 for 7 control and 7 diabetic mice. Results are expressed as a mean and SD of the control and diabetic band intensities after normalization to β actin in each lane. Student's t-test: *p<0.005; ^†p<0.001; ^‡p<0.01.

Figure 3. Immunohistochemical staining of RALDH1.

Representative 5 µm formalin-fixed sections of kidney are shown from control (panels A and C) and db/db (panels B and D) mice. Panels A and B are low magnification images of the entire kidney section scanned using the LI-COR Bioscience Odyssey™ Imaging System while higher power confocal images are shown in Panels C and D. Negative controls included omitting the primary antibody, rabbit monoclonal anti-RALDH1 from Novus Biologicals (data not shown). Asterisks indicate glomeruli. Magnification for panels C and D, 300×.

Figure 4. Quantification of retinoids extracted from tissue and plasma samples.

Retinoids were quantified by LC/MS/MS as described , , while retinol and retinyl ester were quantified by HPLC/UV as described . Tissue retinoids are expressed as mol/g tissue and plasma retinoids are expressed as mol/mL plasma. Student's t-test: *, p<0.05; ^†p<0.005; ^‡p<0.0001.

Figure 5. Western immunoblots of CYP2E1 and CYP26A1.

Total tissue extracts were prepared from renal cortex and liver of 3-month diabetic and age-matched control mouse kidneys. An equal amount of protein (50 µg/lane) was used for all animals, and representative control (lanes 1–4) and db/db (lanes 5–8) tissue extracts are shown on the left. Densitometric quantitation of 7 control and 7 db/db kidneys and livers are shown on the right. Due to group differences in the expression level of β-actin caused by diabetes, results were not normalized to the loading control. Student's t-test: * p<0.02.

Figure 6. Real time-PCR of retinoid binding proteins and atRA-related nuclear receptors.

Relative quantification of select mRNA was performed using a MyiQ Single-Color Real-Time PCR Detection System and iQ SYBR Green Supermix according to the manufacturer's instructions. All primers were purchased from SABiosciences, Inc. Data were analyzed using the ΔCT method in reference to cyclophillin or GAPDH. Controls, n = 7; db/db, n = 6. Significantly different from controls by Student's t-test: * p<0.005.