Inhibition of Aerobic Glycolysis Attenuates Disease Progression in Polycystic Kidney Disease

Abstract

Dysregulated signaling cascades alter energy metabolism and promote cell proliferation and cyst expansion in polycystic kidney disease (PKD). Here we tested whether metabolic reprogramming towards aerobic glycolysis ("Warburg effect") plays a pathogenic role in male heterozygous Han:SPRD rats (Cy/+), a chronic progressive model of PKD. Using microarray analysis and qPCR, we found an upregulation of genes involved in glycolysis (Hk1, Hk2, Ldha) and a downregulation of genes involved in gluconeogenesis (G6pc, Lbp1) in cystic kidneys of Cy/+ rats compared with wild-type (+/+) rats. We then tested the effect of inhibiting glycolysis with 2-deoxyglucose (2DG) on renal functional loss and cyst progression in 5-week-old male Cy/+ rats. Treatment with 2DG (500 mg/kg/day) for 5 weeks resulted in significantly lower kidney weights (-27%) and 2-kidney/total-body-weight ratios (-20%) and decreased renal cyst index (-48%) compared with vehicle treatment. Cy/+ rats treated with 2DG also showed higher clearances of creatinine (1.98±0.67 vs 1.41±0.37 ml/min), BUN (0.69±0.26 vs 0.40±0.10 ml/min) and uric acid (0.38±0.20 vs 0.21±0.10 ml/min), and reduced albuminuria. Immunoblotting analysis of kidney tissues harvested from 2DG-treated Cy/+ rats showed increased phosphorylation of AMPK-α, a negative regulator of mTOR, and restoration of ERK signaling. Assessment of Ki-67 staining indicated that 2DG limits cyst progression through inhibition of epithelial cell proliferation. Taken together, our results show that targeting the glycolytic pathway may represent a promising therapeutic strategy to control cyst growth in PKD.

Fig 1. Dysregulation of the glycolysis and gluconeogenesis pathways in rat polycystic kidney disease.

(A) Microarray analysis showing differential expression of genes coding for glycolysis and gluconeogenesis enzymes in Han:SPRD Cy/+ and wild-type +/+ kidneys. Upregulated genes are shown in red, and downregulated genes are shown in green. (B) Schematic diagram showing the glycolysis/gluconeogenesis cascades. In red, upregulated genes; green, downregulated genes; black, genes unchanged in kidneys from Cy/+ rats compared with wild-type +/+ kidneys. (C) Real-time quantitative PCR analysis of genes coding for key enzymes involved in glycolysis/gluconeogenesis in kidneys from Cy/+ rats and +/+ rats. (D) Real-time quantitative PCR analysis of the hexokinase-1 (Hk1) and hexokinase-2 (Hk2) genes in primary cell cultures of human ADPKD and control NHK cells. The expression levels of β-actin were used as a housekeeping gene.

Fig 2. Increased glycolytic phenotype in polycystic kidney disease.

(A) Intracellular ATP content in primary cell cultures of tubular epithelial cells isolated from kidneys of Cy/+ and +/+ rats. (B) Lactate concentration in the medium of primary cell cultures of tubular epithelial cells isolated from kidneys of Cy/+ and +/+ rats. (C) Cell growth of primary renal Cy/+ cells upon incubation with increasing concentrations of 2DG, as assessed by the MTS assay. (D) Intracellular ATP content in primary cell cultures of ADPKD and NHK cells. (E) Lactate concentration in the medium of primary cell cultures of ADPKD and NHK cells. (F) Effect of 2DG on cell proliferation of human ADPKD cells and control NHK cells, as quantified by BrdU assay. (G) Effect of 2DG on apoptosis of human ADPKD cells and control NHK cells, as analyzed with annexin-V/propidium iodide (PI) staining using flow cytometry.

Fig 3. Effect of 2DG treatment on kidney weight and morphology in Han:SPRD Cy/+ rats.

(A) Representative images of periodic acid-Schiff staining of kidneys from 10 week old +/+ and Cy/+ rats after 5-week treatment with 2DG or vehicle. (B) Ratio of total kidney weight (TKW) to body weight (BW) in 10 week old Cy/+ rats after 5-week treatment with 2DG or vehicle. (C). Cyst index in kidneys from Cy/+ rats after 5-week treatment with 2DG or vehicle. (D) Frequency distribution of the cyst size, and (E) total number of cysts, in kidneys from Cy/+ rats following 5-week treatment with 2DG or vehicle.

Fig 4. Effect of 2DG treatment on parameters of renal function.

Measurement of (A) creatinine clearance, (B) BUN clearance, and (C) uric acid clearance, in +/+ and Cy/+ rats upon treatment with 2DG or vehicle at baseline, 2.5 weeks and 5 weeks. Black, +/+ treated with vehicle; blue, +/+ treated with 2DG; red, Cy/+ treated with vehicle; green, Cy/+ treated with 2DG. *P<0.05, **P<0.01 when comparing Cy/+ 2DG and Cy/+ vehicle at each time point. ^#P<0.05, ^{# #} P<0.01 when comparing Cy/+ and +/+ group. (D) Urine albumin excretion in Cy/+ and +/+ rats after 5-week treatment with 2DG or vehicle. (E) SDS-polyacrylamide gel electrophoresis of urine samples from Cy/+ and +/+ rats after 5-week treatment with 2DG or vehicle. (F) Lactate content in the kidneys of Cy/+ and +/+ rats after treatment with 2DG or vehicle.

Fig 5. Effect of 2DG treatment on cell proliferation and apoptosis in Han:SPRD Cy/+ rats.

(A) Representative images of Ki67 staining of cyst-lining epithelium in kidneys of Cy/+ rats treated with 2DG or vehicle. (B) Quantification of Ki67-positive nuclei in kidneys of Cy/+ rats after 5-week treatment with 2DG or vehicle. (C) Western blot analysis of active caspase-3 in kidneys from Cy/+ rats after treatment with 2DG or vehicle.

Fig 6. Effect of 2DG treatment on cellular signaling pathways in vivo.

Measurements of phosphorylation levels of (A) AMPK, (B) ERK, (C) S6K, and (D) Akt using Western blot analysis in the kidneys of 10 week old +/+ and Cy/+ rats following 5-week treatment with 2DG or vehicle.

Fig 7. Effect of selected inhibitors on the glycolysis pathway in vitro.

Real-time quantitative PCR analysis of the genes coding for glycolytic enzymes (A) Hk1, (B) Hk2, and (C) Pkm2 in primary cell cultures of human ADPKD cells following incubation with rapamycin (50 nM) or metformin (2 mM). (D) Lactate production in the supernatant of NHK and ADPKD cells, and response to inhibitors.

Fig 8. Schematic diagram showing the interplay of various signaling pathways involved in the regulation of glycolysis in polycystic kidney disease.