Liver-Directed Gene Therapy Mitigates Early Nephropathy in Murine Glycogen Storage Disease Type Ia

Abstract

Nephropathy is a complication of glycogen storage disease type Ia (GSD-Ia), a metabolic disorder caused by pathogenic variants in glucose-6-phosphatase-α (G6Pase-α or G6PC1). While maintaining blood glucose homeostasis can delay the progression of renal disease in GSD-Ia, the benefits of liver-directed G6PC1 gene therapy on nephropathy remain unclear. This study evaluates the effects of low- and high-dose G6PC1 liver gene augmentation therapy on kidney function. The G6pc-/- mice, which lack G6Pase-α activity in both liver and kidney, were treated with G6PC1 gene therapy to restore either low or near-normal levels of liver G6Pase-α activity, and renal phenotype was examined at age 12 weeks. Both groups exhibited impaired renal glucose homeostasis, altered renal glucose reabsorption, acute kidney injury, and early signs of renal fibrosis. However, mice with near-normal liver G6Pase-α activity had better renal glucose reabsorption and homeostasis with lower serum levels of cystatin C and blood urea nitrogen, key markers of kidney function. These findings highlight the potential of liver-directed G6PC1 gene therapy to enhance metabolic control and mitigate early kidney disease in GSD-Ia.

Keywords: fibrosis; glucose homeostasis; glucose reabsorption; glucose‐6‐phosphatase‐α; mouse model.

FIGURE 1

The phenotype of L‐G6PC1‐low and L‐G6PC1‐high mice. L‐G6PC1‐low and L‐G6PC1‐high mice were generated from G6pc−/− mice and analyzed at 12 weeks of age. Age‐matched G6pc+/+ and G6pc+/− mice with a similar phenotype served as controls. (A) Liver microsomal G6Pase‐α activity in control (n = 14), L‐G6PC1‐low (n = 30), and L‐G6PC1‐high (n = 19) mice. (B) Enzyme histochemical analysis of G6Pase‐α in the kidneys of 3‐week‐old G6pc−/− and control mice (n = 3 per group) as well as in 12‐week‐old L‐G6PC1‐low and L‐G6PC1‐high mice and age‐matched control littermates (n = 6 per group). (C) Fasting blood glucose levels in 12‐week‐old control (n = 58), L‐G6PC1‐low (n = 26), and L‐G6PC1‐high (n = 28) mice. (D) Serum triglycerides (n = 9 per group), cholesterol (n = 12 per group), lactate (n = 12 per group), and uric acid (n = 12 per group) in 12‐week‐old control, L‐G6PC1‐low, and L‐G6PC1‐high mice. (E) Kidney G6P (n = 16 per group), glycogen (n = 16 per group), lactate (n = 8 per group), and triglyceride (n = 8 per group) levels in 12‐week‐old control, L‐G6PC1‐low, and L‐G6PC1‐high mice. Values represent the mean ± SEM. *p < 0.05, **p < 0.005.

FIGURE 2

Assessment of kidney glucose homeostasis, reabsorption and early injury in L‐G6PC1‐low and L‐G6PC1‐high mice. L‐G6PC1‐low and L‐G6PC1‐high mice were generated from G6pc−/− mice and analyzed at 12 weeks of age. Age‐matched G6pc+/+ and G6pc+/− mice with a similar phenotype served as controls. (A) Oil Red O (neutral triglycerides and lipids) staining of the kidneys in control (n = 6), L‐G6PC1‐low (n = 6), and L‐G6PC1‐high (n = 6) mice. Three sets of representative staining are shown for each group of mice. Scale bar, 20 μm. (B) Ratio of kidney weight (KW) to body weight (BW) in control (n = 26), L‐G6PC1‐low (n = 15), and L‐G6PC1‐high (n = 15) mice. (C) Body mass index (BMI) in control (n = 50), L‐G6PC1‐low (n = 29), and L‐G6PC1‐high (n = 20) mice. (D) H&E staining of the kidneys in control (n = 6), L‐G6PC1‐low (n = 6), and L‐G6PC1‐high (n = 6) mice at magnifications of x200 (top panels, Scale bar, 50 μm) and x400 (lower panels, Scale bar, 20 μm), Arrows point to the area of tubular dilation. Representative sets of staining are shown. (E) Western‐blot analyzes and quantitation of renal levels of GLUT2 and SGLT2 in control (n = 20), L‐G6PC1‐low (n = 23), and L‐G6PC1‐high (n = 23) mice. Densitometric quantification was performed and normalized against β‐Actin. Values represent the mean ± SEM. *p < 0.05, **p < 0.005.

FIGURE 3

Assessment of kidney AKI and fibrosis in L‐G6PC1‐low and L‐G6PC1‐high mice. L‐G6PC1‐low and L‐G6PC1‐high mice were generated from G6pc−/− mice and analyzed at 12 weeks of age. Age‐matched G6pc+/+ and G6pc+/− mice with a similar phenotype served as controls. (A) Western‐blot analyzes and quantitation of renal levels of E‐cadherin and N‐cadherin in control (n = 20), L‐G6PC1‐low (n = 23), and L‐G6PC1‐high (n = 23) mice. (B) Western‐blot analyzes and quantitation of renal levels of Dkk3 and CTGF in control (n = 20), L‐G6PC1‐low (n = 23), and L‐G6PC1‐high (n = 23) mice. (C) Western‐blot analyzes and quantitation of renal levels of total (β‐catenin‐T) and active, dephosphorylated (β‐catenin‐A) β‐catenin in control (n = 20), L‐G6PC1‐low (n = 23), and L‐G6PC1‐high (n = 23) mice. For Western‐blot analyzes, densitometric quantification was performed and normalized against β‐Actin. Values represent the mean ± SEM. *p < 0.05, **p < 0.005. (D) Immunohistochemical analysis of renal levels of active β‐catenin. Scale bar, 50 μm. (E) Quantitation of renal levels of nuclear localized active β‐catenin in control (n = 3), L‐G6PC1‐low (n = 3), and L‐G6PC1‐high (n = 3) mice. Kidney sections were immunostained with HRP‐labeled anti‐active β‐catenin and the nuclei counterstained with hematoxylin. Images were digitized using the Motic EasyScan Infinity 60 scanner and analyzed with QuPath software (v0.4.3). Multiple annotations were selected across the entire renal cortex. The Nucleus DAB OD mean scoring method [19] was used to identify moderate and strong optical density thresholds for nuclear‐stained β‐catenin.

FIGURE 4

Assessment of renal levels of renin, AGT, Snail1, and α‐SMA in L‐G6PC1‐low and L‐G6PC1‐high mice. L‐G6PC1‐low and L‐G6PC1‐high mice were generated from G6pc−/− mice and analyzed at 12 weeks of age. Age‐matched G6pc+/+ and G6pc+/− mice with a similar phenotype served as controls. (A) Western‐blot analyzes and quantitation of renal levels of renin and AGT in control (n = 20), L‐G6PC1‐low (n = 23), and L‐G6PC1‐high (n = 23) mice. (B) Western‐blot analyzes and quantitation of renal levels of Snail1 and α‐SMA in control (n = 20), L‐G6PC1‐low (n = 23), and L‐G6PC1‐high (n = 23) mice. Densitometric quantification was performed and normalized against β‐Actin. Values represent the mean ± SEM. *p < 0.05, **p < 0.005.

FIGURE 5

Assessment kidney fibrosis and dysfunction in L‐G6PC1‐low and L‐G6PC1‐high mice. L‐G6PC1‐low and L‐G6PC1‐high mice were generated from G6pc−/− mice and analyzed at 12 weeks of age. Age‐matched G6pc+/+ and G6pc+/− mice with a similar phenotype served as controls. (A) Western‐blot analyzes and quantification of renal levels of Col‐1α1 and Col‐IV in control (n = 20), L‐G6PC1‐low (n = 23), and L‐G6PC1‐high (n = 23) mice. Densitometric quantification was performed and normalized against β‐Actin. (B) Masson's trichrome staining of kidney sections in 12‐week‐old control, L‐G6PC1‐low, and L‐G6PC1‐high mice at magnifications of ×200 (top panels, Scale bar, 50 μm) and ×400 (lower panels, Scale bar, 20 μm). Representative sets of staining are shown. Renal fibrosis in mice was shown by the blue colored staining of the collagen fibers. (C) Serum levels of cystatin C, BUN, and creatinine in 12‐week‐old control (n = 6), L‐G6PC1‐low (n = 6), and L‐G6PC1‐high (n = 6) mice. Values represent the mean ± SEM. *p < 0.05, **p < 0.005.