Diabetic rats present higher urinary loss of proteins and lower renal expression of megalin, cubilin, ClC-5, and CFTR

Abstract

Diabetic nephropathy (DN) occurs in around 40% of those with diabetes. Proteinuria is the main characteristic of DN and develops as a result of increased permeability of the glomerulus capillary wall and/or decreased proximal tubule endocytosis. The goal of this work was to evaluate renal function and the expression of megalin, cubilin, CFTR (cystic fibrosis transmembrane conductance regulator), and ClC-5 in the proximal tubule and renal cortex of rats with type 1 diabetes. Male Wistar rats were randomly assigned to control (CTRL) and diabetic (DM) groups for 4 weeks. Renal function was assessed in 24-h urine sample by calculating clearance and fractional excretion of solutes. The RNA and protein contents of ClC-5, CFTR, megalin, and cubilin were determined in the renal proximal tubule and cortex using real-time polymerase chain reaction and western blotting techniques, respectively. The results showed higher creatinine clearance and higher urinary excretion of proteins, albumin, and transferrin in the DM group than in the CTRL group. Furthermore, the renal cortex and proximal tubule of diabetic animals showed downregulation of megalin, cubilin, ClC-5, and CFTR, critical components of the endocytic apparatus. These data suggest dysfunction in proximal tubule low-molecular-weight endocytosis and protein glomerulus filtration in the kidney of diabetic rats.

Keywords: CFTR; ClC‐5; cubilin; diabetes; endocytosis; kidney; megalin; proximal tubule.

Figure 1

Timeline representation of the experimental design. Male Wistar rats weighing between 180 and 240 g were divided into two groups: control (CTRL) and diabetes mellitus (DM). The CTRL group received an intraperitoneal injection of 0.1 mol/L citrate buffer (pH 4.7) and the DM group received an intraperitoneal injection of streptozotocin (STZ) 45 mg kg⁻¹ body weight. After 4 weeks, both groups were killed for blood and kidney collection; 24 h before that, the animals were placed in a metabolic cage, which allowed urine collection and measurement of renal function.

Figure 2

Glycemic levels and body weight of rats in the CTRL and DM groups during the 4 weeks of the experiments. (A) After 5 h fasting, the rats in the DM group showed higher glycemia than those in the CTRL group throughout the experimental timeline. (B) The body weight of DM rats was lower than the CTRL rats (n = 6) in the fourth week of the experiment. All values were compared each week and are presented as means ± SEM. Asterisk (*) indicates a significant difference; CTRL vs. DM (P < 0.05). CTRL, n = 6; DM, n = 8.

Figure 3

Analyses of transferrin in urine. (A) Western blot analyses of transferrin excretion in the urine of CTRL and DM rats. (B) Densitometric analyses of transferrin excretion in the urine of CTRL and DM rats normalized by their respective creatinine urinary excretion. MW, molecular weight ladder. CTRL, n = 4; DM, n = 4.

Figure 4

Analyses of ClC‐5, CFTR, TNR‐CFTR, megalin, and cubilin protein content in the renal cortex of CTRL and DM rats. The abundance of key proteins of the endocytic pathway in the renal cortex was measured by western blotting. Graphical representation of the protein content of (A) ClC‐5, (B) CFTR, (C) TNR‐CFTR, (D) megalin, and (E) cubilin. The values are presented as means ± SEM and as fold relative to control. Asterisk (*) indicates a significant difference; CTRL versus DM (P < 0.05). β‐Actin expression was used as an internal control. CTRL, n = 6; DM, n = 6.

Figure 5

Analyses of ClC‐5, CFTR, megalin, and cubilin mRNA level in the renal cortex of CTRL and DM animals. The mRNA content of key components of the endocytic pathway in the renal cortex was measured by real‐time PCR and 36β4 was used as internal control. Graphical representation of the mRNA content of (A) ClC‐5, (B) CFTR, (C) megalin, and (D) cubilin in the renal cortex of CTRL and DM rats. The values are presented as means ± SEM and as fold relative to control. Asterisk (*) indicates a significant difference; CTRL vs. DM (P < 0.05). CTRL, n = 4; DM, n = 7.

Figure 6

Analyses of ClC‐5, CFTR, megalin, and cubilin mRNA level in dissected proximal tubule of CTRL and DM rats. The mRNA content of key components of the endocytic pathway in the proximal tubule was measured by real‐time PCR and 36B4 was used as internal control. Graphical representation of the mRNA content of (A) ClC‐5, (B) CFTR, (C) megalin, and (D) cubilin in the proximal tubule of CTRL and DM rats. The values are presented as means ± SEM and as fold relative to control. Asterisk (*) indicates a significant difference; CTRL versus DM (P < 0.05). CTRL, n = 4; DM, n = 4.