Impaired insulin signaling affects renal organic anion transporter 3 (Oat3) function in streptozotocin-induced diabetic rats

Abstract

Organic anion transporter 3 (Oat3) is a major renal Oats expressed in the basolateral membrane of renal proximal tubule cells. We have recently reported decreases in renal Oat3 function and expression in diabetic rats and these changes were recovered after insulin treatment for four weeks. However, the mechanisms by which insulin restored these changes have not been elucidated. In this study, we hypothesized that insulin signaling mediators might play a crucial role in the regulation of renal Oat3 function. Experimental diabetic rats were induced by a single intraperitoneal injection of streptozotocin (65 mg/kg). One week after injection, animals showing blood glucose above 250 mg/dL were considered to be diabetic and used for the experiment in which insulin-treated diabetic rats were subcutaneously injected daily with insulin for four weeks. Estrone sulfate (ES) uptake into renal cortical slices was examined to reflect the renal Oat3 function. The results showed that pre-incubation with insulin for 30 min (short term) stimulated [3H]ES uptake into the renal cortical slices of normal control rats. In the untreated diabetic rats, pre-incubation with insulin for 30 min failed to stimulate renal Oat3 activity. The unresponsiveness of renal Oat3 activity to insulin in the untreated diabetic rats suggests the impairment of insulin signaling. Indeed, pre-incubation with phosphoinositide 3-kinase (PI3K) and protein kinase C zeta (PKCζ) inhibitors inhibited insulin-stimulated renal Oat3 activity. In addition, the expressions of PI3K, Akt and PKCζ in the renal cortex of diabetic rats were markedly decreased. Prolonged insulin treatment in diabetic rats restored these alterations toward normal levels. Our data suggest that the decreases in both function and expression of renal Oat3 in diabetes are associated with an impairment of renal insulin-induced Akt/PKB activation through PI3K/PKCζ/Akt/PKB signaling pathway.

Figure 1. Effects of diabetes on basal and short term insulin-stimulated ES uptake in renal cortical slices.

Renal cortical slices from control, diabetic (DM) and diabetic with insulin-treated rats (DM-treated) were pre-incubated for 30 min in buffer alone (C), or buffer containing 30 µg/mL insulin (In) at room temperature. After pre-incubation, renal cortical slices were incubated in buffer containing 50 nM [³H]ES for 30 min. Values are expressed as means of T/M ± SEM. from five animals (5 slices/experimental group/animal). ^†† p<0.01, compared to the corresponding control; **p<0.01, compared to the control (non DM rats); ^## p<0.01, compared to the diabetic rats.

Figure 2. Effect of diabetes on Oat3 expression in the renal cortex.

(A) and (C); Western blot analysis for Oat3 in the membrane and whole cell lysate fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Oat3 in membrane and whole cell lysate fractions normalized to β-actin, respectively. Na⁺-K⁺-ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. **p<0.01, compared to control; ^## p<0.01, compared to diabetes.

Figure 3. Effect of Wortmannin on ES uptake in renal cortical slices.

Tissue slices prepared from rat renal cortex were pre-incubated under different experimental conditions as described in the Materials and Methods section. Following pre-incubation, the slices were incubated for 30 min with 50 nM [³H]ES. Values are expressed as means ± SEM from five animals per group (6 slices/animal). **p<0.01, compared to control; ^†† p<0.01, compared to insulin alone.

Figure 4. Effect of PKCζ inhibitor on ES uptake in renal cortical slices.

Tissue slices prepared from rat renal cortex were pre-incubated under different experimental conditions as described in the Materials and Methods section. Following pre-incubation, the slices were incubated for 30 min in 50 nM [³H]ES. Values are expressed as means ± SEM from five animals per group (6 slices/animal). **p<0.01, compared to control; ^††p<0.01, compared to insulin alone.

Figure 5. Effect of diabetes on PI3K expression in the renal cortex.

(A) and (C); Western blot analysis for PI3K in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of PI3K in membrane and cytosolic fractions normalized to β-actin, respectively. Na⁺-K⁺-ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. **p<0.01, compared to control; ^## p<0.01, compared to diabetes.

Figure 6. Effects of diabetes on Akt and phospho-Akt expressions in the renal cortex.

(A) and (C); Western blot analysis for Akt and phospho-Akt in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of Akt and phospho-Akt in the membrane and cytosolic fractions normalized to β-actin, respectively. Na⁺-K⁺-ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. Bar graphs indicate means ± SEM from five independent experiments. **p<0.01, compared to control; ^## p<0.01, compared to diabetes.

Figure 7. Effects of diabetes on PKCζ and phospho-PKCζ expressions in the renal cortex.

(A) and (C); Western blot analysis for PKCζ and phospho-PKCζ in the membrane and cytosolic fractions of renal cortical tissues in control, diabetic (DM) and diabetic with insulin-treated (DM-treated) rats, respectively. (B) and (D); The signal intensity of PKCζ and phospho-PKCζ in the membrane and cytosolic fractions normalized to β-actin, respectively. Na⁺-K⁺-ATPase and β-actin expressions were used as a membrane marker and loading control, respectively. β-actin for the membrane fractions of PKCζ (A) and phospho-PKCζ (C) were obtained from the same membrane/loading protein. Bar graphs indicate means ± SEM from five independent experiments. **p<0.01, compared to control; ^## p<0.01, compared to diabetes.

Figure 8. A hypothetical model for the regulation of renal Oat3 in diabetic condition.

Insulin regulates Oat3 function by activating PKCζ and PKB through PI3K leading to up-regulation of Oat3 function and increased trafficking of Oat3 to plasma membrane and subsequently increased transport function. The impairment of renal insulin signaling in diabetes down-regulates Oat3 function through PI3K/PKCζ/Akt/PKB-mediated pathway. The hyperglycemia-induced activation of PKCα in diabetes leads to internalization of Oat3 to cytoplasm resulting in down-regulation of Oat3 function. Alterations in the internalization and trafficking, the regulatory proteins, and the expression of Oat3 lead to decreased renal Oat3 function in diabetes. These changes can be recovered after insulin treatment for four weeks.