Candesartan Differentially Regulates Epithelial Sodium Channel in Cortex Versus Medulla of Streptozotocin-Induced Diabetic Rats

Abstract

Diabetes is associated with an activated renal renin-angiotensin-aldosterone system (RAAS) and it was shown that streptozotocin (STZ)-induced diabetic rats had increased whole kidney protein levels of the epithelial sodium channel subunits (α-, β- and γ-ENaC). However, the role of the RAAS on the regional, i.e., cortical versus medullary, regulation of ENaC is unclear. Male Sprague-Dawley rats were injected with STZ (intravenous, 65 mg/kg·bw, n=12/group). After 14 days, half of them received drinking water with candesartan (2 mg/kg·bw/day), an angiotensin-II type-1 receptor (AT1R) antagonist, for one week. In the medulla, i.e., inner stripe of the outer medulla (ISOM), base and/or tip of the inner medulla, immunoblotting revealed increased protein abundances of α1 Na-K-ATPase and ENaC subunits with diabetes (200-600% of controls), which were not reversed by candesartan. In fact, candesartan increased all ENaC subunits and α1 Na-K-ATPase in the ISOM and/or base in control rats. In contrast, in the cortex, diabetes did not increase these proteins. However, candesartan reduced cortical β- and γ-ENaC regardless of diabetic state. In summary, diabetes-induced increases in ENaC were seen preferentially in the medulla. These changes appeared to be due to a mechanism clearly distinct from AT1R activation, because they were not abolished by candesartan. In fact, candesartan treatment tended to increase some of these medullary proteins, perhaps in compensation for increased NaCl load. In contrast, cortical β- and γ-ENaC were reduced by candesartan regardless of diabetic state suggesting their regulation by AT1R at this site; however this did not appear to be a site of diabetes-induced ENaC up-regulation.

Fig. 1

Effect of diabetes and candesartan on α-ENaC protein abundance in different kidney regions. A) Representative lanes from immunoblots for the α-ENaC subunit in the cortex homogenate (CTXH), inner stripe of outer medulla homogenate (OMH), inner-medullary base homogenate (IMH (B)) and inner medullary tip homogenate (IMH (T)) from non-diabetic (C) and diabetic (STZ) rats treated with or without candesartan (CAN). B) Densitometric summary of the blots (n = 6/group). For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat sample (confirmed a priori by equal Coomassie-staining of representative protein bands). *indicates mean is significantly different (p<0.05) from that of the untreated non-diabetic (C) by unpaired t-test. Results from two-way ANOVA (STZ X CAN) are given below the graph. Significant (p<0.05) factors are in BOLD.

Fig. 2

Effect of diabetes and candesartan on β-ENaC protein abundance in different kidney regions. A) Representative lanes from immunoblots for the β-ENaC subunit in the cortex homogenate (CTXH), inner stripe of outer medulla homogenate (OMH), inner medullary base homogenate (IMH (B)) and inner medullary tip homogenate (IMH (T)) from non-diabetic (C) and diabetic (STZ) rats treated with or without candesartan (CAN). B) Densitometric summary of the blots (n = 6/group). For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat sample (confirmed a priori by equal Coomassie-staining of representative protein bands). *indicates mean is significantly different (p<0.05) from that of the untreated non-diabetic (C) and τ from untreated diabetic rats (STZ) by unpaired t-test. Results from two-way ANOVA (STZ X CAN) are given below the graph. Significant (p<0.05) factors are in BOLD.

Fig. 3

Effect of diabetes and candesartan on 85kDa band of γ-ENaC protein abundance in different kidney regions. A) Representative lanes from immunoblots for the γ-ENaC subunit in the cortex homogenate (CTXH), inner stripe of outer medulla homogenate (OMH), inner-medullary base homogenate (IMH (B)) and inner medullary tip homogenate (IMH (T)) from non-diabetic (C) and diabetic (STZ) rats treated with or without candesartan (CAN). B) Densitometric summary of the blots (n = 6/group). For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat sample (confirmed a priori by equal Coomassie-staining of representative protein bands). *indicates mean is significantly different (p<0.05) from that of the untreated non-diabetic (C) and τ from untreated diabetic rats (STZ) by unpaired t-test. Results from two-way ANOVA (STZ X CAN) are given below the graph. Significant (p<0.05) factors are in BOLD.

Fig. 4

Effect of diabetes and candesartan on NCC protein abundance in different kidney regions. A) Lane from representative immunoblots for the NCC in the cortex homogenate (CTXH), inner stripe of outer medulla homogenate (OMH), inner medullary base homogenate (IMH (B)) and inner-medullary tip homogenate (IMH (T)) from non-diabetic (C) and diabetic (STZ) rats treated with or without candesartan (CAN). B) Densitometric summary of the blots (n = 6/group). For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat sample (confirmed a priori by equal Coomassie-staining of representative protein bands). * indicates mean is significantly different (p<0.05) from that of the untreated non-diabetic (C) and τ from untreated diabetic rats (STZ) by unpaired t-test. Results from two-way ANOVA (STZ X CAN) are given below the graph. Significant (p<0.05) factors are in BOLD.

Fig. 5

Effect of diabetes and candesartan on protein abundance for α1 subunit of Na-K-ATPase in different kidney regions. A) Representative lanes from immunoblots for the Na-K-ATPase in the cortex homogenate (CTXH), inner stripe of outer medulla homogenate (OMH), inner-medullary base homogenate (IMH (B)) and inner medullary tip homogenate (IMH (T)) from non-diabetic (C) and diabetic (STZ) rats treated with or without candesartan (CAN). B) Densitometric summary of the blots (n = 6/group). For immunoblotting, each lane was loaded with an equal amount of total protein from a different rat sample (confirmed a priori by equal Coomassie-staining of representative protein bands). * indicates a mean is significantly different (p<0.05) from that of the untreated non-diabetic (C) and τ from untreated diabetic rats (STZ) by unpaired t-test. Results from two-way ANOVA (STZ X CAN) are given below the graph. Significant (p<0.05) factors are in BOLD.