Na+,K+-ATPase is modulated by angiotensin II in diabetic rat kidney--another reason for diabetic nephropathy?

Abstract

Angiotensin II (ANGII) plays a central role in the enhanced sodium reabsorption in early type 1 diabetes in man and in streptozotocin-induced (STZ) diabetic rats. This study investigates the effect of untreated STZ-diabetes leading to diabetic nephropathy in combination with ANGII treatment, on the abundance and localization of the renal Na(+),K(+)-ATPase (NKA), a major contributor of renal sodium handling. After 7 weeks of STZ-diabetes (i.v. 65 mg kg(-1)) a subgroup of control (C) and diabetic (D7) Wistar rats were treated with ANGII (s.c. minipump 33 microg kg(-1) h(-1) for 24 h; CA and D7A). We measured renal function and mRNA expression, protein level, Serin23 phosphorylation, subcellular distribution, and enzyme activity of NKA alpha-1 subunit in the kidney cortex. Diabetes increased serum creatinine and urea nitrogen levels (C versus D7), as did ANGII (C versus CA, D7 versus D7A). Both diabetes (C versus D7) and ANGII increased NKA alpha-1 protein level and enzyme activity (C versus CA, D7 versus D7A). Furthermore, the combination led to an additive increase (D7 versus D7A, CA versus D7A). NKA alpha-1 Ser23 phosphorylation was higher both in D7 and ANGII-treated rats in the non-cytoskeletal fraction, while no signal was detected in the cytoskeletal fraction. Control kidneys showed NKA alpha-1 immunopositivity on the basolateral membrane of proximal tubular cells, while both D7 and ANGII broadened NKA immunopositivity towards the cytoplasm. Our study demonstrates that diabetes mellitus (DM) increases the mRNA expression, protein level, Ser23 phosphorylation and enzyme activity of renal NKA, which is further elevated by ANGII. Despite an increase in total NKA quantity in diabetic nephropathy, the redistribution to the cystosol suggests the Na(+) pump is no longer functional. ANGII also caused translocation from the basolateral membrane, thus in diabetic states where ANGII level is acutely elevated, the loss of NKA will be exacerbated. This provides another mechanism by which ANGII blockade is likely to be protective.

Figure 1. mRNA expression, protein level of Na⁺,K⁺-ATPase (NKA) α-1 subunit and enzyme activity in cortical tissue homogenates of angiotensin II(ANGII)-treated and streptozotocin diabetic kidneys

mRNA expression (A), protein level (B) and enzyme activity (C) were determined in kidney samples of control (C), ANGII-treated control (CA), 7 week diabetic (D7) and ANGII-treated D7 (D7A), rats (n = 6 per group). Top panel: representative examples of RT-PCR and Western blot analysis. Results are given as a ratio of intensity of NKA α-1 subunit and GAPDH mRNA, as a ratio of optical density of NKA α-1 subunit and β-actin and the difference between the activity with or without strophantidine in units of μmol fluorescein (mg protein)⁻¹ h⁻¹, respectively. mRNA and protein α-1 subunit: *P < 0.05, C vs D7; **P < 0.05, CA vs D7A. Enzyme activity: *P < 0.01, C vs CA; **P < 0.05, D7 vs D7A; ⁺P < 0.0001, C vs D7.

Figure 2. Protein levels of Na⁺,K⁺-ATPase (NKA) α-1 in (A) detergent-soluble (DS) and (B) detergent-insoluble (DR) fraction of angiotensin II (ANGII)-treated and streptozotocin-diabetic kidneys and (C) DR/DS ratio

Protein level of NKA α-1 subunit was measured in kidney samples of control (C), ANGII-treated control (CA), 7 week diabetic (D7) and ANGII-treated D7 (D7A) rats (n = 6 per group). Top panel: representative examples of Western blot analysis. Results are given as a ratio of optical density of NKA α-1 subunit and β-actin protein level. NKA α-1 protein level ratio of DR (dark hatching)/DS (light hatching) fraction is expressed in the percentage of TOT. *P < 0.05, C vs CA; **P < 0.05, D7 vs D7A; ⁺P < 0.05, C vs D7; ⁺⁺P < 0.05, CA vs D7A.

Figure 3. Serine23 phosphorylation of Na⁺,K⁺-ATPase (NKA) α-1 in angiotensin II (ANGII)-treated streptozotocin-diabetic kidneys

Serin23 phosphorylation was determined in the detergent-soluble fraction of kidney samples of control (C), ANGII-treated control (CA), 7 week diabetic (D7), ANGII-treated D7 (D7A) rats (n = 6 per group). Results are given as a ratio of optical density of Ser23phospho-NKA α-1 and β-actin protein level. No signal was detected in the detergent-insoluble fraction in any of the groups. *P < 0.05, C vs CA; **P < 0.05, D7 vs D7A; ⁺P < 0.05, C vs D7; ⁺⁺P < 0.05, CA vs D7A.

Figure 4. Confocalimages of angiotensin II (ANGII)-treated streptozotocin-diabetic kidney sections labelled with Na⁺,K⁺-ATPase (NKA) α-1 antibody

Representative examples of NKA α-1 (green) localization in kidney sections of (A) control (C), (B) ANGII-treated control (CA), (C) 7 week diabetic (D7) and (D) ANGII-treated D7 (D7A) rats. Nuclei are stained blue. Fluorescence signal intensities of NKA α-1 (green) generated from a scanned horizontal line shown as red arrow in the merged image are shown on the bottom right of each panel. Glom, glomerulus; cTAL, cortical thick ascending limb; PT, proximal tubule. Scale bars: top left images for all panels, 50μm, all other images, 10 μm.