Aldosterone Antagonists in Monotherapy Are Protective against Streptozotocin-Induced Diabetic Nephropathy in Rats

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers

Metabolic and renal parameters of control, diabetic and treated diabetic rats.

<p>Data are means ± SD, n = 8–10/group, ^*p<0.05 vs. C; ^§p<0.05 vs. D, ^**p<0.01 vs. C; ^§§p<0.01 vs. D, ^***p<0.001 vs. C; ^§§§p<0.001 vs. D; respectively. UD–undetectable.</p

Western blot analysis of Na/K ATPase (NKA).

<p>Aldosterone antagonists were the most effective in decreasing diabetes and hyperglycemia induced elevation of tubular NKA protein level. Top panel: Representative examples of Western blot analysis. Lower panels: <i>A</i>: Densitometric analysis of NKA protein levels in kidney homogenates of control, diabetic and treated diabetic rats. <i>B</i>: Densitometric analysis of NKA protein levels in HK-2 tubular cells. Bar graph represents densitometric analysis from multiple experiments. Data represent means ± SD; *p<0.05 <i>vs</i> Control; §p<0.05 <i>vs</i> Diabetes, respectively; (bars show means±SD; n = 8–10/group). IOD – integrated optical density.</p

Confocal images of control, diabetic and treated diabetic rats.

<p>Aldosterone inhibitors prevented the mislocation of NKA induced by diabetes in proximal tubules. Representative pictures of immunofluorescence staining of kidney sections for Na/K ATPase (NKA, green) in control (A), streptozotocin-diabetic (B) and diabetic, Enalapril (C), Losartan (D), Spironolactone (E) and Eplerenone (F) treated rats (63x magnification; scale bar–10 μm). Nuclei are stained blue with Hoechst. PT-proximal tubule, DT-distal tubule, Bm-basal membrane, Lu – apical membrane at the lumen and Nucl – nuclei. Fluorescent signal intensity of NKA (green) generated from a line shown as red arrow in the merged image are shown on the bottom right of each panel.</p

Renal histopathology in control, diabetic and treated diabetic rats.

<p>Aldosterone antagonists were the most effective in attenuating the structural lesions of DN. Representative PAS staining of kidney sections (40x magnification; scale bar –50 μm): non-diabetic control (A), STZ-induced diabetic (B), Enalapril (E), Losartan (F), Spironolactone (G) and Eplerenone (H) treated diabetic rats (n = 8–10/group). Long, wide headed arrows point on mesangial matrix; long, narrow headed arrows on arterioles. Armanni-Ebstein lesions are marked with short, wide headed arrows. <i>C</i>: Mesangial fractional volume values (Vv) are defined by the ratio of mesangial area/glomerular tuft area. The mesangial area is determined by assessment of PAS-positive and nucleus-free areas in the mesangium. *p<0.05 <i>vs</i> Control; §p<0.05 <i>vs</i> Diabetes, respectively; (bars show means ± SD). <i>D</i>: Arteriolar hyalinosis is defined by the average of hyalinized quarters of arterioles. The hyalin is determined by assessment of PAS-positive and nucleus-free areas within the arterioles. *p<0.05 <i>vs</i> Control; §p<0.05 <i>vs</i> Diabetes, respectively; (bars show means ± SD).</p

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

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 μg 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 α-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 α-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 α-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 α-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