Michael S. LaPointe

Increased ACE 2 and Decreased ACE Protein in Renal Tubules From Diabetic Mice: A Renoprotective Combination?

Abstract—Unlike the ubiquitous angiotensin-converting enzyme (ACE), the ACE-related carboxypeptidase 2 (ACE 2) is predominantly expressed in the heart, kidney, and testis. ACE 2 degrades angiotensin (Ang) II to Ang (1–7) and Ang I to Ang (1–9). We investigated the expression of ACE and ACE 2 in a rodent model of type 2 diabetes. ACE and ACE 2 were measured in kidney and heart from 8-week-old no diabetic control (db/m) mice and diabetic (db/db) mice, which at this young age have obesity and hyperglycemia without nephropathy. In renal cortical tissue, ACE mRNA was reduced (db/db 0.31±0.06 versus db/m 0.99±0.05; P <0.005), whereas ACE 2 mRNA was not (db/db 0.94±0.05 versus db/m 1.03±0.11, NS). ACE protein was markedly reduced in kidney cortex of db/db mice (db/db 0.24±0.13 versus db/m 1.02±0.12; P <0.005), and this was associated with a corresponding decrease in renal ACE activity (db/db 12.7±3.7 versus db/m 61.6±4.4 mIU/mg protein; P <0.001). ACE 2 protein, by contrast, was increased in kidneys from diabetic mice (db/db 1.39±0.14 versus db/m 0.53±0.04; P <0.005). An increase in ACE 2 protein and a decrease in ACE protein, respectively, were also seen by immunostaining of renal cortical tubules from the db/db mice. In heart tissue, there were no significant differences between db/db and db/m mice in either ACE mRNA and protein or ACE 2 mRNA and protein. We conclude that in young db/db mice, ACE 2 protein in renal cortical tubules is increased, whereas ACE protein is decreased. We propose that the pattern of low ACE protein coupled with increased ACE 2 protein expression may be renoprotective in early stages of diabetes.

Angiotensin II Increases H+-ATPase B1 Subunit Expression in Medullary Collecting Ducts

Metabolic alkalosis is a common feature of hypokalemic hypertensive syndromes associated with angiotensin II excess. The alkalosis-generating effect of angiotensin II is usually ascribed to its stimulatory effect on aldosterone secretion, a hormone that upregulates collecting duct hydrogen ion secretion. We studied the effect of angiotensin II infusions on the expression of B1 and a4 protein, subunits of the renal H+-ATPase in adrenalectomized rats. Adrenalectomized rats were given either angiotensin II or vehicle for 7 days via osmotic mini-pumps. H+-ATPase B1 protein expression was evaluated by Western blot analysis in isolated medulla and cortex plasma membrane preparations from one kidney, whereas the contralateral kidney was used for immunostaining. By Western blotting, the relative abundance of B1 protein was 2-fold higher in renal medulla membranes from rats with intact adrenal glands (sham surgery) than from adrenalectomized rats (219±47%, n=12; P<0.05). In contrast to renal medulla, adrenalectomy did not significantly alter the relative abundance of B1 protein in renal cortex. Angiotensin II also did not significantly alter the relative levels of B1 protein in the cortex, but it increased it significantly in renal medullary membranes (231±56%, n=8; P<0.005). Moreover, enhanced H+-ATPase B1 subunit protein immunoreactivity was found in medullary collecting duct segments of rats infused with angiotensin II. In contrast to B1, expression of a4, another subunit of the H+-ATPase was not altered by adrenalectomy or angiotensin II. We conclude that adrenalectomy decreases whereas angiotensin II increases H+-ATPase B1 subunit expression in medullary, but not in cortical collecting ducts. By increasing the relative abundance of the B1 subunit of H+-ATPase in the collecting duct, angiotensin II excess may lead to increased hydrogen ion secretion and thus metabolic alkalosis—a common feature of hypertensive syndromes associated with angiotensin II overactivity.

NHE-1 Protein in Vascular Smooth Muscle and Lymphocytes From the Spontaneously Hypertensive Rat

The present study examined the abundance of NHE-1 protein in cultured vascular smooth muscle cells (VSMCs), freshly isolated thymocytes, and fresh aortic tissue from spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. Two sets of affinity-purified antibodies (Ab[765-778] and Ab[698-711]) against different epitopes of the NHE-1 isoform of the Na+-H+ antiporter were used. Each set of antibodies recognized a major protein band at 105 to 110 kD that was more abundant in protein lysates prepared from cultured VSMCs from the SHR than those from WKY rats (Ab[765-778] 0.047 +/- 0.011 vs 0.010 +/- 0.002 O.D. units/10 microg protein, P<.001 for SHR and WKY, respectively; and Ab(698-711) 0.173 +/- 0.026 vs 0.087 +/- 0.028 O.D. units/10 microg protein, P<.05, for SHR and WKY, respectively). The increase in NHE-1 protein abundance in cultured VSMCs from the SHR was associated with a greater Vmax of the Na+-H+ antiporter as compared to those from WKY rats (17.93 +/- 2.07 vs 8.16 +/- 1.05 mmol H+/min, P<.001, respectively). In contrast to cultured VSMCs, there was no difference in the relative abundance of NHE-1 protein in fresh aortic tissue (0.075 +/- 0.018 vs 0.083 +/- 0.017 O.D. units/10 microg protein, from SHR and WKY, respectively) or in freshly isolated thymocytes (0.158 +/- 0.046 vs 0.226 +/- 0.054 O.D. units/10 microg protein, from SHR and WKY, respectively). We conclude that the increase in the Vmax of the Na+-H+ antiporter in cultured VSMCs from the SHR, compared to those from WKY rats, is due, at least in part, to increased levels of NHE-1 protein.

Free cytosolic calcium in renal proximal tubules from the spontaneously hypertensive rat.

Free intraceUular calcium was measured in renal proximal tubules obtained from spontaneously hypertensive rats (SHR) and from age-matched Wistar-Kyoto rats (WKY) ingesting a normal diet. Experiments were performed on renal proximal tubule suspensions using fura-2 to monitor cytosolic calcium. In 4-week-old rats, when systolic blood pressure was not significantly different between the two groups, renal proximal tubule cytosolic calcium was similar (143 ± 28 and 144 ± 15 nM, respectively). By the age of 5 weeks, cytosolk calcium increased significantly in both SHR and WKY (214 ± 24 and 262 ± 34 nM, respectively, p < 0.05). Calcium, however, was not significantly different between the two groups, even though at this age blood pressure was higher in SHR than in WKY. As compared with values in 4-week-old rats, cytosolic calcium was also found increased in tubules from both SHR and WKY aged 10 to 12 weeks (261 ± 42 and 279 ± 30 nM, respectively) and 20 to 24 weeks (263 ± 42 and 308 ± 28 nM, respectively). However, no significant differences in cytosolic calcium were found between SHR and WKY even though at these ages systolic blood pressure increased markedly in the SHR. Moreover, regression analysis failed to reveal a correlation between cytosolic calcium and blood pressure when data from either group of rats of all ages studied were pooled. Exposure to ouabain (10−3 M) to inhibit Na+,K+ – adenosine triphosphatase and increase intracellular sodium had no significant effect on cytosolic calcium in tubules from either SHR or WKY (260 ± 69 and 250 ± 45 nM, respectively). This finding suggests that a circulating ouabainlike factor is not likely to increase cytosolic calcium in renal proximal tubules from te SHR. Our data indicate that cytosolic calcium is not elevated in renal proximal tubules obtained from the SHR either before or during sustained hypertension.