Aldosterone Blockade Attenuates Urinary Monocyte Chemoattractant Protein-1 and Oxidative Stress in Patients with Type 2 Diabetes Complicated by Diabetic Nephropathy

Abstract

Context: Aldosterone causes organic impairment by enhancement of oxidative stress and subsequent induction of proinflammatory cytokines and chemokines.

Objective: This study was designed to investigate the effect of spironolactone, an aldosterone blocker, on oxidative stress and the level of urinary monocyte chemoattractant protein (MCP)-1, a cysteine-cysteine chemokine that may contribute to progression of various nephropathies in type 2 diabetic patients with diabetic nephropathy.

Design, Setting, Patients and Other Participants, and Intervention: The patients were randomly assigned to two groups in which they received either spironolactone (50 mg/d; n = 23) or amlodipine (2.5 mg/d; n = 14).

Main Outcome Measures: Urinary 8-iso-prostaglandin (PG) F2α (a marker of oxidative stress), urinary MCP-1, and urinary albumin excretion (UAE) were measured at the start of administration (0 months) and after 3 months in each group. Baseline levels of these variables were also measured in 25 age-matched healthy subjects.

Results: There were significant positive correlations between log₁₀-transformed (log) 8-iso-PGF2α and log MCP-1 levels in control and diabetic subjects and all subjects combined, but no correlations between log UAE and log 8-iso-PGF2α or log MCP-1 were found in any group. Significant decreases in 8-iso-PGF2α, MCP-1, and UAE were observed with spironolactone (P = 0.0001, P = 0.0041, and P = 0.0037, respectively), and systolic blood pressure significantly decreased after both spironolactone and amlodipine therapy (P = 0.00011 and P = 0.0051, respectively).

Conclusions: Our data suggest that urinary MCP-1 is correlated with oxidative stress as measured by urinary 8-iso-PGF2α and that spironolactone can decrease urinary MCP-1 and oxidative stress.

ALTHOUGH PARTICIPATION OF angiotensin II (A-II) in progression of diabetic nephropathy has been proposed (1, 2), recent reports (3, 4) have demonstrated that the effect of A-II on impairment of various organs, including the kidney, is partially mediated by aldosterone, which is induced by A-II. The mechanism underlying the effect of aldosterone is not fully understood, but increased oxidative stress and subsequent induction of proinflammatory cytokines and chemokines might be responsible (3, 5, 6).

Recent reports demonstrated that administration of an angiotensin-converting enzyme inhibitor (ACE-I) in patients with diabetic nephropathy causes a decrease in not only urinary albumin excretion (UAE) but also the level of urinary monocyte chemoattractant protein (MCP)-1 (7), which belongs to the family of cysteine-cysteine chemokines (8) and induces macrophage migration to the lesion (9). The urinary MCP-1 level reflects MCP-1 produced in kidney (10) and may play an important role in progression of nephropathy, including diabetic nephropathy (10, 11), although prospective evidence that MCP-1 is associated with worsening nephropathy has yet to be obtained. Although the decrease in urinary MCP-1 caused by ACE-I administration may be attributable directly to inhibition of the effect of A-II, a decrease in aldosterone due to reduction of A-II activity may also have had an influence.

Given the above background, we investigated the effects of spironolactone, a representative aldosterone blocker, on urinary MCP-1, UAE, and oxidative stress in patients with type 2 diabetes complicated by diabetic nephropathy. The urinary 8-iso-prostaglandin (PG) F2α level was used as a measure of oxidative stress (12, 13). We hypothesized that spironolactone may decrease urinary MCP-1 and UAE and that these effects may be associated with inhibition of oxidative stress by spironolactone.

Patients and Methods

Patients

Initially 40 type 2 diabetic outpatients with diabetic nephropathy (25 in the spironolactone group and 15 in the amlodipine group) and 25 age-matched healthy subjects were enrolled in the study. Diabetic nephropathy was defined as having UAE of more than 30 mg/g creatinine (Cr), based on American Diabetes Association Guidelines (14). Of the diabetic patients, two subjects receiving spironolactone were subsequently excluded from the study because they showed apparent symptoms of a common cold on their second visit. One patient treated with amlodipine was also excluded because of poor compliance at follow-up. Therefore, the final study included 23 patients receiving spironolactone, 14 receiving amlodipine, and 25 healthy subjects. Patients were excluded if they had been treated with any antihypertensive drug except for an α₁-adrenaline-receptor blocker (doxazosin), which had been administrated to four patients in the spironolactone group and one in the amlodipine group. In both groups, patients exhibiting evidence of liver dysfunction or findings of infectious or autoimmune disease were also excluded from the study. One patient in the spironolactone group had a Cr level of 1.2 mg/dl, and two patients in the amlodipine group had Cr levels of 1.5 and 1.1 mg/dl, respectively. All other patients had Cr values of less than 1.0 mg/dl. All patients had a serum potassium concentration of less than 5.0 mEq/liter at baseline.

Methods

Patients were assigned randomly at a ratio of 5:3 to the spironolactone group (50 mg/d) or amlodipine group (2.5 mg/d); we hypothesized that amlodipine would have little effect on major findings such as UAE, MCP-1, and 8-iso-PGF2α levels, and therefore, we decided to assign more patients to the spironolactone group because of the small sample size of the study.

Patients received spironolactone or amlodipine over a 3-month period, and blood and urine tests were performed at the beginning of this period (0 wk) and after 12 wk (3 months). No change in administration of any drug occurred for any patient during the 12-wk investigational period. For all patients and healthy subjects, urine and blood were sampled in the outpatient department from 0830–0930 h after at least 10 h of overnight fasting.

Urinary MCP-1 assay.

Urinary MCP-1 was measured with an ELISA kit (Quantikine; R&D Systems, Minneapolis, MN). The intra- and interassay coefficients of variation for MCP-1 were 4.2–5.9% and 4.5–5.9%, respectively.

Urinary 8-iso-PGF2α assay.

Urinary 8-iso-PGF2α was measured with an enzyme immunoassay kit (ACE enzyme immunoassay; Cayman Chemical Co., Ann Arbor, MI). The intra- and interassay coefficients of variation were both less than 10%, based on actual values. This assay has been reported to show no differences in measurements of morning urine samples analyzed immediately after collection or after storage for 24 h (15).

Ethical considerations

All subjects gave informed consent to their inclusion in the study, which was performed according to the guidelines proposed in the Declaration of Helsinki and was approved by the Dokkyo University School of Medicine Ethics Committee (Koshigaya, Japan).

Statistical methods

All data are presented as means ± sd, except those for UAE, MCP-1, 8-iso-PGF2α, plasma renin activity (PRA), and PRA to aldosterone ratio, which are expressed as geometric means with interquartile ranges (25th and 75th percentiles) because of their skewed distribution. Data for the two measurement time points for each individual were compared by paired t test. Comparisons between data for the two groups were made by unpaired t test. Data for UAE, MCP-1, 8-iso-PGF2α, PRA, and PRA to aldosterone ratio were log transformed before each comparison.

Results

Significant elevation of UAE, MCP-1, and 8-iso-PGF2α levels occurred in the spironolactone-treated patients, compared with these levels in age-matched healthy subjects [19.3 (10.6, 38.2) vs. 543.7 (170, 1146) mg/g Cr, P < 0.0001; 194.3 (104.95, 303.5) vs. 350.0 (216, 517) pg/ml, P = 0.0092; 154.8 (92, 625) vs. 221.2 (200, 340) pg/ml, P = 0.0465, in controls and patients, respectively].

Changes in variables from baseline to 3 months after spironolactone and amlodipine therapy are presented in Table 1, and these data for UAE, MCP-1, and 8-iso-PGF2α are also shown in Fig. 1. No significant differences in MCP-1 and 8-iso-PGF2α were observed between healthy subjects at baseline and patients in the spironolactone group after 3 months (P = 0.9615 and P = 0.4279, respectively). Clinical characteristics and laboratory data did not differ significantly between the spironolactone and amlodipine groups (data not shown).

Fig. 1.

The changes in UAE (A), MCP-1 (B), and 8-iso-PGF2α (C) levels in spironolactone group and the changes in UAE (D), MCP-1 (E), and 8-iso-PGF2α (F) levels in amlodipine group.

TABLE 1.

Changes in various variables at baseline and 3 months after spironolactone or amlodipine therapy

	Spironolactone (50 mg/d; n = 23)			Amlodipine (2.5 mg/d; n = 14)			P1
	Baseline	3 months	P	Baseline	3 months	P
UAE (mg/g Cr)	543.7	376.7	0.00372	403.7	340.5	0.3816	0.3455
	(170, 1146)	(135, 794)		(149.75, 1058.5)	(85.25, 1702)
MCP-1 (pg/ml)	350.0	196.5	0.00412	259.7	345.8	0.1746	0.00382
	(216, 517)	(104, 350)		(141.25, 501.5)	(201, 571)
8-iso-PGF2α (pg/ml)	221.2	136.2	0.00012	162.8	181.3	0.4273	0.00112
	(200, 340)	(96, 200)		(117.5, 225)	(135, 242.5)
FPG (mg/dl)	142.6 ± 45.6	155.4 ± 41.7	0.1859	158.3 ± 51.8	157.4 ± 57.1	0.9318	0.3542
HbA1C (%)	7.6 ± 1.4	8.2 ± 1.4	0.00592	7.7 ± 1.8	7.5 ± 1.4	0.7021	0.0624
TC (mg/dl)	211.9 ± 36.5	215.5 ± 29.8	0.5683	197.4 ± 38.7	219.2 ± 43.7	0.1091	0.1621
HDL-C (mg/dl)	54.6 ± 19.6	53.5 ± 14.4	0.6480	49.5 ± 19.3	52.1 ± 16.3	0.3513	0.3168
LDL-C (mg/dl)	124.9 ± 29.3	130.2 ± 27.1	0.2631	112.3 ± 39.7	132.1 ± 44.7	0.0633	0.1371
TG (mg/dl)	131.1 ± 58.2	122.3 ± 41.4	0.3610	190.1 ± 233.3	174.9 ± 151.4	0.7028	0.8454
SBP (mm Hg)	135.3 ± 8.1	128.8 ± 8.8	0.000112	137.6 ± 8.9	127.8 ± 11.0	0.00512	0.2561
DBP (mm Hg)	71.7 ± 5.7	69.7 ± 4.5	0.1206	74.2 ± 8.9	68.9 ± 8.1	0.03492	0.1722
BW (kg)	59.9 ± 9.6	59.8 ± 9.7	0.7650	64.0 ± 9.3	63.5 ± 9.3	0.3977	0.3973
Creatinine (mg/dl)	0.74 ± 0.17	0.93 ± 0.24	<0.00012	0.69 ± 0.34	0.74 ± 0.29	0.1874	0.00212
Potassium (mEq/liter)	4.09 ± 0.40	4.51 ± 0.45	0.00262	4.14 ± 0.39	4.03 ± 0.24	0.3433	0.00612
Aldosterone (ng/dl)	10.51 ± 4.95	12.76 ± 5.70	0.02222	10.42 ± 5.47	11.12 ± 6.11	0.5690	0.3078
PRA (ng/ml·h)	0.66	1.33	0.00052	0.78	0.82	0.8905	0.03862
	(0.3, 1.3)	(0.5, 3)		(0.3, 1.6)	(0.375, 2.225)
Aldosterone/PRA	14.4	8.76	0.00492	11.3	11.5	0.9930	0.02182
	(9.4, 27.8)	(4.3, 19.2)		(6.4, 28.4)	(5.6, 21.0)
ACTH (pg/ml)	37.52 ± 14.62	36.44 ± 19.46	0.8083	36.01 ± 16.25	40.10 ± 21.72	0.3772	0.4409

P value by paired t test. Data are expressed as means ± sd, except for UAE, MCP-1, 8-iso-PGF2α, PRA, aldosterone/PRA, which are expressed as geometric means (interquartile range, 25th and 75th percentiles). FPG, Fasting plasma glucose; HbA_1C, glycosylated hemoglobin; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglyceride; DBP, diastolic blood pressure; BW, body weight.

¹

P values for comparison of changes between spironolactone and amlodipine groups by Student t test.

²

P < 0.05 defined as statistically significant.

A strong and significant positive correlation was obtained between log MCP-1 and log 8-iso-PFG2α in the 37 diabetic patients, in the 25 healthy subjects and in the 62 total subjects (r = 0.5327, P < 0.0001; r = 0.6477, P 0.0005; r = 0.6010, P < 0.0001, respectively). However, no correlation was obtained between log UAE and log 8-iso-PGF2α or log MCP-1 in healthy, diabetic, or total subjects. A significant correlation between the MCP-1 and 8-iso-PFG2α ratios (calculated as the value of each variable after 3 months divided by the respective value at baseline) was observed in the spironolactone group (r = 0.6682, P = 0.0005), but no such correlation was found in the amlodipine group (r = 0.4533, P = 0.1037).

In a linear regression model of treatment effect on change in (log transformed) MCP-1, using baseline systolic blood pressure (SBP) and baseline MCP-1 as covariates, the effects of treatment and baseline MCP-1 were statistically significant (P = 0.0100 and P = 0.0190, respectively), but SBP was not significant (P = 0.9690). A least square means estimate of the change in MCP-1 (expressed as the ratio of the 3-month value to the baseline value) adjusted by baseline SBP and baseline MCP-1, showed a decrease in the spironolactone group (ratio 0.60) but an increase in the amlodipine group (ratio 1.20).

Discussion

In the current study, we found a strong positive correlation between urinary MCP-1 and 8-iso-PGF2α in diabetic and healthy subjects, which to our knowledge is the first report of such a correlation. Although the promotion of cardiac MCP-1 production based on oxidative stress has been observed in an animal model (5), our data suggest a close association between MCP-1 and oxidative stress in human kidney in vivo because the urinary MCP-1 level reflects MCP-1 produced in the kidney (10).

A recent report (7) demonstrated that administration of an ACE-I decreases not only UAE but also urinary MCP-1, independently of blood pressure, in patients with diabetic nephropathy. The influence of oxidative stress on MCP-1 was not investigated, but the inhibitory effect of the ACE-I on MCP-1 production may at least in part have been due to a decrease in oxidative stress caused by a reduction of A-II, given that A-II has been shown to increase oxidative stress (16). However, it has also been reported that oxidative stress resulting from A-II is partially mediated by aldosterone (3, 4) and that administration of an ACE-I cannot completely suppress elevation of aldosterone because of the so-called aldosterone breakthrough phenomenon (17). The evidence from these reports therefore suggests that both ACE-I and aldosterone blockers may be effective for decreasing MCP-1; in fact, the effect of an aldosterone blocker on MCP-1 or oxidative stress in kidney has been shown in animal models (18, 19).

To our knowledge, the current study is the first investigation of the effect of spironolactone treatment on urinary MCP-1 and 8-iso-PGF2α in type 2 diabetic patients with diabetic nephropathy. As expected, spironolactone significantly attenuated urinary MCP-1, as well as urinary 8-iso-PGF2α, to levels found in normal subjects. Notably, the changes in MCP-1 before and after treatment were closely positively correlated with changes in 8-iso-PGF2α. Furthermore, we also found that spironolactone administration caused a significant decrease in UAE, which is consistent with other results found for patients with diabetic nephropathy (20). Although spironolactone also decreased blood pressure significantly, the changes in SBP or diastolic blood pressure did not correlate with the changes in 8-iso-PGF2α or MCP-1. Furthermore, as a control we also investigated the effects of amlodipine, a representative long-acting calcium channel blocker, on urinary 8-iso-PGF2α and MCP-1. Despite causing a significant decrease in blood pressure, amlodipine did not have a significant effect on urinary 8-iso-PGF2α, MCP-1, or UAE.

Taken together, these results suggest that spironolactone inhibits production of MCP-1 in kidney and reduces oxidative stress independently of blood pressure in diabetic patients with nephropathy. In the current study, we noted that the antihypertensive agent doxazosin was more widely prescribed in the spironolactone group. Enrollment of patients treated with doxazosin was permitted because this agent is likely to have only a very modest effect on the renin-angiotensin system; however, we cannot formally exclude the possibility of an influence of doxazosin on the results. Furthermore, an unbalanced design was adopted for the drug-treatment groups; it would have been preferable to have used a balanced design and a larger number of patients, and this is one of the major limitations of the study. Nonetheless, the significant positive correlation between the levels of urinary MCP-1 and 8-iso-PGF2α is of importance, and our data clearly show that in patients with type 2 diabetes complicated by diabetic nephropathy, spironolactone decreases urinary MCP-1 and 8-iso-PGF2α to their respective levels in healthy subjects.

Acknowledgments

Received August 1, 2005. Accepted March 22, 2006.

Abbreviations:

 
• A-II,

  Angiotensin II;

 
• ACE-I,

  angiotensin-converting enzyme inhibitor;

 
• Cr,

  creatinine;

 
• 8-iso-PGF2,

  8-iso-prostaglandin F2α;

 
• MCP,

  monocyte chemoattractant protein;

 
• PG,

  prostaglandin;

 
• PRA,

  plasma renin activity;

 
• SBP,

  systolic blood pressure;

 
• UAE,

  urinary albumin excretion.