J Vet Intern Med 2008;22:317–324
P l a s m a A s y m m e t r i c D i m e t h y l a r g i n i n e , Sy m m e t r i c Dim ethyl ar ginine, L - A r g i n i n e , a n d N i t r i t e / N i t r a t e C o n c e n t r a t i o n s i n C a t s w i t h C h r o n i c Ki d n e y Di s e a s e and Hypertension R.E. Jepson, H.M. Syme, C. Vallance, and J. Elliott Background: Chronic kidney disease (CKD) and hypertension have been associated with decreased bioavailability of nitric oxide (NO) and endothelial dysfunction. Increased concentrations of the endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) are implicated. Hypothesis: Plasma ADMA concentration is increased in cats with CKD and systemic hypertension corresponding to a decrease in total plasma nitrate/nitrite (NOx) availability. Decrease in systolic blood pressure (SBP) and proteinuria during treatment of hypertension with amlodipine besylate may be associated with increased NOx availability. Animals: Sixty-nine client-owned normotensive and hypertensive cats with variable azotemia. Methods: Plasma ADMA, symmetric dimethylarginine (SDMA), and L-arginine were measured simultaneously by hydrophilic-interaction liquid chromatography-electrospray tandem mass spectrometry in cats from 6 groups: normotensive nonazotemic (n 5 10), normotensive mildly azotemic (n 5 10), hypertensive mildly azotemic with hypertensive retinopathy (n 5 20), hypertensive mildly azotemic without hypertensive retinopathy (n 5 10), normotensive moderately azotemic cats (n 5 10), and hypertensive nonazotemic cats (n 5 9). Plasma NOx concentrations were measured. Results: A moderate correlation between plasma creatinine and ADMA (n 5 69, r 5 .608, P o .001), SDMA (n 5 69, r 5 .741, P o .001), and NOx concentrations (n 5 69, r 5 .589, P o .001) was observed. There was no association among plasma ADMA, SDMA, and NOx concentrations and SBP. Conclusions and Clinical Importance: Plasma ADMA and SDMA concentrations are increased in cats with CKD and correlate with plasma creatinine concentration. This may imply the presence of endothelial dysfunction in cats with CKD. Plasma ADMA concentrations were not associated with systemic hypertension. Treatment of systemic hypertension with amlodipine besylate did not affect plasma ADMA or NOx concentrations. Key words: Amlodipine; Nitric oxide; Proteinuria.
itric oxide (NO), produced in the vasculature by the constitutive endothelial nitric oxide synthase (eNOS), is an important vasodilator with a role in the control of vascular tone, blood pressure, and regional blood flow. Experimental studies in rats indicate that chronic inhibition of eNOS results in systemic and glomerular hypertension, proteinuria, and glomerulosclerosis.1 NO concentrations also may be decreased in human patients with chronic kidney disease (CKD) and end-stage renal failure.2–4 Reduced bioavailability of nitrate and nitrite (NOx) results in endothelial dysfunction and has been implicated in the pathogenesis of renal hypertension and progression of kidney disease in humans.5,6 NOx may be decreased because of decreased availability of the substrate arginine because the kidney is an important site for its synthesis.7 Recently, there has been interest in the role of plasma asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor.
N
From the Department of Veterinary Basic Science (Jepson, Elliott); Department of Veterinary Clinical Science, Royal Veterinary College, Royal College Street, Camden, London, UK (Syme); and the WALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds, LEICS, UK (Vallance). Results of this study were presented at the 16th annual ECVIM-CA congress, Amsterdam, The Netherlands, 2006. Corresponding author: R.E. Jepson, Department of Veterinary Basic Science, Royal Veterinary College, Royal College Street, Camden, London NW1 0TU, UK; e-mail:
[email protected].
Submitted March 28, 2007; Revised August 2, 2007; Accepted November 28, 2007. Copyright r 2008 by the American College of Veterinary Internal Medicine 10.1111/j.1939-1676.2008.0075.x
Plasma concentrations of ADMA are increased in human patients with kidney disease and have been associated with increased cardiovascular morbidity and mortality.8,9 Primarily ADMA is degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), with only a small proportion being excreted by the kidneys.10 In kidney disease, therefore, increased ADMA concentrations are independent of decreased renal clearance. DDAH has been colocalized in the kidney with eNOS, and it has been proposed that changes in the availability and activity of DDAH may result in altered ADMA concentrations in patients with kidney disease.11 Plasma symmetric dimethylarginine (SDMA), the inactive stereoisomer of ADMA, is also increased in kidney disease. Unlike ADMA, SDMA is excreted by the kidneys and hence is affected by changes in glomerular filtration rate (GFR). SDMA may play a role in decreasing NOx availability by competition with arginine for cellular uptake mechanisms (Fig 1).12 In studies of humans, infusion of physiologically relevant concentrations of ADMA has been shown to result in concentration-dependent increases in blood pressure, decreases in effective renal plasma flow, and concomitant increases in renovascular resistance.13,14 In experimental studies in rats, administration of L-arginine antagonists resulted in a substantial increase in systolic blood pressure (SBP) and increased glomerular capillary pressure.5 These changes were associated with development of proteinuria and glomerulosclerosis.5 Rats with subtotal nephrectomy to which L-arginine was administered had significantly higher GFR, a greater number of normal glomeruli, decreased tubulointerstitial damage, and a decrease in proteinuria in comparison with rats that did
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Jepson et al Protein
L-arginine
PRMT Methylated Proteins Hydrolysis i SDMA
NOS
ADMA
Renal Excretion i Possible DDAH Inhibitors: -Oxidative stress -Hyperglycaemia -Hypertension -Hypercholesterolaemia
i
Metabolism by DDAH
NOx + Citrulline
Dimethylamine + citrulline
Fig 1. Diagram representing production and degradation of ADMA and SDMA and the synthesis of nitric oxide. NOS, nitric oxide synthase; PRMT, protein arginine methyltransferase; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; NOx, nitric oxide; DDAH, dimethylarginine dimethylaminohydrolase; i, potential inhibition. 15
not receive L-arginine supplementation. Proteinuria may therefore be an indicator of endothelial cell dysfunction in the kidney. Plasma ADMA concentrations also are increased in human patients with essential hypertension when compared with healthy normotensive controls. In this situation, ADMA concentrations have been correlated inversely with NOx and the degree of endothelium dependent vasodilatation and hence endothelial dysfunction.16–18 CKD is a commonly diagnosed condition in the geriatric feline population and has been associated with systemic hypertension in approximately 20% of cases, although prevalence figures as high as 65% have been reported in some studies.19,20 Approximately 20% of cats with systolic hypertension may be nonazotemic and euthyroid, although variable urinary concentrating ability may be present.a Hypertensive retinopathy or choroidopathy is documented in 50–80% of hypertensive cats and may be considered a clinical marker of severe hypertensive vascular damage and endothelial dysfunction.19,20 Our group recently has reported that proteinuria is an independent variable significantly associated with survival in cats with CKD and also in those with systemic hypertension.21,22 We have also documented a substantial decrease in proteinuria with treatment of hypertension using amlodipine besylate in those cats that were mildly to severely proteinuric (urine protein to creatinine ratio [UP/C] 40.2) at the time of diagnosis of hypertension. Calcium channel blockers (CCB) act primarily by inhibition of calcium channels in vascular smooth muscle cells, resulting in vascular relaxation and a decrease in blood pressure. Dihydropyridine CCB (eg, felodipine, nifedipine, diltiazem) but not phenylalkylamine CCB (eg, verapamil) can cause up-regulation of NO production and eNOS expression and activity in cultured endothelial cells.23 In a chronic NO inhibition model in rats treated with the NO antagonist L-NAME, co-administration of nifedipine prevented hypertension and renal morphologic abnormalities.24 Also, the 3rd generation CCB lercanidipine significantly decreased plasma
ADMA concentrations in a transgenic rat model with overexpression of renin and angiotensin genes.25 We hypothesized that a potential mechanism for the decrease in proteinuria seen with treatment of hypertension in cats may be a reduction in ADMA concentrations, increased bioavailability of NOx and resolution of endothelial dysfunction at the glomerular level. The aim of this study was to validate a technique for the measurement of plasma ADMA, SDMA, L-arginine, and NOx concentrations in cats and to evaluate these variables in both normotensive and hypertensive cats with variable azotemia. In the 2nd part of this study, we investigated the change in plasma ADMA and NOx concentrations after treatment of systemic hypertension with the dihydropyridine CCB, amlodipine besylate, with particular reference to those cats in which a clinically relevant decline in proteinuria was seen.
Methods ADMA, SDMA, L-Arginine, and NOx in Normotensive and Hypertensive Cats with Variable Azotemia For the evaluation of plasma ADMA, SDMA, L-arginine, and total NOx in CKD and hypertension, geriatric (4 9 years) cats were recruited from clinics held at 2 first opinion practices in central London (Beaumont Animals’ Hospital and Peoples’ Dispensary for Sick Animal in Bow). In the 1st part of the study, cats were selected in accordance with the following 6 groups: normotensive nonazotemic (creatinine o2.00 mg/dL; normal), normotensive mildly azotemic cats (creatinine 2.00–3.96 mg/dL; Azo), hypertensive mildly azotemic cats with evidence of hypertensive choroidopathy or retinopathy (HT-Ret), hypertensive mildly azotemic cats with no evidence of hypertensive choroidopathy or retinopathy (HT), normotensive moderate to severely azotemic cats (creatinine 4 3.96 mg/ dL; Sev-Azo), and hypertensive nonazotemic cats (creatinine o2.00 mg/dL; Non-Azo HT). Classification of cats into these groups was based on blood pressure measurement and plasma creatinine concentrations obtained at a single time point. However, in all cats classified with CKD, serial measurements of plasma creatinine concentration were available to demonstrate persistence of azotemia. All groups of cats were matched for age at entry into the study. Cats in the normal group were considered to be clinically healthy on the basis of a full history, clinical examination, plasma biochemistry, total thyroxine concentrations, and urinalysis. There was no substantial difference in plasma creatinine concentration among the Azo, HT-Ret, and HT groups or between the normal and Non-Azo HT groups. At entry into the study, all cats received a full physical examination and measurement of SBP using the Doppler techniqueb as previously described.19 A period of acclimatization was allowed for each cat to decrease the likelihood of white coat hypertension.26 A series of 5 measurements were taken on each occasion and subsequently averaged to give a mean SBP for each cat at each visit. Systolic hypertension was defined as SBP 4170 mmHg on 2 separate visits or SBP 4170 mmHg on 1 occasion in association with clinical manifestations of hypertension, most commonly hypertensive retinopathy, or choroidopathy. When present, systolic hypertension was treated with amlodipine besylatec (0.625 mg PO q24h). Hypertensive cats were routinely reexamined 7–21 days after starting antihypertensive medication and until stabilization of blood pressure. In some cases, the dose of amlodipine besylate was increased to 1.25 mg PO q24h in order to attain a target SBP of o160 mmHg. After adequate control of blood pressure, all clients were offered reexamination visits every 8 weeks.
ADMA in Feline Renal Disease The Ethics and Welfare Committee of the Royal Veterinary College and the WALTHAM ethics committee approved the study protocol. Collection and storage of blood and urine samples were performed with the informed consent of the cats’ owners. Blood samples were obtained by jugular venipuncture. Routinely, owners were asked to withhold food for 8 hours before visiting the practice. Blood samples were collected into lithium heparin and EDTA tubes and centrifuged to produce heparinized plasma for immediate biochemical analysis. EDTA plasma was stored at 80 1C and ADMA, SDMA, L-arginine, and NOx concentrations were evaluated retrospectively in these cats. Total thyroxine concentration was measured in all nonazotemic cats at entry into the study to exclude the diagnosis of hyperthyroidism. Plasma ADMA, SDMA, and L-arginine concentrations were measured simultaneously by hydrophilic-interaction liquid chromatography-electrospray tandem mass spectrometry as described by Martens-Lobenhoffer and Bode-Bo¨ger.27 This technique for measurement of ADMA, SDMA, and L-arginine was validated for use in the cat by evaluation of intra- and interassay coefficient of variance (CV). Plasma total NO was measured with a commercially available kitd by the Griess reaction and was performed in accordance with the manufacturer’s instructions. The assay was validated for use with feline plasma by evaluation of intra- and interassay CV, demonstration of dilutional parallelism, and substrate recovery of a spiked sample.
Effect of Amlodipine Besylate Treatment on Plasma ADMA and NOx Concentrations Hypertensive cats recruited for the 1st part of the study also were used to evaluate a potential change in ADMA and total NOx concentrations with amlodipine besylate treatment. These hypertensive cats were stratified by their degree of proteinuria as estimated by their UP/C at the time of diagnosis of hypertension in accordance with the International Renal Interest Society (IRIS)e staging of proteinuria (nonproteinuric o 0.2, borderline proteinuric 0.2–0.4, proteinuric 4 0.4). In all hypertensive cats, a urine sample was collected by cystocentesis both at the diagnosis of hypertension before starting antihypertensive medication and at the 1st available visit after stabilization of SBP. This enabled the evaluation of change in UP/C with antihypertensive medication. UP/Cs were measured at an external laboratoryf by a colorimetric pyrogallol red method to determine urine protein concentration and a colorimetric picric acid method to determine urine creatinine concentration. Pre- and posttreatment UP/C and plasma measurements of ADMA and NOx concentrations were therefore available for the same date in each cat.
Statistical Analysis Computerized statistical softwareg,h was used for all analyses. Probabilities .05 were considered significant. Data are reported as median (range) unless otherwise stated. The Kolmogorov-Smirnov test was used to assess normality of ADMA, SDMA, L-arginine, and UP/C data. ADMA, SDMA, and UP/C data were logarithmically transformed to obtain normality. A one-way ANOVA was used to compare baseline plasma creatinine concentration among groups to demonstrate that the Azo, HTRet, and HT groups and the Normal and Non-Azo HT groups were comparable in terms of their renal function. A one-way ANOVA also was used to compare log ADMA, log SDMA, L-arginine, and total NO concentrations among hypertensive, normotensive, and kidney disease groups. Where necessary the Bonferroni posttest was applied. A Pearson’s correlation was used to evaluate the relationship between log ADMA and log SDMA with SBP and plasma
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creatinine concentration. A Pearson’s correlation also was used to evaluate the relationship between log ADMA and L-arginine and to examine the relationship of total NOx concentrations with log ADMA and creatinine concentrations. For all Pearson’s correlations, the distribution of the combined data sets was normal. Baseline clinical data, including log ADMA, log SDMA, L-arginine, and total NOx concentrations from the hypertensive cats grouped according to severity of proteinuria (nonproteinuric UP/C o0.2, borderline proteinuric UP/C 0.2–0.4, proteinuric UP/C 40.4), were compared by a one-way ANOVA with a Bonferroni posttest. A paired t-test was used to evaluate the change in UP/C, log ADMA, and plasma NOx concentrations pre- and posttreatment of hypertension. A Pearson’s correlation was used to evaluate the relationship between log UP/C and log ADMA and also log UP/ C and total NOx concentrations before starting antihypertensive medications.
Results ADMA, SDMA, L-Arginine, and NOx in Normotensive and Hypertensive Cats with Variable Azotemia In total, 69 cats were included in the study with a mean (SD) age of 14.0 (2.5) years. Clinical variables, plasma L-arginine, and NOx concentrations at baseline are compared between normotensive and hypertensive cats with variable azotemia in Table 1. The mean (SD) ADMA, SDMA, and L-arginine concentrations for the cats used in the assay validation were 6.5(0.3), 2.3(0.1), and 73.5(3.4) mmol/L, respectively. The intra-assay CVs (n 5 6) for ADMA, SDMA, and L-arginine were 5.0, 4.5, and 4.6%, respectively. The interassay CV (n 5 3) for ADMA, SDMA, and L-arginine were 6.8, 14.2, and 4.4%, respectively. The intra-assay CVs for NOx (n 5 5) were 5.67, 6.73, and 2.80% for samples with low (20.18 [1.14] mmol/L), medium (69.93 [4.71] mmol/L), and high (183.71 [5.14] mmol/L) concentrations, respectively. The interassay CVs for NOx (n 5 4) were 19.73, 15.38, and 4.68% for the low, medium, and high controls, respectively. Dilutional parallelism was shown for 1 high concentration sample, and a low concentration sample that was spiked with nitrate showed a 96.85% recovery. A significant difference in log ADMA concentration was found among groups (P o .001) (Fig 2a). Using the Bonferroni posttest, log ADMA concentrations in the normal group were found to differ significantly from both the HT-Ret (P 5 .004) and Sev-Azo groups (P o .001). The difference in log ADMA concentration between the normal group and the HT and Azo groups was not significant (P 5 .068 and .056, respectively). Log ADMA concentrations in the Non-Azo HT group were found to differ significantly from those in the HT-Ret (P 5 .024) and Sev-Azo groups (P 5 .001). Log SDMA concentrations also were found to differ significantly among groups (P o .001) (Fig 2b). Using the Bonferroni posttest, the log SDMA concentrations in the normal group were found to differ significantly from the HT (P 5 .025), Azo (P 5 .026), and Sev-Azo (P o .001) groups. The difference in log SDMA concentrations was not significant between normal and HT-Ret groups (P 5 .06). No significant difference in log SDMA concentrations was found between the normal and NonAzo HT groups. However, the log SDMA concentration
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Table 1. Demographic data, plasma L-arginine, and NOx concentration for normotensive and hypertensive cats with variable azotemia
n Age (years) SBP (mmHG) Creatinine (mg/dL) L-arginine
(mmol/L)
Total NOx (mmol/L)
Normal
Azo
Sev-Azo
HT-Ret
HT
Non-Azo HT
10
10
10
20
10
9
13.6a [11.6–16.7] 125.0a [102.0–160.8] 1.40a [1.05–1.78] 66.2 [31.6–107.4] 10.73a [0.75–34.60]
14.1a [10.5–17.9] 150.0a [141.2–165.5] 2.79b [2.14–3.88] 67.7 [28.7–176.3] 27.52a [1.82–97.20]
13.2a [9.4–18.7] 142.4a [106.4–158.0] 5.50 [4.05–13.7] 84.9 [31.3–210.2] 46.69 [7.42–195.80]
15.1a [10.3–21.0] 208.9b [173.2–276.8] 2.45b [1.66–4.15] 78.3 [38.1–146.4] 31.80a [3.50–109.60]
13.5a [11.0–17.0] 191.4b [174.4–248.5] 2.67b [1.88–6.32] 79.3 [33.2–131.1] 23.52a [5.99–157.60]
13.0a [9.5–16.4] 188.8b [179.6–230.0] 1.57a [1.01–1.83] 75.8 [44.9–125.2] 14.07a [7.42–42.57]
Data are reported as median [range]. n indicates the number of cats in each group. a no significant difference between groups. b no significant difference between groups. Normal, nonazotemic normotensive cats; Azo, normotensive azotemic cats (creatinine 2.00–3.96 mg/dL); Sev-Azo, normotensive severely azotemic cats (creatinine 43.96 mg/dL); HT, hypertensive azotemic cats with no evidence of hypertensive retinopathy or choroidopathy; HT-Ret, hypertensive azotemic cats with evidence of hypertensive retinopathy or choroidopathy; Non-Azo HT, hypertensive nonazotemic cats.
in the Non-Azo HT group differed significantly from all remaining groups (HT-Ret [P 5 .003], HT [P 5 .002], Azo [P 5 .002], Sev-Azo [P o .001]). In addition, there was a significant difference in log SDMA concentrations between the HT-Ret and Sev-Azo (P 5 .011) groups. No significant differences in L-arginine concentrations were found among groups (P 5 .75). A significant difference in plasma NOx was found between groups (P 5 .037). Using the Bonferroni posttest this difference was shown to be between the normal and Sev-Azo group (P 5 .048) (Table 1). No correlation was found between SBP and log ADMA (r 5 .059, P 5 .631). As would be expected, however, a moderate positive correlation was found between plasma creatinine concentration and log ADMA (r 5 .608, P o .001) and log SDMA (r 5 .741, P o .001) (Fig 3a and 3b). A single cat included in this data set had a particularly increased creatinine concentration of 13.7 mg/dL. When this cat was excluded from the data analysis, the correlation coefficient between creatinine concentration and log ADMA was 0.498 (P o .001), between creatinine concentration and log SDMA was 0.685 (P o .001), and between creatinine concentration and NOx was 0.340 (P 5 .005). A weak correlation was found between log ADMA and L-arginine concentrations (r 5 .317, P 5 .008) and also between log ADMA and plasma NOx concentrations (r 5 .391, P 5 .001). A moderate correlation was found between creatinine concentration and plasma NOx (r 5 .589, P o .001) (Fig 4).
Effect of Amlodipine Besylate Treatment on Plasma ADMA and NOx Concentrations Thirty-four hypertensive cats were included in the study to evaluate change in ADMA and NOx with treatment of hypertension. The median number of days between diagnosis of hypertension and the posttreatment urine and blood samples was 35 (7–190) days. Clinical
variables and ADMA and NOx data are summarized for these cats in Table 2 according to their stage of proteinuria at diagnosis of hypertension. As expected, we found a significant decrease in SBP with treatment in all 3 groups of cats (P o .001). A significant decrease in proteinuria was found in the mild proteinuria (P 5 .036) and proteinuric group (P o .001). No significant changes in plasma creatinine concentrations (P 5 .310), log ADMA (P 5 .183), or NOx (P 5 .296) concentrations were found with treatment of hypertension. No correlation was found between log UP/C and log ADMA concentration (r 5 .096, P 5 .541) or between log UP/C and plasma NOx (r 5 .098, P 5 .481).
Discussion Hydrophilic-interaction liquid chromatography-electrospray tandem mass spectrometry has provided a reliable and repeatable method for simultaneous evaluation of ADMA, SDMA, and L-arginine concentrations in feline plasma. The higher interassay CV for SDMA using this technique has been reported in previous studies in humans and relates to the fact that there currently is no isotopically labeled analog available to act as a control.27 Concentrations of plasma SDMA and L-arginine in normal cats appear comparable with normal baseline measurements in humans using this technique.27 However, feline ADMA concentrations in our normotensive nonazotemic euthyroid group appear to be considerably higher than those observed in healthy humans. An association between increasing age and ADMA concentration has been found in human patients with renal disease, and therefore this higher feline ADMA concentration may reflect the elderly population of cats that we have examined.28 However, a recent paper by Pedersen et al29 reported plasma ADMA concentrations in both normal young mixed breed dogs and Cavalier King Charles spaniels with asymptomatic mitral regurgitation.
ADMA in Feline Renal Disease
Fig 2. (a) Plasma ADMA concentration in normotensive and hypertensive cats with variable renal function. (b) Plasma SDMA concentration in normotensive and hypertensive cats with variable renal function. Represents a significant difference between groups when evaluating log transformed data (P o .05). Horizontal bar represents median concentration. ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; Normal, nonazotemic normotensive cats; Azo, normotensive azotemic cats; Sev-Azo, normotensive severely azotemic cats; HT, hypertensive azotemic cats with no evidence of hypertensive retinopathy or choroidopathy; HT-Ret, hypertensive azotemic cats with evidence of hypertensive retinopathy or choroidopathy; Non-Azo HT, hypertensive nonazotemic cats.
Although this paper used a different methodology for measurement, the plasma ADMA concentrations in healthy young dogs were of the same order of magnitude as in the normal elderly cat samples. Unfortunately, no plasma samples from young cats have been analyzed to resolve this discrepancy. The biological relevance of higher circulating concentrations of ADMA in the cat remains to be determined but could relate to a lower sensitivity or specificity of ADMA as an inhibitor of eNOS or to species differences in L-arginine metabolism and hence NO production. Similar to evidence from the human literature, we found that plasma ADMA and SDMA concentrations were significantly increased in cats with CKD. However, there was considerable overlap in ADMA and SDMA concentrations among the normal, mildly, and moderate to severely azotemic groups. A substantial positive correlation was present between both plasma ADMA and SDMA and creatinine concentration. The stronger relationship between SDMA and creatinine concentration
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Fig 3. (a) Correlation between ADMA and plasma creatinine concentration. (b) Correlation between SDMA and plasma creatinine concentration. ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine.
supports the fact that SDMA is primarily excreted by the kidneys. The slightly poorer correlation between ADMA and creatinine concentration suggests, as in the human literature, that ADMA may be only partially excreted by the feline kidney. It is therefore possible that an enzyme equivalent to DDAH may exist in the cat for degradation of ADMA. However, the proportions of ADMA metabolized by an enzyme such as DDAH as opposed to renal excretion cannot be ascertained from this study, and there may be important differences between handling of ADMA in humans and cats. The positive association between plasma ADMA and L-arginine concentrations indicates that cats with CKD are able to maintain adequate L-arginine concentrations. There was no evidence to suggest that plasma ADMA concentrations contribute to the development of hypertension in cats in association with CKD. Nor was there any evidence that plasma ADMA concentrations were higher in those cats with signs of hypertensive vascular damage, namely hypertensive retinopathy or choroidopathy. Interestingly, unlike in human patients, the prevalence of feline hypertension does not increase with increasing severity of kidney disease. Syme et al.19 documented relatively mild azotemia in cats with hypertension, a finding that is complemented by the present study. It is difficult from the current study to implicate ADMA or SDMA in the pathophysiology of feline renal hypertension.
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Fig 4. Correlation between plasma creatinine and total nitric oxide concentrations.
Similarly, despite evidence from the human literature that plasma ADMA concentrations are increased and may contribute to the development of essential hypertension, we were unable to document any significant difference in plasma ADMA concentrations between our normotensive nonazotemic group and the hypertensive nonazotemic cats. Plasma NOx concentrations in cats in this study were positively correlated with the degree of azotemia. We may have hypothesized a decreased availability of NO with increasing severity of renal disease if plasma ADMA concentrations were limiting the productivity of eNOS. However, there are controversial reports in the human literature with regard to NO production and consumption in renal disease.30 There are studies that have evaluated plasma NOx and, similar to our study, have reported increased plasma NOx in human patients with
CKD and also end-stage renal failure patients undergoing hemodialysis.2,31 This finding is supported by the study by Lau et al32 that assessed the activity of the Larginine-NO pathway using radio-labeled arginine in hemodialysis patients and found a significantly increased NO production in end-stage renal failure patients. These studies suggest that whereas NO production and total plasma NOx may be increased in renal failure, there may be a simultaneous increase in consumption and hence decreased bioavailability of NO.30 It is also possible that inflammatory cytokine-mediated upregulation of inducible NOS (iNOS) may contribute to NO production during kidney disease. In the future, there is a role for the development of methods to directly assess endothelial dysfunction in cats with both CKD and hypertension and also to evaluate the L-arginine-NO pathway more completely in the cat. The limitations of evaluating plasma total NOx concentration also must be considered.33 First, there was no dietary control in our population of cats. Although many of the cats with CKD were consuming a low protein, low phosphorus diet provided by the clinics, often this was not the sole source of nutrition. Despite all owners being advised to withhold food for 8 hours before sampling, it is still possible that dietary or water sources of NOx may have contributed to increased NOx concentrations. Second, the measurement of plasma total NOx has considerable limitations because it is not a direct measurement of the functional bioavailability of NO at the endothelial level or more specifically at the level of the kidney.33,34 Some studies have suggested that measurement of plasma nitrite may better reflect constitutive NOS activity or that measurement of cyclic guanosine monophosphate (cGMP) may imply activity of NO.35
Table 2. Clinical variables pre- and posttreatment of hypertension divided according to IRIS classification of proteinuria na Creatinine (mg/dL) Pre Post SBP (mmHg) Pre Post UP/C Pre Post ADMA (mmol/L) Pre Post na Plasma NOx (mmol/L) Pre Post
Nonproteinuric
Mild Proteinuria
Proteinuric
12
12
10
2.53 [1.57–5.25] 2.42 [1.47–4.54] 185.8 [174.4–216.2] 151.6 [129.0–169.0]
2.47 [1.43–4.14] 2.39 [1.30–3.57] 207.8 [177.2–276.8] 142.2 [124.0–157.6]
2.47 [1.73–6.32] 2.74 [1.92–6.74] 212.0 [173.2–259.2] 146.8 [139.2–167.6]
0.12 [0.04–0.19] 0.10 [0.07–1.41]
0.27 [0.21–0.38] 0.20 [0.11–0.47]
0.69 [0.46–1.98] 0.35 [0.26–1.13]
2.89 [1.76–4.74] 3.24 [2.07–4.93] 10
3.20 [1.66–7.58] 3.01 [2.08–5.76] 10
3.01 [1.88–4.92] 2.88 [2.20–4.87] 10
19.56 [5.99–157.63] 26.90 [16.87–212.12]
26.84 [5.66–97.66] 30.58 [3.83–98.24]
37.18 [3.50–109.57] 36.55 [20.37–63.74]
Data are presented as median [Range]. a n indicates the number of cats in each group. pre/post indicates parameters either preantihypertensive medication or after stabilization of systolic blood pressure postantihypertensive medication. A significant change was found between pre- and postantihypertensive medication. SBP, systolic blood pressure; UP/C, urine protein to creatinine ratio; ADMA, asymmetric dimethylarginine; NOx, total nitric oxide.
ADMA in Feline Renal Disease
In hypertensive cats, we found a substantial decrease in proteinuria after initiating treatment with amlodipine besylate. In the human literature, there has been little evidence that amlodipine besylate is able to modulate proteinuria despite providing adequate blood pressure control. In the 20-week study by Praga and coworkers, the angiotensin receptor blocker losartan was compared with amlodipine besylate treatment in nondiabetic proteinuric renal disease patients. Despite similar control of hypertension, amlodipine failed to have any significant impact on proteinuria.36 When amlodipine was compared with the angiotensin converting enzyme inhibitor (ACEi) lisinopril in human patients with nondiabetic renal failure, again amlodipine showed no antiproteinuric effect over 16 weeks.28,37 For this reason, an ACEi or angiotensin receptor blocker either independently or in combination with a CCB is the preferred treatment for hypertension in association with renal disease. The pathophysiologic mechanisms that result in hypertension in cats with renal disease are not fully understood, and although comparisons with studies in humans can be made, the most effective antihypertensive and antiproteinuric drugs for treatment of hypertension and renal disease in cats may not necessarily be equivalent to those utilized in human medicine. Proteinuria was evaluated immediately before and after implementing antihypertensive treatment with amlodipine besylate. The cats included in this study were client owned and it would therefore have been unethical to provide a control group of cats that were not treated with amlodipine besylate after diagnosis of hypertension. The use of selfcontrols and paired samples may have created bias, particularly because we have not performed studies to evaluate the day-to-day variability of either ADMA or NOx concentrations in normal or hypertensive cats. However, we were unable to document any change in plasma ADMA or NOx concentrations with amlodipine besylate treatment. We are therefore unable to confirm whether a change in bioavailability of NO at the glomerular level could be contributing to the reduction of proteinuria apparent in hypertensive cats treated with amlodipine besylate. More recently, there has been interest in human medicine about the use of ADMA not just as a marker of endothelial cell dysfunction but also as a marker of progression of renal disease.17,38 Plasma ADMA concentrations were increased in cats with kidney disease, and in future studies it may prove interesting to evaluate ADMA as a potential predictive marker of progression of renal disease and survival in both normotensive and hypertensive cats. In humans, it is possible to directly evaluate endothelial dysfunction by use of flow-mediated vasodilatation of the brachial artery with resultant reactive hyperemia and subsequently to correlate these clinical findings with potential plasma-based markers of endothelial dysfunction. A clinical technique for functional evaluation of endothelial cell dysfunction would seem to be an important step in determining the presence of endothelial dysfunction in companion animals, and preliminary use of a flow-mediated vasodilatation technique recently has been reported in dogs.39 Although this technique may not be directly transferable to the cat, it would seem im-
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portant for a clinical technique to be developed to determine whether endothelial dysfunction does indeed accompany CKD and hypertension in the cat.
Footnotes a
Elliott, Fletcher M, and Syme HM. Idiopathic feline hypertension: Epidemiological study. Proceedings 13th ECVIM-CA Congress, Uppsala, 2003 (abstract) b Parks Electronic Doppler flow probe—Model 811B; Perimed UK, Bury St Edmunds, UK c Amlodipine 0.625–1.25mg/cat/d, Istin; Pfizer, Sandwich, Kent, UK d Parameter, Total NO/Nitrite/Nitrate; R&D Systems, Abingdon, UK e IRIS staging—(2006) International Renal Interest Society as accepted by the European Society for Veterinary Nephrology and Urology (ESVNU) f Idexx Laboratories, Wetherby, West Yorkshire, UK g SPSS 13.0 for Windows, SPSS Inc, San Diego, CA h GraphPad Prism version 3.02 for Windows, GraphPad Software, San Diego, CA
Acknowledgment This work was funded by WALTHAM Centre for Pet Nutrition.
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