Short-term effects of dietary supplementation with amino acids in dogs with proteinuric chronic kidney disease

Abstract

This retrospective study investigated the impact of amino acid supplementation on body weight, serum albumin, creatinine and urea concentrations, and urine protein-to-creatinine (UPC) ratio in proteinuric dogs with chronic kidney disease (CKD). Forty-six client-owned azotemic dogs with spontaneous proteinuric CKD already on a renal diet and in therapy with enalapril were included. After approximately 1 month of treatment (baseline), 29 dogs received oral amino acid supplementation daily (group A) and 17 dogs did not (group B). The parameters under investigation were determined at baseline and after 4 to 8 weeks in both groups. Compared to baseline, body weight and serum albumin increased (P < 0.01, P < 0.05, respectively) at follow-up in group A, but did not change in group B. Serum creatinine concentration did not change in both groups; urea concentration (P < 0.05) and UPC ratio (P < 0.01) decreased in group B, but not in group A. Supplementation with amino acids increased body weight and serum albumin concentration in these dogs but it might have prevented a decrease in proteinuria and urea concentration.

Introduction

In dogs, proteinuria is often associated with chronic kidney disease (CKD) and studies in this species led to the hypothesis that proteinuria may promote the progression of renal damage, as it does in humans (1–8). In endemic areas for certain vector-borne diseases, such as leishmaniosis, the prevalence of dogs with proteinuria, azotemia, or both, has been reported to be up to 50% (8,9). Among dogs at risk for developing proteinuric nephropathy, other than those living or having lived in endemic areas, there are also breeds that are genetically predisposed to proteinuric CKD (3,4,6,8). Early identification and treatment of proteinuria appears to be crucial in dogs, as its management slows the progression of renal disease, risk of uremic crisis, and renal-related death (1,3,4). Together with treatment of the underlying disease, the major cornerstones of therapy in proteinuric dogs with CKD are angiotensin converting enzyme inhibitors (ACEI), dietary intervention, and omega-3 fatty acids which slow the progression of renal disease, minimize clinical signs of uremia and, at least for the diet, maintain an optimal body weight (BW) and body condition score (1–3,6,9–14). However, in some dogs, anti-proteinuric therapy may not reduce proteinuria despite concurrent administration of diets lower in protein compared to maintenance diets (2–4,8–11,13,14). Furthermore, in proteinuric dogs renal diets may not adequately meet protein requirements, thus possibly leading to low BW, hypoalbuminemia, and malnutrition. One study on a model of spontaneous proteinuric nephropathy in dogs showed that a low protein content diet (14% dry matter), similar to the renal diets commonly recommended in dogs with kidney disease, caused a significant reduction in BW and plasma albumin concentration that were noticeable at 4 wk of administration (8).

In humans with CKD, nutritional status is helpful to identify patients with increased risk of morbidity and mortality: a significant association was observed between decreased baseline BW and subsequent risk of hospital admission (15–18). For these reasons, oral supplementation with amino acids (AA) or intradialytic AA administration has been proposed in malnourished humans with CKD (15–19).

In dogs with CKD and severe proteinuria, either low BW or hypoalbuminemia is frequent and can be associated with increased morbidity and risk of mortality (20). Indeed, albumin hypercatabolism and its down-regulated synthesis can contribute to glomerular disease-associated hypoalbuminemia, possibly leading to marked hypoalbuminemia in dogs with CKD with subnephrotic range proteinuria, thus worsening the prognosis (21). Based on this premise, it seems plausible that the amount of protein needed should be individually tailored in dogs, depending on the stage of CKD and the extent of proteinuria (8). As in humans, also in dogs, AA supplementation may represent the easiest means to correct an insufficient daily intake of proteins. Thus, the aim of this retrospective case-control study was to investigate the impact of an oral AA supplementation during a short period of time on BW, serum concentrations of albumin, creatinine, and urea, and on the urine protein-to-creatinine (UPC) ratio in proteinuric dogs with CKD treated with enalapril and fed a commercial renal diet (RD).

Materials and methods

Animals and inclusion criteria

Medical records of proteinuric dogs in IRIS stages ≥ 2 (14) admitted in 2007 and 2008 at one of the authors’ institutions (AZ, PDI) were reviewed. All the data available on clinical history, physical examination, BW, complete blood (cell) count (CBC), serum biochemical profile, urinalysis, UPC ratio, indirect blood pressure, abdominal ultrasonographic findings, ongoing treatments, and follow-up examinations were collected. Dogs without stable renal function were excluded; stable renal function was defined by serum creatinine concentration that did not increase or decrease by ≥ 20% within 1 mo of initial determination (10). Dogs were considered to be proteinuric if the UPC ratio was above 0.5 (IRIS substage P) (14) in 2 urine samples collected at 1-month interval; dogs that did not fulfil this criterion were excluded. Furthermore, to be included in the study, dogs had to receive enalapril (Enacard; Merial Italia spa, Milano, Italy) at 0.5 mg/kg BW, q12h, and a commercial renal diet (Hill’s Prescription Diet Canine k/d; Hill’s Pet Nutrition, Topeka, Kansas, USA or Royal Canin Renal Canine; Royal Canin SA, Aimargues, France); the amount of diet was according to the recommendation of the companies and corrections were not made if dogs received or did not receive AA supplementation.

Information was collected from each record, to identify dogs that received or did not receive oral AA supplementation [IT IS pet; ACME srl, Cavriago (RE), Italy; formulation shown in Table 1]. Among dogs on AA supplementation, only those taking the daily amount (X mg) of AA arbitrarily calculated using the following formula, BW (kg) × UPC ratio × 20 = X (22), were included. One tablet provided approximately 675 mg of AA. Dogs that had received oral or intravenous AA supplementation within 1 mo from the time of admission were excluded. Finally, dogs were excluded if the diagnostic workup identified inflammation or infection of the genitourinary tract (based on ultrasonography and urinalysis), a pre-renal cause of proteinuria (based on serum biochemistry), and if cardiac disease, neoplasia, or endocrinopathies were diagnosed or suspected. All dogs had been tested for leishmaniosis, ehrlichiosis, and babesiosis, and were not included if an active form of infection was identified or suspected.

Table 1.

Composition of the amino acid supplement per 100 grams.

Branched-chain aliphatic amino acids
isoleucine, leucine, valine	26 g
Aliphatic amino acids
threonine, arginine, lysine	24 g
Sulfur-containing amino acids
cysteine, methionine	7 g
Aromatic amino acids
tyrosine, phenylalanine	11 g
Heterocyclic amino acids
tryptophan, histidine	6 g
Carrier
glucose	10 g
sucrose	10 g
pregelatinized rice	6 g

Additional treatments and follow-up

As a standard of care at the authors’ institution, dogs classified as “severely hypertensive” (systolic arterial pressure ≥ 180 mmHg) according to the IRIS staging system (14) were treated with oral amlodipine (Norvasc; Pfizer Italia srl, Latina, Italy), 0.1 to 0.5 mg/kg BW, q24h, in order to reduce systolic arterial pressure to < 160 mmHg (substage “normotensive” or “borderline hypertensive”). In addition, dogs with severe hypoalbuminemia received oral acetylsalicylic acid at 2.0 mg/kg BW, q24h, to prevent thrombosis. Based on the reference range of serum albumin (28 to 38 g/L), dogs were considered hypoalbuminemic if the albumin concentration was ≤ 27 g/L; severe hypoalbuminemia was arbitrarily defined as a value < 20 g/L.

As stated, dogs with proteinuric CKD were reassessed after 1 mo to check if the renal disease was stable; all throughout the manuscript this time-point will be called baseline. After baseline, dogs were re-evaluated between 4 and 8 wk.

Blood sampling and assay

During each examination, blood samples were collected in dogs fasted overnight, and serum was obtained within 30 min, stored at 4°C and analyzed within 24 h. Results from CBC and serum biochemical analysis, including albumin, total protein, glucose, bilirubin, cholesterol, amylase, alanine transferase, alkaline phosphatase, urea nitrogen, creatinine, sodium, potassium, chloride, and phosphate, were obtained by the same methods (BC-2800Vet, MINDRAY, Mindray Co., Shenzhen, China; Cobas Mira, Roche Diagnostic AG, Basel, Switzerland) in all samples.

Urine collection and urinalysis

An ultrasound-guided cystocentesis was performed in all dogs using a 5-mL syringe connected to a 23-G needle. All urine samples were placed in 10 mL, sterile, evacuated collection tubes, and analyzed by the same operator. Urine samples were examined within 60 min from collection if samples were stored at room temperature (~20°C), or within 4 h if stored at 4°C to 8°C. Urine sediment was obtained by centrifugation (10 min at 900 × g) of 5 mL of urine, followed by removal of 4.5 mL of supernatant, and resuspension of the remaining 0.5 mL of sediment. A sample of 12 μL of the resuspended urine sediment was microscopically assessed. The supernatant was transferred into separate tubes and stored at −20°C to determine the UPC ratio within 7 d. Red blood cells and white blood cells were expressed as mean number of cells/10 high power fields (hpf, 40 × magnification). Urine sediment with bacteriuria, and/or > 5 red blood cells or white blood cells/hpf, was considered indicative of active inflammation and excluded from the UPC ratio evaluation (23).

UPC ratio

To calculate the UPC ratio, protein concentration was measured with pyrogallol red, and creatinine was measured using the Jaffé method on undiluted urine supernatant that was thawed before analysis. Analytes were measured in an automated spectrophotometer (Cobas Mira, Roche Diagnostic AG) in each case.

Statistical analysis

For data evaluation, BW, serum albumin, creatinine and urea concentrations, and the UPC ratio were retrieved from baseline and after 4 to 8 wk in all dogs. Dogs that received the AA supplementation were included in group A, those that did not receive the AA supplementation belonged to group B. To verify whether population characteristics were similar in the 2 groups, baseline age, BW, serum albumin, creatinine and urea concentrations, and UPC ratio were compared with unpaired t-test. Gender distribution, and frequency and severity of hypoalbuminemia were compared between groups with Chi-squared test or Fisher’s exact test.

To study the effect of AA supplementation, BW, serum albumin, creatinine and urea concentrations, and UPC ratio at baseline and after 4 to 8 wk were compared between the 2 groups with paired t-test. Because severe hypoalbuminemia may be associated with morbidity and mortality in dogs (3,20,21), the effect of AA supplementation was also explored in the subset of cases with serum albumin concentration < 20 g/L by paired comparisons between baseline and 4 to 8 wk for the discussed parameters. Normality of all data sets was investigated with Kolmogorov-Smirnov test and non-normally distributed variables were log-transformed to achieve Gaussian distribution before using parametric tests. Results are reported as mean ± standard deviation or as percentages. A P < 0.05 was considered statistically significant. Statistical analysis was performed with commercial software (GraphPad Prism version 4.0; GraphPad Software, La Jolla, California, USA).

Results

Baseline

Forty-six proteinuric CKD dogs in IRIS stages 2, 3, or 4 were included; 29 of them received AA supplementation (group A), while 17 did not (group B).

Age and BW of both groups are reported in Table 2. In group A, 20 (69%) dogs were males (17 intact and 3 castrated) and 9 (31%) were females (8 intact and 1 spayed). Regarding dog breeds, 7 were boxer, 2 of each were German shepherd, dogue de Bordeaux, epagneul Breton or Italian pointer, and 1 of each was cocker spaniel, dachshund, Dalmatian, doberman, dogo Argentino, German pointer, golden retriever, Jack Russell terrier, pit bull and rottweiler; the remaining 4 dogs were cross-breed. In group B, 11 (65%) dogs were intact males and 6 (35%) were females (5 intact and 1 spayed). Regarding dog breeds, 3 were boxer, 2 were Great Dane, and 1 of each was American Staffordshire, Dalmatian, dogo Argentino, dogue de Bordeaux, English setter, German pointer, German shepherd, Irish wolfhound, Labrador and Pomeranian; the remaining 2 dogs were cross-breed. Age, gender distribution and BW did not significantly differ between groups.

Table 2.

Age, body weight (BW), serum concentration of creatinine, urea and albumin, and urine protein to creatinine (UPC) ratio at baseline in dogs receiving amino acid (AA) supplementation (group A) and in dogs not receiving AA supplementation (group B).

	Group A (mean ± SD)	Group B (mean ± SD)
Age (years)	6 ± 3	6 ± 3
BW (kg)	30 ± 14	28 ± 15
Creatinine (μmol/L)	256 ± 115	407 ± 248
Urea (mmol/L)	24 ± 11	25 ± 16
Albumin (g/L)	24 ± 7	24 ± 7
UPC ratio	4.1 ± 4.5	4.3 ± 4.6

SD — standard deviation.

Serum concentration of creatinine, urea and albumin, as well as UPC ratios are reported in Table 2. In group A, 18 (62%) dogs were in IRIS stage 2, 9 (31%) were in IRIS stage 3, and 2 (7%) were in IRIS stage 4. In group B, 7 (41%) dogs were in IRIS stage 2, 5 (29%) were in IRIS stage 3, and 5 (29%) were in IRIS stage 4. Serum concentration of creatinine was significantly lower in dogs in group A (P < 0.05), whereas albumin and urea, and the UPC ratio did not significantly differ between groups. In group A, 20 (69%) dogs had low albumin concentration, 11 of which showed severe hypoalbuminemia; in group B, 11 (65%) dogs had low albumin concentration, 4 of which showed severe hypoalbuminemia. The frequency of dogs with hypoalbuminemia or severe hypoalbuminemia was not significantly different between groups A and B.

Follow-up at weeks 4 to 8

In group A at follow-up, BW increased in 16 (55%) dogs (range: 0.5 to 4 kg), remained unchanged in 11 (38%), and decreased in 2 (7%); by arbitrarily considering BW as stable if it increased or decreased by ≤ 2.5%, 14 (48%) of the 29 dogs had stable BW. The mean BW of dogs (32 ± 15 kg) significantly increased by 6.2% (P < 0.01) compared to baseline. Body weight was available for 10 out of 17 dogs of group B and was increased in 1 dog, equal in 5, and decreased in 4; BW was stable in 5 of the 10 dogs. The mean value did not differ from baseline (26 ± 12 kg; P > 0.05) (Figure 1).

Figure 1.

Dot plot of body weight (BW) in dogs in groups A and B at baseline and 4 to 8 weeks later. After 4 to 8 weeks, BW significantly increased in group A, but did not change in group B.

In group A, serum albumin concentration increased in 19 (65%) dogs, was equal in 2 (7%), and decreased in 8 (28%); by arbitrarily considering albumin concentration as stable if it increased or decreased by ≤ 5%, 8 (27%) of the 29 dogs had stable albumin concentration. The mean albumin concentration (26 ± 7.0 g/L) significantly increased by 2.0 g/L (P < 0.05), compared to baseline. None of the 19 dogs with higher than baseline albumin had concentrations above the reference range. In group B, albumin concentration increased in 8 (47%) dogs, was equal in 2 (12%), and decreased in 7 (41%); albumin was stable in 4 (23%) of the 17 dogs. The mean value (24 ± 8.0 g/L) did not differ from baseline (P > 0.05) (Figure 2).

Figure 2.

Dot plot of serum albumin concentration in dogs in groups A and B at baseline and 4 to 8 weeks later. After 4 to 8 weeks, albumin concentration significantly increased in group A, but did not change in group B.

Serum concentration of creatinine in group A increased in 6 (21%) dogs and decreased in the remaining 23 (79%); by considering creatinine as stable if it increased or decreased by ≤ 20% (10), 13 (45%) of the 29 dogs had stable creatinine. In group B, creatinine concentration increased in 4 (23%) dogs, was equal in 2 (12%), and decreased in 11 (65%); creatinine was stable in 6 (35%) of the 17 dogs. In both groups, mean serum concentration of creatinine measured at 4 to 8 wk did not statistically differ from baseline (group A: 177 ± 177 μmol/L; group B: 301 ± 292 μmol/L; P > 0.05).

In group A, serum concentration of urea increased in 9 (31%) dogs, was equal in 1 (3%), and decreased in 19 (65%); by arbitrarily considering urea as stable if it increased or decreased by ≤ 20%, 13 (45%) of the 29 dogs had stable urea concentration. The mean urea concentration did not statistically differ from baseline (18.9 ± 27.1 mmol/L; P > 0.05). In group B, urea concentration increased in 5 (29%) dogs, was equal in 2 (12%) and decreased in 10 (59%); urea was stable in 6 (35%) of the 17 dogs. The mean urea concentration significantly decreased by 5.7 mmol/L (18.9 ± 12.5 mmol/L; P < 0.05) (Figure 3).

Figure 3.

Dot plot of serum urea concentration in dogs in groups A and B at baseline and 4 to 8 weeks later. After 4 to 8 weeks, urea concentration significantly decreased in group B but did not change in group A.

In group A, the UPC ratio increased in 9 (31%) dogs and decreased in 20 (69%); by arbitrarily considering UPC ratio as stable if it increased or decreased by ≤ 20%, 7 (24%) of the 29 dogs had stable UPC ratio. The mean UPC ratio did not differ from baseline (3.9 ± 4.9; P > 0.05). In group B, the UPC ratio increased in 2 (12%) dogs, was equal in 1 (6%), and decreased in 14 (82%); the UPC ratio was stable in 5 (29%) of the 17 dogs. The mean UPC ratio significantly decreased by 1.9 (2.4 ± 3.5; P < 0.01) (Figure 4).

Figure 4.

Dot plot of urine protein to creatinine (UPC) ratio in dogs in groups A and B at baseline and 4 to 8 weeks later. After 4 to 8 weeks, the UPC ratio significantly decreased in group B, but did not change in group A.

The time at which the examination was performed within the 4- to 8-week interval did not differ between groups (5.5 ± 1.0 wk, both groups).

Dogs with hypoalbuminemia

In group A, serum albumin concentration at 4 to 8 wk was increased compared to baseline in all 11 dogs with severe hypoalbuminemia (serum albumin < 20 g/L). The mean albumin concentration significantly increased by 7.0 g/L (P < 0.001). None of these dogs had detectable subcutaneous edema or ascites, based on physical examination or abdominal ultrasonography, respectively. No significant differences were observed for BW, serum creatinine and urea concentrations, or the UPC ratio. At 4 to 8 wk, the 9 dogs with hypoalbuminemia between 20 and 27 g/L had no significant change compared to baseline in BW, serum albumin, creatinine and urea concentrations, or UPC ratio.

In group B, the 4 dogs with severe hypoalbuminemia had albumin concentration that was decreased in 2 of them and was equal and increased in one of each at 4 to 8 wk compared to baseline. Due to the limited number of cases (4 dogs), statistical analyses were not performed for BW, serum albumin, creatinine and urea concentrations, or the UPC ratio. The 7 dogs with hypoalbuminemia (serum albumin between 20 and 27 g/L) had no significant change in BW, serum albumin, creatinine and urea concentrations, or the UPC ratio at 4 to 8 wk compared to baseline.

Discussion

A significant increase in serum albumin concentration compared to baseline was evident in proteinuric dogs with CKD showing severe hypoalbuminemia (serum albumin < 20 g/L) when receiving AA supplementation. Along with a beneficial effect on serum albumin level, supplementation with AA also increased dogs’ BW, albeit mildly. The effect on BW was evident in the whole group of proteinuric dogs with CKD but not in those with severe hypoalbuminemia, possibly due to the worse nitrogen balance of the latter cases. On the other hand, even though supplementation with AA increased BW and serum albumin concentration in proteinuric dogs with CKD, it prevented the decrease in proteinuria and lowering of urea.

Indeed, at follow-up, proteinuria and urea significantly decreased in dogs that did not receive AA while they did not differ in dogs supplemented with AA. It is therefore possible that in these dogs the reduced efficacy of enalapril and the commercial renal diet on either proteinuria or azotemia was a direct consequence of the positive nitrogen balance and increased protein synthesis induced by the AA supplementation. In fact, diets lower in protein compared to maintenance diets offer a chance to reduce the overall renal trafficking of protein, and if serum protein can be lowered then there is less risk of protein overload across the glomerular barrier, thus leading to less tubular protein reabsorption and inflammation (3). The amount of protein in the diet has a well-known effect on the magnitude of proteinuria, and dogs fed a diet lower in protein compared to maintenance diets have reduced proteinuria, which can in turn improve serum albumin concentration despite the reduction of albumin synthesis that can occur in dogs with CKD (2,3,8). Too strict restriction of protein intake can lead to loss of BW and decreased plasma albumin concentration; therefore, in proteinuric dogs with CKD the protein amount administered daily with food should be tailored to the degree of proteinuria. In these patients, dietary therapy should minimize proteinuria and control plasma albumin concentration while not compromising the nutritional status (8,24). The correct amount of protein might differ depending on the dog’s stage of renal disease and extent of proteinuria (8). The commercial renal diets currently available for dogs have lower protein compared to maintenance diets, and it is possible they do not meet the minimum requirements in the case of severe proteinuria (thus leading to hypoalbuminemia and loss of BW). Meanwhile, the degree of proteinuria is strictly associated with survival and CKD progression in dogs (1,3,11). In light of these findings, it is the authors’ opinion that supplementation with AA should be carried out with caution in proteinuric dogs with CKD, but it might be considered as an adjunctive therapy in severely hypoalbuminemic dogs in which the anti-proteinuric treatment has failed to control proteinuria and maintain plasma albumin concentration within normal limits.

This study has some limitations including its retrospective nature and consequent lack of blinding. It is therefore possible that some of the effects would have been different if cases were randomly allocated to receive or not receive the AA supplementation and the 2 groups were more homogeneous. Indeed, serum concentration of creatinine at baseline was significantly higher in dogs that did not receive the AA supplementation. Then, it cannot be excluded that administering AA supplementation to dogs with higher creatinine concentration is associated with detrimental effects on renal function. In addition, follow-up time of all dogs included in the study was short. A longer follow-up period might have allowed detection of additional differences between the groups.

Additionally, even though owners were instructed to feed their dogs with just 1 of the 2 renal diets available, sometimes they switched to the other. However, the effect of this potential bias was probably minor because both commercial renal diets were expected to be randomly provided to dogs. Furthermore, studies comparing the effect of different diets in dogs with CKD have not been published, but it is likely that the 2 commercial renal diets used for the present study provided similar beneficial effects. The IRIS simply suggests the use of a renal diet, without offering specific guidance on a particular brand on the market (14).

Another limitation is represented by the fact that from medical records it was possible to retrieve BW but not the body or the muscle condition score of the dogs; the latter might have provided more information regarding the potential beneficial effect of AA supplementation. Furthermore, the increase of BW in dogs receiving supplementation of AA at follow-up might have been biased by the concurrent presence of subcutaneous edema or abdominal effusion; however, none of the dogs with severe hypoalbuminemia in the present group developed any of the above. With regard to the same group of dogs, it is worth noting that at follow-up BW increased on average by only 6.2%, thus the beneficial effect of AA supplementation would be questionable. However, by considering the 16 dogs that had an increase of BW, the increase was from 0.5 to 4 kg, possibly suggesting a more relevant gain.

The re-evaluation at 4 to 8 wk may be considered a rather large interval, which might have affected the results. Although this hypothesis is conceivable, the potential bias was evenly distributed in the 2 groups, likely limiting the source of error.

Another factor that might have affected the study results is that the AA provided with the supplementation were predominantly essential AA. Even though it has been demonstrated that humans with CKD have a decrease in circulating essential AA relative to non-essential AA (25,26), there are no data available on the AA blood profile of dogs with renal disease, particularly in those affected by spontaneous proteinuric CKD. Determining the AA profile of these dogs might prove useful in identifying the specific AAs that are needed to correct their imbalance. Finally, even though BW and serum albumin concentrations have been historically considered as insensitive and late indicators of malnutrition, in a previous study these values were considered clinically useful in assessing the adequacy of the nutritional status in dogs with renal proteinuria (8). Our results support the notion that serum albumin concentration represents a helpful indicator to plan dietary modification in proteinuric dogs affected by spontaneous CKD.

In conclusion, proteinuric dogs with CKD treated with enalapril and fed commercial renal diets that received supplementation with AA had improved BW and serum albumin concentration, while maintaining stable serum creatinine. However, administration of AA appeared to prevent the reduction of proteinuria and lowering of urea. In light of these findings, the authors propose the use of AA supplementation in proteinuric dogs with severe hypoalbuminemia that are not adequately controlled with standard treatments consisting of renal diets and ACEI. Relying on serum albumin was useful to identify the benefits of dietary changes in proteinuric dogs with CKD. Further clinical trials are expected to be valuable in order to evaluate the impact of different AA formulations on BW, hypoalbuminemia, and survival time of dogs affected by CKD and severe proteinuria.