Nutritional and laboratory parameters affect the survival of dogs with chronic kidney disease.

Chronic kidney disease is a common disease in dogs, and factors such as serum concentrations of creatinine, albumin, and phosphorus at the moment of diagnosis may influence the survival of these patients. The present retrospective study aimed to evaluate the relationship between survival in dogs with chronic kidney disease and laboratory parameters (creatinine, phosphorus, albumin, and hematocrit) and nutritional parameters (body condition score, muscle mass score, type of food, appetite and feeding method). A total of 116 dogs with chronic kidney disease stages 2 to 4 were included, and survival was calculated considering the time between diagnosis and death. Survival curves were configurated by Kaplan-Meier analysis and a comparison between survival curves was performed by the log-rank test. Factors related to survival were disease stage (p<0.0001), serum phosphorus concentration (p = 0.0005), hematocrit (0.0001), body condition score (p = 0.0391), muscle mass score (p = 0.0002), type of food (p = 0.0009), feeding method (p<0.0001) and appetite (p = 0.0007). Based on data obtained in this study, it is possible to conclude that early diagnosis, as well as nutritional evaluation and renal diet intake, are determinant strategies to increase survival in dogs with chronic kidney disease.

Introduction

Chronic kidney disease (CKD) is considered one of the most common diseases in dogs, and its prevalence can vary between 0.5 to 3.0% of the general population and can be up to 10.0% on the hospitalized canine population [1,2]. It is a progressive disease caused by morphological and functional changes of the kidneys, and clinical signs usually occur when there is more than 70.0% of nephrons compromised, and it can be congenital or acquired, the latter being more common in animals of more than 7 years of age [3-7].

The diagnosis is based on the animal’s history, clinical signs and laboratory exams. The main clinical signs in dogs with CKD are polyuria, polydipsia, emesis, and muscle weakness, which are the result of the decrease in the glomerular filtration capacity and the accumulation of toxic substances [6,7]. The most common laboratory changes in this disease are azotemia, hyperphosphatemia, metabolic acidosis, non-regenerative anemia, decreased urinary specific gravity (isosthenuria) and proteinuria [8,9]. Changes in imaging exams, such as ultrasound, are also an important diagnostic tool, especially for the characterization of congenital alterations [6,7]. To focus on the needs of different CKD stages, the International Renal Interest Society [10] suggests four stages.

Several factors can influence the progression rate of CKD and, therefore, can influence survival. One of these factors is the increase of serum phosphorus, which leads to calcium phosphate deposit in soft tissues, including renal cells, and thus can increase the loss of functional cells [3]. Another factor that can influence progression rate is the normocytic normochromic non-regenerative anemia, which occurs mainly because of decreased synthesis of erythropoietin by the kidneys, and can lead to hyporexia or anorexia and reduce general health and survival [6,11].

Another change common in CKD is hypoalbuminemia, and the main cause in this disease is glomerular protein loss inflammation, and chronic malnutrition [6, 12]. Previous studies have correlated serum albumin concentration with survival in dogs. Michel [13] observed that hospitalized animals with albumin concentrations below 2.7g/dL presented lower hospital discharge rates than animals with concentrations above this value. Regarding survival in dogs with CKD, two studies observed decreased survival in patients with hypoalbuminemia when compared to those with values between the reference ranges of each study [14,15].

Associated with conservative treatment, nutrition is considered an important tool in the management of CKD and, therefore, there is a recommendation to introduce a therapeutic renal diet as of stage 2 [6,8,16]. Nutritional key aspects for patients with CKD are to help control clinical signs of the disease, to reduce electrolytic and mineral disturbances, and to maintain muscle mass score (MMS) and body condition score (BCS) [7]. However, it must be taken into consideration that anorexia and hyporexia are common consequences of CKD and they occur because of the series of metabolic alterations caused by the disease [7,8]. For dogs that, even with support treatment, do not voluntarily ingest sufficient food quantities to supply their energy and nutritional requirements, feeding tubes should be recommended [1,17,18].

There is scarce evidence about factors that influence survival in dogs with CKD. In a retrospective study conducted by Parker and Freeman [14], 100 dogs with CKD were evaluated and it was observed that dogs diagnosed in stage 2 had increased survival than those diagnosed in stages 3 and 4. Furthermore, it was observed that underweight dogs presented decreased survival than those of ideal weight or those who were overweight. A recent prospective study conducted by Rudinsky et al. [15] evaluated the influence of factors on the survival of 27 dogs with CKD. Factors such as hypoalbuminemia, hyperphosphatemia, low BCS and muscle mass loss were associated with decreased survival in these animals.

As for the influence of the type of diet on the survival of dogs with CKD, one study assessed two groups for 24 months, one that received a therapeutic renal diet and one that received a maintenance diet [19]. During the period of the study, dogs that received the renal diet took 2.5 times more time to develop uremic crisis, and survival of this group was almost three times higher than the group that received a maintenance diet.

Based on the current scarce evidence regarding parameters like type of diet, BCS, MMS, and serum concentrations of substances linked to CKD and the survival of dogs with this disease, the present study aimed to evaluate the influence of laboratory and nutritional parameters in the survival of dogs with CKD.

Materials and methods

This study was approved by the Animal Use Ethics Committee from the School of Veterinary and Animal Science of the University of Sao Paulo (FMVZ/USP), protocol number 3138/2013. Information was obtained retrospectively from records of dogs assessed between February 2013 and December 2018 by the Veterinary Nutrology Service of the Teaching Hospital of the School of Veterinary Medicine and Animal Science from the University of Sao Paulo, Brazil. Inclusion criteria were dogs with 12 months of age or older, with serum creatinine concentrations above 1.4mg/dL for more than three months and urine specific gravity of 1.030 or lower, which characterizes animals diagnosed with CKD stages 2 and up [10]. Exclusion criteria were dogs with comorbidities at the moment of diagnosis and animals with incomplete records or with records that did not contain the information necessary to this study, and dogs that were already treated before the assessment. Furthermore, gestating and lactating bitches and dogs diagnosed with congenital kidney diseases were not included.

Information obtained from the records at the time of diagnosis were: age; breed; body weight; BCS [20]; MMS [21]; hyporexia or anorexia; if feeding tubes were used; serum concentrations of creatinine, albumin, phosphorus, and urea; and percentage of hematocrit.

Animals were considered in hyporexia if the daily intake of food was reduced in 25% or more, and were considered in anorexia if the owner reported no food intake and the animal did not accept food (dry and/or wet) offered by the staff of the Nutrition Service at the first assessment.

Data regarding the type of food consumed from the time of diagnosis until death was also collected.

For calculation of survival time, the date of death was obtained from the hospital records or, if not presented in the records, after a telephone contact with the owners. When the date of death was impossible to obtain or if the animal was still alive after the end of the data collection period, this information was considered as censored data and the date considered for the survival curve was from the last assessment performed in the hospital.

Statistical analysis was performed with GraphPad Prism 6.0 (GraphPad Software, USA). Survival curves were calculated with Kaplan-Meier analysis and log-rank test (Mantel-Cox) was used to compare curves. Values of p ≤ 0.05 were considered significant.

Results

A total of 271 dog records were assessed, and 116 animals were included according to the study’s criteria, 59 females and 57 males, with a mean age of 11.4±3.5 years (range 3 to 20 years) and a mean body weight of 13.9±11.4kg (range 1,8 to 54,1 kg) at the moment of diagnosis. The 155 animals that were not included either had comorbidities at the time of diagnosis (n = 134; 86.45%) or did not have complete record information (n = 21; 13.55%). Breeds included mixed breed (n = 40), Poodle (n = 17), Labrador Retriever (n = 9), English Cocker Spaniel (n = 7), Lhasa Apso (n = 6), Miniature Schnauzer (n = 6), Pinscher (n = 5), Dachshund (n = 4), Golden Retriever (n = 4), Yorkshire Terrier (n = 4), Shih Tzu (n = 3), Weimaraner (n = 2) and one of each of the following: Beagle, Boxer, Brazilian Terrier, Bull Terrier, German Shepherd, Kuvasz, Maltese, Pug, and White Swiss Shepherd.

Six dogs were still alive at the time of the data analysis and 16 dogs did not have an established date of death. Of the 94 dogs with death dates, 83 were euthanized due to complications of the disease and 11 died at their household. The distribution of animals according to parameters is described in Table 1.

Table 1

Distribution of dogs with chronic kidney disease according to laboratory and nutrition parameters and their influence on survival.

Item		N. dogs (%)	Survival median (days)	p
Stage1	2	50 (43.1)	475a	<0.0001
	3	43 (37.1)	187b
	4	23 (19.8)	13c
BCS2	1 to 3	48 (41.4)	125a	0.0391
	4 and 5	35 (30.2)	103a
	6 to 9	33 (28.4)	327b
MMS3	0	9 (7.8)	45a	0.0002
	1	34 (29.2)	122a
	2	51 (44.0)	206b
	3	22 (19.0)	255c
Serum phosphorus4	≤ 5.5mg/dL	31 (48.4)	573	0.0005
	> 5.5mg/dL	33 (51.6)	136
Hematocrit4	≤ 37%	59 (53.6)	99	0.0001
	> 37%	51 (46.4)	415
Serum albumin4	≤ 2.3g/dL	18 (17.5)	128	0.2260
	> 2.3g/dL	85 (82.5)	263
Appetite	Anorexia	38 (32.8)	33a	0.0007
	Hyporexia	38 (32.8)	246b
	No alteration	40 (34.4)	429b
Feeding method	Feeding tube	41 (35.4)	32	<0.0001
	Voluntary	75 (64.6)	309
Type of food5	Renal6	63 (54.3)	309	0.0009
	Other	53 (45.7)	92

1Staging according to IRIS [10]

2Body condition score according to Laflamme [20]

3Muscle mass score according to Michel et al. [21]

4Data not present for all dogs in the study

5Type of food consumed for 75% of the survival time or more

6Commercial or homemade diet formulated by the veterinarians of the Nutrology Service team of the hospital (16% crude protein and 0.3% phosphorus in dry matter basis)

a,b,cMedians with more than two variables with different letters were statistically different (p≤0.05).

Stage of the disease, serum phosphorus concentration, BCS, MMS, and hematocrit at the moment of diagnosis influenced survival (Figs 1–5). Serum albumin concentration at diagnosis, however, did not influence survival time (Fig 6).

Fig 1

Survival curve according to CKD stage [10] at diagnosis (p<0.0001).

Fig 5

Survival curve according to the hematocrit at diagnosis (p = 0.0001).

Fig 6

Survival curve according to serum albumin at diagnosis (p = 0.2260).

The feeding method and appetite at diagnosis also influenced survival, although there was no difference between dogs with hyporexia and no appetite alteration (p = 0.1080) but a difference between dogs with anorexia and all the others (p = 0.0088) (Figs 7 and 8).

Fig 7

Survival curve according to appetite at diagnosis (p = 0.0007).

Fig 8

Survival curve according to the feeding method at diagnosis (p<0.0001).

Type of food prescribed at the time of diagnosis were therapeutic renal diets (n = 28; 24.1%), homemade diets with phosphorus and protein restriction (n = 29; 25.0%), commercial senior diets (n = 12; 10.4%), homemade diet for maintenance (n = 9; 7.8%), and other commercial diets (n = 15; 12.9%). Furthermore, milk replacers (n = 6; 5.2%) and a commercial powdered enteral feeding diet (n = 17; 14.6%) were prescribed for animals that had feeding tubes placed at the time of diagnosis. As for the type of diet consumed for 75% or more of the time between diagnosis and death, animals that consumed renal diets (commercial or homemade) presented increased survival time when compared to those who consumed other diets (Fig 9).

Fig 9

Survival curve according to the type of food of 75% or more of the period from diagnosis until death (p = 0.0009).

The distribution of dogs by appetite and feeding method at diagnosis according to CKD stage is presented in Table 2.

Table 2

Distribution of dogs by appetite and feeding method according to the stage of chronic kidney disease at the time of diagnosis.

Item		Appetite			Feeding method
		Anorexia (%)	Hyporexia (%)	No alteration (%)	Voluntary (%)	Feeding tube (%)
Stage1	2	9 (18.0)	17 (34.0)	24 (48.0)	41 (82.0)	9 (18.0)
	3	15 (34.9)	15 (34.9)	13 (30.2)	28 (65.1)	15 (34.9)
	4	14 (60.9)	6 (26.1)	3 (13.0)	9 (39.1)	14 (60.9)

1Staging according to IRIS [10].

Discussion

In the present study, the factors that were associated with increased survival in dogs with CKD were early stages of the disease, higher BCS, higher MMS, phosphorus levels and hematocrit in the reference ranges, along with the intake of renal diets, no alteration in appetite or hyporexia and voluntary feeding.

The stage of the disease had a negative correlation with survival in the studied population, which is similar to previous studies [14,15]. This is an important fact because it demonstrates that early diagnosis of CKD increases considerably the survival of the animal. This may be due to early conservative treatment, which can help correct dehydration, metabolic acidosis, and hyperphosphatemia, as well as the change to a renal diet, which was also associated with increased survival.

The body weight was not evaluated in the present study, but instead, BCS and MMS were evaluated, considering that even if the body weight changes, it can underestimate cachexia or include ascites or edema. Therefore, body weight is not considered to be specific, as observed by Parker and Freeman [14]. Overweight and obese dogs (BCS 6 to 9) presented increased survival than dogs with ideal body weight (BCS 4 and 5) or underweight dogs (BCS 1 to 3) [20], similar to studies conducted by Parker and Freeman [14] and Rudinsky et al. [15]. This reverse relationship between body condition and survival time is known as the obesity paradox, and is been previously observed in dogs with heart failure [22] and in humans, both with heart failure and advanced chronic kidney disease [23,24]. Among the potential reasons for this paradox in patients with heart conditions is the effect of endocrine factors secreted by the adipose tissue [24]. However, the cause of this paradox in dogs with CKD is yet to be determined.

Several chronic diseases can lead to weight loss and also muscle mass loss, including CKD [18]. The BCS is used to evaluate body fat deposits and, therefore, it must be used together with a tool to evaluate muscle mass, once there is no clinical correlation between these two evaluations [20,21]. In the present study, dogs at the time of diagnosis of CKD that presented mild, moderate or severe muscle loss had lower survival when compared to dogs that did not present muscle loss. Another study evaluated the association between MMS and survival in dogs with CKD, with similar results as the present study [15]. This study, however, divided animals into only two groups according to muscle mass: animals with muscle loss and animals without muscle loss. The present study presents an analysis according to the MMS published by Michel et al. [21], which enables a more precise evaluation of the correlation between MMS and survival in dogs with CKD.

Hypoalbuminemia was not correlated to survival in the present study, which differs from previous studies conducted on dogs with CKD [14,15]. The difference observed between the studies may be attributed to the laboratory reference ranges used. In the present study, the minimum reference value for albumin was considered as 2.3g/dL, which is the reference value used in the teaching hospital where the study took place. Rudinsky et al. [15] used a minimum reference value of 2.9g/dL, and in the study conducted by Parker and Freeman [14], this information was not available.

Another parameter that had a negative correlation with survival was the serum phosphorus concentration, similar to the results observed by Rudinsky et al. [15]. The post-treatment serum phosphorus concentrations for dogs with CKD recommended by IRIS [16] are 4.6mg/dL, 5.0mg/dL, and 6.0mg/dL for stages 2, 3 and 4, respectively. To achieve these concentrations, diets with controlled or restricted phosphorus are recommended, as well as the use of phosphate binders [8,16].

Regarding hematocrit percentage, dogs that had values under 37.0% in the present study had 4 times lower survival time than animals with hematocrit values of 37.0% or higher. This differs from results observed by Rudinsky et al. [15], and may be due to two reasons: first, the previous study considered 36.0% of hematocrit; and second, the number of animals in the previous study was 27, as opposed to 116 dogs included in the present study.

As for the influence of diet, Jacob et al. [19] conducted a prospective study for 24 months and observed that dogs with CKD that consumed a therapeutic renal diet had survival time 3 times higher than those that consumed maintenance diets. The results of the present study corroborate these findings. It is important to state that some of the animals in the present study were prescribed diets that were not therapeutic. This occurred when dogs did not accept the renal diet or owners could not afford to purchase the therapeutic diets. However, as seen in the present study and in the study conducted by Jacob et al. [19], the renal diet is the best option for dogs with CKD in terms of survival.

Another factor related to nutrition observed in the present study is that dogs that were anorexic at the moment of diagnosis had lower survival time than dogs with hyporexia or with no changes in appetite. This can be justified considering that anorexia is more common in dogs with advanced stages of CKD, as observed in the present study, which has also been correlated to lower survival [1,8,15]. The feeding method prescribed at the moment of diagnosis did also influence survival. Animals that had feeding tubes placed were mostly in stages 3 and 4, and similar to appetite, the need to use feeding tubes is associated with disease stage [18]. Therefore, the influence of the appetite of the dog at the time of diagnosis, and consequently the need for feeding tubes, are important parameters that can influence survival in dogs with CKD.

Conclusions

Based on the results of the present study it is possible to conclude that early diagnosis is important, so the proper treatment and clinical assessment can be established and, therefore, extend survival. Factors such as BCS and MMS at diagnosis influenced survival, which makes the assessment of these parameters of uttermost importance in dogs with CKD. Furthermore, the intake of a renal diet was determinant to increase survival and should be part of the recommendations for patients with this diagnosis. The present study brings to light important information to better understand the prognosis of CKD and also identify key nutritional points to provide a better quality of life and increase survival in dogs with chronic kidney disease.

30 Apr 2020

PONE-D-20-08705

Nutritional and laboratory parameters affect the survival of dogs with chronic kidney disease

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Reviewer #1: This is a well-written succinct manuscript that will add nicely to the canine CKD literature. I have just a few comments/questions for the authors to address.

1. Since the cases recruited were from 2013-2018, it seems as though they should be staged according to the IRIS guidelines from 2017 and earlier, as the cut-offs between stages 2 and 3 changed in the 2019 version. Also, I am not convinced that Table 1 is necessary. Most readers should be familiar with IRIS staging, and if not, it is referenced accordingly. I will defer to the Editor on these comments.

2. Did you have IRIS substage information for these dogs? UPC? Blood pressure measurement? Please add this if so or comment otherwise if not. Especially since proteinuria and hypertension have also been shown to correlate with survival in CKD patients.

3. It might be nice to include additional data on the dogs included in another table, including medians and ranges or means and standard deviations. I defer to the Editor on this.

4. Since this was a retrospective study, please comment further on how MMS was determined. Is it standard for this to be recorded in all records? This is unfortunately not true for many academic institutions. Were specific scores provided or did the authors extrapolate MMS scores from the records?

5. Please comment on the use of a numerical feline MMS score (Michel) vs. a canine muscle condition score (eg, AAHA & WSAVA recommendations for using descriptive terms (eg, normal, mild, moderate, severe atrophy). It would be ideal to use a descriptive score here.

6. Please comment further on how dogs were determined to be completely anorexic vs. hyporexic. Was their intake compared to RER or MER? How subjective was it? How many days did that history entail? If this is too muddy, I might suggest combining anorexia and hyporexia into one category for statistical analysis.

7. For dogs that had feeding tubes, how long were they in place?

8. Would you care to comment further on why dogs with higher BCS and MMS lived longer than the underweight and muscle wasted dogs? This relates to the obesity paradox and there is a great deal of literature in people with more coming out in dogs and cats with various diseases.

9. Were there any other differences in the dogs regarding medication and/or dietary supplement use? Please comment further on these results.

10. It is important to recognize that not all “senior” diets are appropriate for dogs with CKD, and their nutrient profiles can vary tremendously. It might be nice to make a statement regarding this so that it is not inferred otherwise.

Specific comments:

1. Lines 69-70. In the context of CKD, it seems prudent to list inflammation and malnutrition as additional factors that could influence hypoalbuminemia.

2. Line 80. Please abbreviate body condition score (BCS) here and then use this abbreviation subsequently (eg, line 116)

3. Line 100. Please denote that MMS refers to muscle mass score here before its first abbreviation. See line 116.

Reviewer #2: You have written a very nice paper here with some very interesting results. I thoroughly enjoyed reading it. One thing which does spring to mind which maybe a study worth undertaking is to define reference ranges for these biochemical parameters which will allow for comparison of studies between countries. I have only made minor points as I think the manuscript is good, and well written, so I both commend you, and thank you.

Line 34- influenced survival?

Line 46- perhaps in the hospitalised dog population?

Line 49- replace ‘with’ with ‘of’

Table 1- is this the most up to date details? I believe that this has been updated (but could be wrong)

I am also not convinced that you need this, but If you wish you can leave it in

Line 75- Maybe state what you class as the reference range as in my experience this seems to vary slightly between countries and even between clinics

Line 89- those of ideal weight

Line 99- you perhaps need to define BCS earlier?

Line 100- possibly also need to define MMS?

Line 110- did you have all the clinical stages of disease for each animal in the study?

Line 116- you have the above two terms defined here- these would be better prior to this

Line 117- maybe better reworded as ‘if feeding tubes were used’ ?

Line 127- p = <0.05

Line 130- looks like a space missing between ‘and 116’

Line 132- I wonder if the mean for the body weight is the best, or would a range be better? As a miniature schnauzer would be much less than a golden retriever? Were any differences seen in biochemical parameters between breeds? Or any difference in survival?

Line 140- at the time of data analysis?

Table 2- did you take into account variation among people for BSC and MMS? That may be very different between animals

Also, how did you define hyporexic and anorexic?

Line 171- also influenced survival?

Line 180- at the time of diagnosis?

Line 180-181- Would it be possible to have more details on the contents of these foods? Perhaps as a supplementary table? If not, I think somewhere it would be nice to define what you mean by a renal diet and what that will include.

I do wonder if the figures would be better as a large block rather than individual? But am happy either way so will leave that up to you

Line 203- no alteration was seen in appetite and …

Line 221- at the time of diagnosis?

Line 229- 235- I think this is an important study which needs to be done at some point, to define exact parameters for this disease which allows for cross study comparison, and similar treatment and diagnostics of dogs between surgeries

Line 243- haematocrit values of 37.0% of higher….

Line 258- I think this section ends very abruptly. Perhaps a nice summary line may be useful?

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26 May 2020

Dear Editor,

The authors wish to thank you for the attention given to our research. We have considered all the suggestions and comments by the reviewers, which are addressed below. Thank you once again for considering our work for publishing.

Reviewer #1: This is a well-written succinct manuscript that will add nicely to the canine CKD literature. I have just a few comments/questions for the authors to address.

1. Since the cases recruited were from 2013-2018, it seems as though they should be staged according to the IRIS guidelines from 2017 and earlier, as the cut-offs between stages 2 and 3 changed in the 2019 version. Also, I am not convinced that Table 1 is necessary. Most readers should be familiar with IRIS staging, and if not, it is referenced accordingly. I will defer to the Editor on these comments.

Thank you for your comment. We changed the version of IRIS to that of 2017, and removed Table 1.

2. Did you have IRIS substage information for these dogs? UPC? Blood pressure measurement? Please add this if so or comment otherwise if not. Especially since proteinuria and hypertension have also been shown to correlate with survival in CKD patients.

The information regarding UPC and blood pressure was not obtained because most records were not clear on this information, this information in most cases is in the referral’s records. Unfortunately, as this is a retrospective study, we are only able to use what is on our records.

3. It might be nice to include additional data on the dogs included in another table, including medians and ranges or means and standard deviations. I defer to the Editor on this.

Thank you for your comment. We opted to include ranges for age and body weight in the manuscript (lines 140-141), and not as a table.

4. Since this was a retrospective study, please comment further on how MMS was determined. Is it standard for this to be recorded in all records? This is unfortunately not true for many academic institutions. Were specific scores provided or did the authors extrapolate MMS scores from the records?

The MMS is standard for all the patients assessed by the Nutrology Service at our teaching hospital, and that is why we only included animals that were assessed by the nutrition staff. Specific scores were provided according to the scale between 0 and 3 published by Michel et al. (2011).

5. Please comment on the use of a numerical feline MMS score (Michel) vs. a canine muscle condition score (eg, AAHA & WSAVA recommendations for using descriptive terms (eg, normal, mild, moderate, severe atrophy). It would be ideal to use a descriptive score here.

We determined that the numerical score described by Michel et al. (2011) would be standard for our practice. We believe it does not impact the results of the present study, since the WSAVA recommendations of muscle condition score are based on the research of Michel and coworkers, which was not published then, as cited in the WSAVA Global Nutrition Guidelines (Freeman et al., 2011).

6. Please comment further on how dogs were determined to be completely anorexic vs. hyporexic. Was their intake compared to RER or MER? How subjective was it? How many days did that history entail? If this is too muddy, I might suggest combining anorexia and hyporexia into one category for statistical analysis.

Dogs were determined to be anorexic if the owner reported that the animal did not eat anything at least for a day. When this occurs, it is standard in our practice to offer the animal some types of food (dry and/or wet) to evaluate appetite. If the animal does not accept any food, it is considered anorexic. The dogs were considered hyporexic if the daily intake of food was reduced in 25% or more. We added this to the text to make it more clear to the readers (lines 12-126).

7. For dogs that had feeding tubes, how long were they in place?

It varied greatly between individuals, but it ranged from 1 to 12 days.

8. Would you care to comment further on why dogs with higher BCS and MMS lived longer than the underweight and muscle wasted dogs? This relates to the obesity paradox and there is a great deal of literature in people with more coming out in dogs and cats with various diseases.

Thank you for your comment. We added a paragraph in the discussion section regarding the obesity paradox (lines 229-234).

9. Were there any other differences in the dogs regarding medication and/or dietary supplement use? Please comment further on these results.

There were many differences regarding treatment, especially regarding correction of dehydration and phosphorus chelation drugs. However, as the data compared is that obtained at the time of diagnosis, the treatment did not interfere in the results. As this was not made clear, we added a sentence in the material and methods section (line 115).

10. It is important to recognize that not all “senior” diets are appropriate for dogs with CKD, and their nutrient profiles can vary tremendously. It might be nice to make a statement regarding this so that it is not inferred otherwise.

Thank you for the comment. We included a sentence in the discussion to address this information (lines 268-272).

Specific comments:

1. Lines 69-70. In the context of CKD, it seems prudent to list inflammation and malnutrition as additional factors that could influence hypoalbuminemia.

Thank you for your comment. We included this information and a reference (line 70).

2. Line 80. Please abbreviate body condition score (BCS) here and then use this abbreviation subsequently (eg, line 116)

Thank you for pointing this out. We included the abbreviation (line 81).

3. Line 100. Please denote that MMS refers to muscle mass score here before its first abbreviation. See line 116.

Thank you for pointing this out. We included the abbreviation (line 81).

Reviewer #2: You have written a very nice paper here with some very interesting results. I thoroughly enjoyed reading it. One thing which does spring to mind which maybe a study worth undertaking is to define reference ranges for these biochemical parameters which will allow for comparison of studies between countries. I have only made minor points as I think the manuscript is good, and well written, so I both commend you, and thank you.

Line 34- influenced survival?

Thank you for pointing this out. We corrected the sentence (lines 34).

Line 46- perhaps in the hospitalised dog population?

Thank you. We changed the writing (line 46).

Line 49- replace ‘with’ with ‘of’

Thank you for pointing this out. We corrected the sentence (line 49).

Table 1- is this the most up to date details? I believe that this has been updated (but could be wrong)

I am also not convinced that you need this, but If you wish you can leave it in

Thank you for your comment. As also stated by the other reviewer, we have accepted the suggestion and removed the table.

Line 75- Maybe state what you class as the reference range as in my experience this seems to vary slightly between countries and even between clinics

In this specific sentence, we refer to the reference ranges cited in the references. We included some words in the sentence to make it more clear to the readers (line 75-76).

Line 89- those of ideal weight

Thank you for pointing this out. We corrected the sentence (line 91).

Line 99- you perhaps need to define BCS earlier?

Thank you for pointing this out. We corrected this and cited BCS earlier in the text (line 81).

Line 100- possibly also need to define MMS?

Thank you for pointing this out. We corrected this and cited MMS earlier in the text (line 81).

Line 110- did you have all the clinical stages of disease for each animal in the study?

We are not sure if we understand this questioning. If you mean if we followed-up on animals and recorded if they progressed in the disease, the answer is no. As this was a retrospective study, and the main point was to evaluate the animals at the time of diagnosis, information regarding staging and progression of the disease was not obtained. If you ask if we had animals in all CKD stages, we included only animals stages 2, 3 and 4.

Line 116- you have the above two terms defined here- these would be better prior to this

Thank you for pointing this out. We corrected it, as they are cited earlier in the text (line 119).

Line 117- maybe better reworded as ‘if feeding tubes were used’?

Thank you for pointing this out. We corrected the sentence (line 120).

Line 127- p = <0.05

Thank you for pointing this out. We corrected the sentence (line 136).

Line 130- looks like a space missing between ‘and 116’

Thank you for pointing this out. We corrected the sentence (line 139).

Line 132- I wonder if the mean for the body weight is the best, or would a range be better? As a miniature schnauzer would be much less than a golden retriever? Were any differences seen in biochemical parameters between breeds? Or any difference in survival?

Thank you for your comment. We included the range of age and body weight in the manuscript (lines 141-141). As for the comparison of breeds, we did not analyze this parameter because there is a great variance between the group sizes.

Line 140- at the time of data analysis?

Thank you for pointing this out. We corrected the sentence (line 149).

Table 2- did you take into account variation among people for BSC and MMS? That may be very different between animals

We obtained the BCS and MMS from the records, and evaluations of these parameters were performed by different veterinarians. Although we understand that these parameters may be subjective, we have highly trained veterinarians in our Nutrology Service with experience, and therefore we trust our records to be as close to reality as possible.

Also, how did you define hyporexic and anorexic?

We included a sentence to make it more clear as how we defined anorexic and hyporexic animals (lines 123-126).

Line 171- also influenced survival?

Thank you for pointing this out. We corrected the sentence (line 180).

Line 180- at the time of diagnosis?

Thank you for pointing this out. We corrected the sentence (line 194).

Line 180-181- Would it be possible to have more details on the contents of these foods? Perhaps as a supplementary table? If not, I think somewhere it would be nice to define what you mean by a renal diet and what that will include.

As this was a retrospective study including cases from 2013 to 2018, it is not possible to include a table with the guaranteed analysis of all the diets used since manufactures change their formulation from time to time, and this information could not be obtained. However, we included the information in the legend of Table 1 (line 160).

I do wonder if the figures would be better as a large block rather than individual? But am happy either way so will leave that up to you

Thank you for your comment. We, however, opted to keep the figures as individuals.

Line 203- no alteration was seen in appetite and …

We opted to maintain the sentence as it was. In this sentence, we are referring to “no alteration in appetite” as one of the factors that were associated with survival.

Line 221- at the time of diagnosis?

Thank you for pointing this out. We corrected the sentence (line 238).

Line 229- 235- I think this is an important study which needs to be done at some point, to define exact parameters for this disease which allows for cross study comparison, and similar treatment and diagnostics of dogs between surgeries

We agree. Studies to better define important parameters, especially in common illnesses, are necessary to improve our diagnostics and therefore treatment in veterinary medicine.

Line 243- haematocrit values of 37.0% of higher….

Thank you for pointing this out. We corrected the sentence (line 260).

Line 258- I think this section ends very abruptly. Perhaps a nice summary line may be useful?

Thank you for your comment. We added one sentence to summarize the paragraph (lines 280-282).

References

IRIS. Sataging of CKD. Disponível em: iris-kidney.com/pdf/IRIS_2017_Staging_of_CKD_09May18.pdf>. Access in: 2 dez. 2019.

MICHEL, K. E. et al. Correlation of a feline muscle mass score with body composition determined by dual-energy X-ray absorptiometry. British Journal of Nutrition, v. 106, p. S57–S59, 2011.

FREEMAN, L. M. et al. Nutritional Assessment Guidelines. Journal of Small Animal Practice, v. 00, p. 1–12, 2011.

Submitted filename: Reviewer response[8669].docx

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2 Jun 2020

Nutritional and laboratory parameters affect the survival of dogs with chronic kidney disease

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19 Jun 2020

PONE-D-20-08705R1

Nutritional and laboratory parameters affect the survival of dogs with chronic kidney disease

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