Exposure of urine of domestic cats to different substrates: impact on urine specific gravity

Abstract

Objectives

Urine specific gravity (USG) is a crucial indicator of renal function and is integral in the monitoring of renal disease progression. Although USG is a readily quantifiable parameter, the process of urine collection in cats can induce stress. Utilizing both hydrophobic and non-hydrophobic substrates in litter trays may reduce this stress and enhance owner compliance with urine collection protocols. This study investigated changes in USG as a result of exposing urine to different substrates over different time periods. A second objective was to assess the impact of other urine parameters, including microhematuria, proteinuria, pyuria, glycosuria, crystalluria and cylindruria, on USG within the context of the substrates used.

Methods

A total of 34 cat urine samples were exposed to different substrates (a hydrophobic substrate, waterproof sand and aquarium gravel). Urine absorption and USG values were evaluated 10, 30, 60 and 120 mins after exposure.

Results

The use of aquarium gravel as a substrate was deemed unsuitable because it absorbed the urine samples, thereby hindering sequential evaluations. In contrast, the hydrophobic substrate (Kit4Cat) showed no significant influence on USG. The non-absorbent waterproof sand (Fantastic Sand) significantly increased the USG values of cat urine during exposure periods of 60 mins and 120 mins.

Conclusions and relevance

The hydrophobic substrate used in this study did not interfere with USG monitoring for up to 120 mins.

Introduction

Urine specific gravity (USG) is an easily measurable and clinically significant parameter that can indicate early changes in the urinary system.^{1 –4} The inability to concentrate urine, even without azotemia, can indicate early renal pathology or polyuria-causing conditions. ² Conversely, an increase in USG levels has been associated with reduced spontaneous water intake and may be correlated with diseases such as feline idiopathic cystitis ⁵ and urolithiasis.^3,6,7 Other diseases outside the urinary tract can lead to poorly concentrated urine, such as hypercalcemia, diabetes mellitus, hyperthyroidism, diuretic therapy, diabetes insipidus, hypoadrenocorticism or hyperadrenocorticism. ³

USG levels may be influenced by glycosuria and proteinuria and are susceptible to physiological variations.^4,8,9 As such, serial collections are recommended to confirm the persistence of any alteration.^4,8 In addition, USG values may be slightly higher in home-collected urine samples compared with cystocentesis samples from the same cat. The underlying reason for this discrepancy remains unclear, and the influence of litter box substrate is one potential explanation. ¹⁰

Manipulating cats for urine collection can be challenging and is often considered a stressful event for the animal. ¹⁰ Collecting samples directly from the litterbox for USG monitoring is recommended, given the simplicity of the procedure, as it causes less stress to the cat and improves owner acceptance of the procedure.^{10 –12} The use of hydrophobic substrates and aquarium gravel have both been reported for this purpose in the literature.^2,10,13 However, no studies have yet evaluated the effects on USG when urine is exposed to different substrates. Therefore, the aim of the present study was to investigate the effects of varying exposure times to different substrates on the USG of domestic cats.

Materials and methods

Approval for this study was deemed to not be required by the Ethics Committee for Animal Use at the Federal University of Uberlandia (UFU) as all analyses were performed on urine collected from routine procedures.

A total of 34 urine samples with a USG of <1.060 routinely collected from cats at the Veterinary Hospital of the UFU were evaluated. Clinical indications, method of urine collection and reason for the hospital visit were not considered for selection. Samples with macroscopically identifiable alterations, such as hematuria, were not included. The samples underwent physical, chemical and sediment evaluations following the procedures of the Clinical Pathology Laboratory of UFU. For the analysis of urinary sediment, the samples were centrifuged at 400 g for 5 mins. The majority of the supernatant was carefully removed and the sediment was resuspended in the remaining supernatant, placed on a slide and covered with a coverslip. The preparation was then analyzed immediately under a light microscope, employing dry objectives of 10× and 40× to count each element observed in the microscopic field. The initial USG (pre-exposure) was determined during the physical evaluation of the samples using an optical refractometer (RZ; Contec, 0–12 g/dl or 1000–1060 specific gravity [sg]; resolution 0.2% or 0.002 sg with automatic temperature compensation).

Approximately 20 g of different substrates (aquarium gravel [basalt, soil (Discus Soil; Mbreda) and aragonite], hydrophobic collection substrate [Kit4Cat; Coastline Global] and waterproof sand [Fantastic Sand]) were added to universal collection flasks up to the 10 ml mark. Subsequently, 2 ml of urine from different cats was added to each flask to ensure exposure to the substrate. The USG was then determined at intervals of 10, 30, 60 and 120 mins after contact with the substrate.

The flasks were kept open at ambient temperature (approximately 28°C) during the analysis intervals. At the time of each USG analysis, a drop (50 µl) of urine in contact with the substrate was removed with a pipette.

The results were categorized based on changes detected during urinalysis as follows: microhematuria (<5 red blood cells per 40× field); pyuria (>5 leukocytes per 40× field); proteinuria (⩾0.06 g/l, corresponding to ⩾1 unit on Combur-10 Test dipstick [Cobas]); cylindruria (presence of any type of cast); glycosuria (⩾17 mmol/l, corresponding to ⩾2 units on Combur-10 Test dipstick [Cobas]); and crystalluria (any type of crystal identified).

SAS software (SAS OnDemand; SAS Institute) was used for data analysis. The USG data were subjected to the Shapiro–Wilk and Bartlett tests to verify the normality of errors and homogeneity of variances, respectively. Baseline USG values were compared between groups using ANOVA (GLM Procedure) according to the following model:

$$Y_{ij}=\mu +T_i+e_{ij}$$

where Y _ij indicates the response, μ is a constant, T _i is the effect of substrate and e _ij is the error. Subsequently, data were analyzed using linear mixed models with repeated measures (mixed procedures) according to the following model:

$$Y=X\beta +Zu+e$$

where Y is the data vector, β is the coefficient vector corresponding to the fixed effects, X is the design matrix for the fixed effects, u is the coefficient vector corresponding to the random effects, Z is the design matrix for the random effects part of the model and e is the error vector.

The fixed effects were time (10, 30, 60 and 120 mins), substrate (hydrophobic substrate and waterproof sand), proteinuria (positive or negative), pyuria (positive or negative), microhematuria (positive or negative), glycosuria (positive or negative), crystalluria (positive or negative), cylindruria (positive or negative) and their interaction with time.

The intercept per animal was considered a random effect. The repeated-measures factor was defined as the time in each level of the animal (subject) and a covariance matrix of spatial power structure (SP[POW]) was used. ¹⁴ The baseline USG values (pre-evaluation) were considered as covariates. The least squares means were compared using Tukey’s test. Percentage data were arranged in contingency tables and analyzed using Fisher’s exact test (the FREQ Procedure). A significance level of α = 0.05 was adopted.

Results

The average age of the evaluated cats was 1.96 years (range 1–4). The cohort comprised 16 (47.05%) unneutered female and 18 (52.95%) unneutered male cats. The mean (± SD) pre-evaluation USG value of the samples (baseline values) was 1.044 (0.011) (range 1.024–1.060). Urine absorption in the basalt, soil and aragonite substrates prevented the evaluation of USG, as it made subsequent collection for optical refractometer analysis impossible (Figure 1). Only the hydrophobic substrate and waterproof sand were impermeable, allowing sequential sample collection for analysis (Figure 1). The abnormalities in the urine samples exposed to both substrate groups are presented in Table 1. Overall, we observed no association between the frequency of alterations and substrate (P >0.05).

Figure 1.

Urine exposed to the substrates (a) basalt, (b) soil (Mbreda), (c) aragonite, (d) hydrophobic substrate (Kit4Cat) and (e) waterproof sand (Fantastic Sand). Urine absorption was observed in samples (a–c), making the collection of urine for evaluation impossible

Table 1.

Primary alterations determined by analysis of cat urine samples

Variables	Hydrophobic substrate (n = 17)	Waterproof sand (n = 17)	Total (n = 34)
Microhematuria	10 (58.8)	6 (35.3)	16 (47.0)
Pyuria	1 (5.9)	5 (29.4)	6 (17.6)
Proteinuria	13 (76.5)	12 (70.6)	25 (73.5)
Cylindruria	2 (11.8)	6 (35.3)	8 (23.5)
Glycosuria	4 (23.5)	2 (11.8)	6 (17.6)
Crystalluria	3 (17.6)	4 (23.5)	7 (20.6)

Data are n (%)

The mean (SEM) baseline USG values (pre-evaluation) were 1.045 for the waterproof sand group and 1.043 for the hydrophobic substrate group (0.0028), with no significant differences (P = 0.6191) between groups; however, there was sufficient evidence (P <0.0001) of an association between the baseline covariate values and the subsequent USG responses over time (see Tables 1 and 2 in supplementary material).

Overall, a significant interaction was observed between the group and time (P <0.05). USG was higher in the waterproof sand group at 60 and 120 mins compared with baseline values, whereas there was no significant difference (P >0.05) between groups at 10 and 30 mins (Table 2).

Table 2.

Urine specific gravity means according to group and time of evaluation in feline urine samples

Time of evaluation (mins)	Hydrophobic substrate (n = 17)	Waterproof sand (n = 17)
10	1.044 ± 0.02	1.046 ± 0.02
30	1.044 ± 0.02	1.047 ± 0.02
60	1.044 ± 0.02	1.050 ± 0.02 *
120	1.044 ± 0.02	1.052 ± 0.02 *

Data are mean ± SD

*Significantly different from values at 10 and 30 mins according to Tukey’s test (P <0.05)

Discussion

USG levels increased in the waterproof sand group but remained constant in the hydrophobic substrate group. Alterations in USG values did not lead to any further changes in clinical management. However, in the waterproof sand group, 2 (11.7%) samples showed alterations in USG values that may affect cat clinical management, increasing from <1.030 to >1.035 (Figure 2). The change in USG from <1.030 to >1.035 would initiate different therapeutic approaches as the <1.030 USG finding would initiate further workup for renal or other diseases that result in diluted urine, while a finding of 1.035 would support adequate renal concentrating ability.

Figure 2.

Variation of urine specific gravity over time in samples exposed to the hydrophobic substrate (Kit4Cat) and waterproof sand (Fantastic Sand) substrates

One possible explanation for this variation is the chemical composition and/or lack of standardization of the product, as it was not designed for urine collection and may have affected the results. Some samples of waterproof sand contained a higher number of suspended particles. These particles could have absorbed urine, altering the USG. This is because specific gravity is influenced by the size, weight and quantity of particles in a solution. In addition, we observed significant time-dependent changes in all urinary parameters within the waterproof sand group, further supporting the contraindication of this substrate for urine collection and USG evaluation.

Different urine constituents were evaluated because of the possibility that alterations could have an impact on USG over time. The small number of samples with these abnormalities (<10) limited the ability to draw meaningful conclusions.

The use of an inert aquarium substrate for urine collection is mentioned in the literature;^2,12,14 however, no studies have evaluated its effects on USG. Despite the limited references regarding its indication, we evaluated its use as a potential low-cost option for urine collection. However, as urine was absorbed by the substrate, it was impossible to collect urine for USG measurement.

This study has certain limitations. The impact of various substrates on USG was evaluated under controlled conditions. In addition, we had a small number of cases. The substrate-to-urine ratio used in the experiments may not fully represent real-world scenarios, such as when a cat urinates in a litter box. Furthermore the 120 mins exposure period is short, especially when considering the time it would take an owner to collect a sample and transport it to a veterinary clinic for evaluation. Thus, it is recommended to use only the substrates specifically indicated for urine collection and USG evaluation to avoid variability that may affect clinical management.

Conclusions

Although inert aquarium gravel has been suggested as a potential substrate for urine collection in the literature, it absorbs urine and is not suitable for USG evaluation. Moreover, impermeable sand promotes an increase in USG over time. Further studies are needed to determine the best substrate for home urine collection. Until then, hydrophobic substrates are recommended to minimize the impact on clinical management of the cat.