The Risk Factors And Implications Associated With Renal Mineralization in Chronic Kidney Disease in Cats

ABSTRACT

1. Background

Nephrocalcinosis is a pathological feature of chronic kidney disease (CKD). Its pathophysiological implications for cats with CKD are unexplored.

2. Objectives

Identify nephrocalcinosis risk factors and evaluate their influence on CKD progression and all-cause mortality.

3. Animals

Fifty-one euthyroid client-owned cats with International Renal Interest Society (IRIS) stages 2-3 azotemic CKD.

4. Methods

Retrospective cohort study. Histopathological kidney sections were assessed for nephrocalcinosis (von Kossa stain). Nephrocalcinosis severity was determined by image analysis (ImageJ). Ordinal logistic regressions were performed to identify nephrocalcinosis risk factors. The influence of nephrocalcinosis on CKD progression and mortality risk were assessed using a linear mixed model and Cox regression, respectively. Cats were categorized by their owner-reported time-averaged phosphate-restricted diet (PRD) intake, where PRD comprised ≥50%, 10-50%, or none of the food intake.

5. Results

Nephrocalcinosis was rated as mild-to-severe in 78.4% and absent-to-minimal in 21.6% of cases. Higher baseline plasma total calcium concentration (tCa; odds ratio [OR] = 3.07 per 1 mg/dL; P = .02) and eating a PRD (10%-50%: OR = 8.35; P = .01; ≥50%: OR = 5.47; P = .01) were independent nephrocalcinosis risk factors. Cats with absent-to-minimal nephrocalcinosis had increasing plasma creatinine (0.250 ± 0.074 mg/dL/month; P = .002), urea (5.06 ± 1.82 mg/dL/month; P = .01), and phosphate (0.233 ± 0.115 mg/dL/month; P = .05) concentrations over 1 year, and had shorter median survival times than cats with mild-to-severe nephrocalcinosis.

6. Conclusion and Clinical Importance

Higher plasma tCa at CKD diagnosis and PRD intake are independently associated with nephrocalcinosis. However, nephrocalcinosis is not associated with rapid CKD progression in cats.

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KEYWORDS

calcification, CKD-MBD, hypercalcemia, nephrocalcinosis.

INTRODUCTION

Disturbances in mineral and bone metabolism are present in cats with chronic kidney disease (CKD), even in the early stages of the disease.1,2 The kidneys play a fundamental role in calcium and phosphate homeostasis. A gradual decrease in functioning nephrons in CKD results in phosphate retention,3 stimulating phosphaturic hormones, fibroblast growth factor 23 (FGF23), and subsequently parathyroid hormone (PTH), to maintain physiological plasma phosphate concentrations.4-6 However, not only does this adaptive response influence calcium metabolism regulation but also bone remodeling. When this adaptive response eventually fails to prevent plasma phosphate concentration from increasing, it promotes ectopic calcification. These adaptive responses are collectively referred to as chronic kidney disease-mineral and bone disorder (CKD-MBD).7

Nephrocalcinosis is characterized by tubulointerstitial calcium phosphate (CaP) or calcium oxalate (CaOx) crystal deposition,8 in humans almost exclusively in the renal medulla.9 This process begins from Randall's plaques formation in the renal papillae, which act as a nadir for progressive calcification.9,10 Observation of this phenomenon on light microscopy is termed microscopic nephrocalcinosis (referred to herein as nephrocalcinosis).8,11 Spontaneous ectopic calcification can be explained, in part, by calcium and phosphate salts precipitating from supersaturated fluid when the calcium phosphate product (CaPP) exceeds the solubility product. Previous studies showed that increased serum phosphate concentration and CaPP correlated positively with renal calcium content,12,13 and increased serum calcium concentration was an independent risk factor for nephrocalcinosis in human CKD patients.11 Nephrocalcinosis is prevalent in humans and cats with CKD.14,15 Renal calcium deposition was found on histology in ≥50% of cats with International Renal Interest Society (IRIS) Stage 2 to 4, compared to 21% in non-azotemic cats.15 In vivo in rats, renal calcium content was negatively correlated with creatinine clearance, suggesting a deleterious impact on renal function.13,16

Cats with CKD are at increased risk of developing plasma total hypercalcemia, with prevalence increasing with advancing azotemia.17 In CKD cats eating a phosphate-restricted diet (PRD), increasing plasma phosphate and total calcium (tCa) concentrations are associated with CKD progression.18 However, it remains to be determined whether calcium and phosphate homeostasis dysregulation plays a role in nephrocalcinosis pathogenesis. Furthermore, the implications of nephrocalcinosis associated with CKD in cats have not been investigated. Our study objectives were to: (a) explore risk factors for nephrocalcinosis in CKD cats and (b) assess associations of nephrocalcinosis with changes in CKD-MBD parameters, CKD progression, and all-cause mortality.

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METHODS

1. Case selection

Records of 2 London-based first-opinion practices between 1 January 1992 and 31 December 2017 were reviewed and azotemic CKD cats that underwent complete necropsy examinations were identified. Azotemic CKD diagnosis was defined as a plasma creatinine concentration ≥2 mg/dL with a urine specific gravity (USG) <1.035, or plasma creatinine concentration ≥2 mg/dL on 2 consecutive occasions 2-4 weeks apart without evidence of a pre-renal cause. All cats with a CKD diagnosis were offered a PRD. A variety of PRD was used throughout the study (Feline Low Protein Diet [wet]; Masterfoods, Bruck, Austria; Waltham Veterinary Diet, Feline Low Phosphorus Low Protein [dry and wet]; Effem, Minden, Germany [dry] and Masterfoods, Bruck, Austria [wet]; Feline Veterinary Diet Renal [dry and wet], Royal Canin SAS, Aimargues, France [dry] and Masterfoods, Bruck, Austria [wet]) with a phosphorus content of 0.7-1.1 g/Mcal and calcium-to phosphorus ratio (Ca:P) of 1.3-2.1. Cats not accepting a PRD continued on their maintenance diets.

Inclusion required a formalin-fixed paraffin-embedded (FFPE) kidney block for histopathological evaluation. Cats were excluded if they had clinically suspected hyperthyroidism and their plasma total thyroxine (TT4) concentration was >40 nmol/L, they were being medically managed for hyperthyroidism, had evidence of another concurrent disease, or were being treated with corticosteroids, furosemide, or bisphosphonates. Cats with IRIS CKD stage 4 at diagnosis and cats with no follow-up visit after CKD diagnosis also were excluded. Cats receiving amlodipine besylate for systemic hypertension were included.

2. Clinicopathological data

Blood, urine, and necropsy samples were collected with owner informed consent and Royal Veterinary College Ethics and Welfare Committee approval (URN20131258E). Blood samples were collected by jugular venipuncture into heparinized and ethylenediaminetetraacetic acid (EDTA) tubes and urine was obtained by cystocentesis. Samples were stored at 4
C for <6 hours before centrifugation and separation. Heparinized plasma was analyzed biochemically at an external laboratory (IDEXX laboratories, Wetherby, UK). Inhouse urinalyses, including USG measurement by refractometry, dipstick chemical analysis, and microscopic urine sediment examination, were performed on the day of collection. Urinary tract infection was confirmed by bacterial culture (Royal Veterinary College Diagnostic Laboratory Services, Hatfield, UK).

Systolic blood pressure (SBP) measurements were made as previously described by Doppler.19 Indirect ophthalmoscopy was performed using a retinal camera (ClearView, Optibrand, Fort Collins, Colorado) in cats with an average SBP >160 mm Hg. Systemic hypertension was defined as an average SBP >160 mm Hg in conjunction with ocular pathology consistent with hypertensive damage, or SBP >170 mm Hg on 2 consecutive occasions.

Clinical records were reviewed to extract the following: age, sex, breed, body weight, SBP, tCa, ionized calcium (iCa), plasma creatinine, urea, phosphate, potassium, sodium, chloride, total protein, albumin, and TT4 concentrations, plasma alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities, PCV, USG, and urine culture results. The proportion of PRD recorded as being fed at every visit (until death) from each cat with PRD prescribed after CKD diagnosis was reviewed from our clinical records. Owners were asked at each visit to estimate the proportion of PRD by volume of the total quantity of food fed. A time-averaged proportion of PRD fed by volume of the total ration was calculated from this estimate for each cat. Where the proportion fed was missing from the record at a particular visit, the proportion stated at the previous visit was imputed, except if it was the visit at which euthanasia was recommended and the cat had deteriorated clinically from the previous visit. Cats were categorized according to the time-averaged ingestion of PRD, where PRD comprised ≥50%, >10%, <50%, or none of their food intake for their CKD duration.

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3. Histopathological data

Necropsy examinations were offered to all clients when their cats were euthanized and informed consent was obtained from those who agreed. At necropsy, each kidney was dissected longitudinally and transversely and fixed (10% neutral buffered formalin). Formalin-fixed renal tissues (including cortex and medulla) were paraffin-embedded for future histopathological evaluation. A 4-μm section of FFPE tissue was stained with von Kossa to identify hydroxyapatite deposits.20 Stained sagittal kidney sections were imaged, blinded to case data, using a digital microscope (Leica DM4000, Wetzlar, Germany) for microscopic nephrocalcinosis assessment (Figure 1). ImageJ (Version 2.1.0, National Institutes of Health, Bethesda, Maryland) was used to quantify nephrocalcinosis from representative images captured at the medullary region. Nephrocalcinosis was graded according to the average proportion of positively stained tissue from 5 medullary images captured at 10 magnification. They were graded as follows: <0.06% = grade 0, 0.06% to 1% = grade 1, and >1% = grade 2 (Figure 1). This grading system, pre-determined before statistical analyses, provided an objective quantification of nephrocalcinosis severity (0.06% and 1% are equivalent to 1  10 5 cm2 and 1.4  10 4 cm2, respectively). An adjacent FFPE section from each case was stained with Alizarin red (at pH 4.2), allowing CaP and CaOx crystal differentiation.20

FIGURE 1 Kidney, von Kossa stain,x10 magnification. An example of each nephrocalcinosis grade (0-2) is shown as follows: (A) and (B) cat no. 7 with grade 0 nephrocalcinosis (0.004%); (C) and (D) cat no. 34 with grade 1 nephrocalcinosis (0.327%); (E) and (F) cat no. 39 with grade 2 nephrocalcinosis (5.51%). Von Kossa positive staining is outlined in black coloring in the original images (A, C, and E), or bright red after processing using ImageJ (B, D, and F; 8-bit color; threshold 0-92) for the quantification of the proportional area of nephrocalcinosis in each case (n = 51)

4. Micro-computed tomography (micro-CT) for macroscopic nephrocalcinosis

The FFPE kidney blocks were scanned for macroscopic nephrocalcinosis using a micro-computed tomography (CT) scanner (Skyscan 1172, Bruker, Kontich, Belgium) using a small (4000 x 2672 pixels) camera without a filter. Images were obtained using the following scanning parameters: isotropic voxel size 5 μm per pixel, source voltage 50 kV, source current 200 μA, exposure time 670 ms, and imaging rotation scan 180
with a 0.4
rotation step. Projection images were reconstructed into tomograms using NRecon 1.7.5.9 (Bruker, Kontich, Belgium) and repositioned using Dataviewer 1.5.6.6 (Bruker, Kontich, Belgium). Tomograms were analyzed using Bruker analysis software CT-Analyzer (CTAn) 1.20.3 (Bruker, Kontich, Belgium) and volume-rendered 3-dimensional (3D) visualizations were created using CTVox 3.3 (Bruker Kontich, Belgium). Briefly, for each block, tissue volume was calculated by multiplying the depth by the average area from 5 tomograms spaced at equal distances throughout the sample; nephrocalcinosis volume was calculated using the 3D analysis tool in CTAn. The nephrocalcinosis volume-to-kidney tissue ratio (VN: KT) was calculated using the formula:

5. Statistical analysis

Statistical analyses were performed using R software (Version 4.1.1 GUI 1.77 High Sierra build, R Foundation for Statistical Computing, Vienna, Austria). Type I error rate was set at .05. Continuous variables were assessed for normality by visual inspection of histograms and using the Shapiro-Wilk test. Levene's test was used to test if the groups had equal variances. Most data were not normally distributed and therefore numerical data are presented as median (25th, 75th percentile) for consistency. Categorical data are presented as percentages.

5.1 Nephrocalcinosis risk factors

Baseline variables were compared between groups by either 1-way analysis of variance (ANOVA) followed by the Tukey posthoc test or Kruskal-Wallis and Dunn's posthoc test for continuous variables with normal or skewed distributions, respectively. Proportions of categorical outcomes were compared using Fisher's exact test.

Baseline microscopic nephrocalcinosis risk factors were assessed using ordinal logistic regression. Age, body weight, tCa, creatinine, urea, phosphate, potassium, sodium, chloride, total protein, albumin, ALT, ALP, PCV, USG, and CKD survival time were entered as continuous variables, whereas sex and proportion of PRD ingested (“Not eating PRD” vs “Eating 10%–50% PRD” vs “Eating ≥50% PRD”) were entered as categorical variables for univariable analyses. Variables associated with nephrocalcinosis at P < .10, and with data available for at least half of the cats (n > 25), were entered into a multivariable model. Manual backward elimination was applied to obtain the final model with P < .05. Ordinal version of the Hosmer-Lemeshow test was used to evaluate the final model’s goodness-of-fit, and presence of co-linearity among the significant independent risk factors (P < .05) was assessed by variance inflation factor. Residual for outliers and influential observations were checked by Quantile-Quantile plot visual inspection. A linear relationship between the continuous predictor and the logit was assessed by categorizing the variables into equal intervals of “low,” “medium,” and “high” in the logistic regression analysis and evaluating the increase or decrease trend of the coefficient. Results are reported as odds ratio (OR; 95% confidence interval [CI]).

5.2 Changes in CKD-MBD parameters over time about nephrocalcinosis

Linear mixed-effects models were used to assess changes in continuous clinicopathological variables over time. Longitudinal data from all available visits during the first 365 days after CKD diagnosis were included for the following: body weight, tCa, creatinine, urea, phosphate, potassium, sodium, chloride, total protein, albumin, ALT, ALP, and PCV. Group (“grade 0” vs “grade 1” vs “grade 2”), time (in months [30.4 days]), and the interaction between group and time were treated as fixed effects. Each cat's case number and time nested within individual cats were included as 2 uncorrelated random effects. Residuals were assumed to be independent in the model, and normality was checked. No attempt was made to impute missing data. Results are reported as coefficient (β) ± SE.

5.3 Association of nephrocalcinosis and other factors with survival

The date of azotemic CKD diagnosis was defined as a baseline, whereas the death of all-cause was the event of interest. Survival times were depicted with a Kaplan-Meier curve and were compared among groups using the log-rank test and Kruskal-Wallis with Dunn's posthoc tests because all cats reached the study endpoint. Baseline variables associated with survival were explored using Cox proportional hazard analysis. Martingale residuals were used to assess the assumption of linearity of the continuous variables in the Cox model. Residuals for outliers and influential observations were checked by Quantile-Quantile plot visual inspection. Continuous variables were transformed into categorical variables based on tertiles (age, sodium) if the assumption of proportional hazards, as evaluated by Kaplan-Meier curve inspection, and assessment of the independence between each variable and time were not met. Variables associated with survival with P < .10 in univariable analyses were entered into a multivariable Cox model. The final Cox regression model was derived by manual backward elimination with P < .05. Results are reported as hazard ratio (HR; 95% CI).

5.4 von Kossa with Alizarin red staining correlation and macroscopic nephrocalcinosis

For each case, proportional nephrocalcinosis areas in the renal medulla at X 2.5 magnification, as stained by von Kossa and Alizarin red separately, were calculated. The relationship between these 2 staining techniques for microscopic nephrocalcinosis was evaluated using Spearman's correlation. In a subset of 49 cases, macroscopic nephrocalcinosis was assessed using micro-CT. Spearman's correlation was used to evaluate the relationship between proportional nephrocalcinosis volume (micro-CT) and proportional nephrocalcinosis area (von Kossa, overview X 0.14- X 0.36 magnification).

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DISCUSSION

Our results indicated that higher baseline plasma tCa concentration and PRD ingestion were independent of nephrocalcinosis risk factors. Cats with grade 0 nephrocalcinosis were associated with increasing plasma creatinine, urea, and phosphate concentrations over the first 365 days after azotemic CKD diagnosis. This cohort of cats also had significantly shorter survival times compared to those with more severe nephrocalcinosis (grades 1 and 2). A significant independent positive association between dietary phosphate restriction and survival was observed.

Renal mineralization is a complicated and multifaceted process and its pathogenesis in association with CKD remains unclear. However, increasing evidence suggests that mineral metabolism disturbances, particularly calcium, and phosphate, are likely to contribute to nephrocalcinosis.11,12 In our study, higher plasma tCa at CKD diagnosis, even with tCa within the reference interval, was an independent nephrocalcinosis risk factor; only 1 (2%) cat had total hypercalcemia at baseline. This finding suggests that mild perturbations in calcium homeostasis may promote renal mineralization in CKD cats. Consistent with our findings, a CKD study in humans showed that serum tCa concentration was an independent nephrocalcinosis risk factor.11 Furthermore, an association between total hypercalcemia and nephrocalcinosis was found in human patients after kidney transplantation,21 and patients with macroscopic nephrocalcinosis by CT imaging had higher tCa and iCa concentrations than those without.22 However, a histopathological study in humans found no association of serum tCa concentration with renal calcium content and renal tubular calcium deposition.12 The discrepancies in calcium involvement in nephrocalcinosis between studies are attributable to the study population heterogenicity, sample size, and renal calcification detection methods.

Ingestion of a PRD was identified as an independent nephrocalcinosis risk factor in CKD cats in our study. Dietary phosphate restriction significantly influences mineral and hormonal regulation in CKD-MBD including decreases in FGF23 and PTH.23,24 A recent study showed that certain CKD cats developed an increasing trend in tCa and iCa concentrations, together with higher urinary calcium excretion, after a PRD initiation.18 Lower dietary phosphate content and higher dietary Ca:P ratio, when compared with commercially available foods formulated for healthy adult cats,25 potentially could enhance intestinal calcium absorption and increase plasma calcium concentration.26 In human patients, hypercalciuria is a common nephrocalcinosis risk factor.27 It is postulated, therefore, that nephrocalcinosis may be driven by the increase in plasma tCa concentration and enhanced urinary calcium excretion after transitioning to a PRD in CKD cats. However, direct evidence supporting this hypothesis could not be obtained in our study and prospective studies measuring urinary electrolytes are required.

Our results showed that nephrocalcinosis is not positively associated with CKD progression and all-cause mortality. This finding is intriguing because it is somewhat contrary to a previous study in humans that suggested a detrimental role of nephrocalcinosis on renal function by identifying a positive correlation between renal calcium content and serum creatinine concentration.12 Nephrocalcinosis identified by imaging (CT, ultrasonography, or radiography) also has been associated with an increased risk of end-stage kidney disease in human patients.28 A study in CKD cats showed renal mineralization association with more severe interstitial inflammation and fibrosis, suggesting its role in accelerating CKD progression.15 However, recent cohort studies involving human patients with kidney CT imaging found a lack of association between renal function and renal calcification.22,29 This observation is consistent with our results and supports our hypothesis that nephrocalcinosis, to a certain extent, may not exert a direct deleterious effect on kidney function and contribute to CKD progression. Nonetheless, future prospective studies are required to better understand nephrocalcinosis implications for CKD deterioration in cats.

Although PRD ingestion was an independent nephrocalcinosis risk factor, our survival analysis showed that dietary phosphate restriction (when ≥50% PRD was ingested) was associated with longer MST and increased the odds of survival by up to 3-fold for CKD cats when compared to cats that consumed their maintenance diets (Table 4 and Figure 4). These findings support previous studies that found a survival benefit resulting from dietary phosphate restriction in CKD cats.30-32 Moreover, results from our sub-analysis (Tables S2 and S3) showed that CKD cats that continued to be fed maintenance diets had greater increases in plasma creatinine, urea, and phosphate concentrations and a decrease in PCV and body weight over the first year after CKD diagnosis, further reinforcing current evidence on the salutary effects of dietary phosphate restriction for alleviating disease progression.23,24,32,33 Hyperphosphatemia is associated with a decrease in renal function,34 and phosphate binders have been shown to protect against the progression of CKD.35,36

Plasma phosphate concentration and PCV were independent predictors of all-cause mortality. Hyperphosphatemia and anemia are associated with more progressive CKD and poorer prognosis in cats and humans.37-42 Phosphate retention occurs as glomerular filtration rate (GFR) decreases but, in the early stages of CKD, plasma phosphate concentrations usually are maintained within physiological limits as a result of adaptive hormonal regulatory mechanisms, including increased FGF23 and PTH production and decreased calcitriol production.6,43 Therefore, higher plasma phosphate concentration at CKD diagnosis could be suggestive of more severely deranged phosphate homeostasis and associated with a higher risk of death. Consistent with our results, a previous study found that higher serum phosphate concentration was independently associated with survival in 773 CKD cats.42 The pathogenesis of anemia in CKD is multifactorial but decreased erythropoietin production is a main contributing factor.42,44 In support of our findings, other studies also found an association between lower PCV and increased mortality in CKD cats,38,39,45 but not all.42 Interestingly, neither plasma creatinine concentration nor IRIS staging was a predictor of death in the present study, which is inconsistent with previous studies.38,39,42,45,46 Plasma creatinine concentration reflects GFR and is a surrogate biomarker commonly used to assess kidney function.47 The discrepancy could be explained by the exclusion of IRIS stage 4 CKD cats in our study, and the comparatively small sample size, with only 17 IRIS stage 3 CKD cats. Additionally, 29% (n = 15) of cats with azotemic CKD in our study did not receive dietary management for CKD, which may have affected the outcome and precluded an accurate assessment of the predictive value of plasma creatinine concentration, and potentially other variables at the time of CKD diagnosis on survival in our study.

Detection of calcium deposition was performed with both the von Kossa method using silver nitrate and Alizarin red staining. The von Kossa method principle is based on the binding of silver ions with anions, such as phosphate, oxalate, and carbonate, from calcified tissues and the reduction of silver salts, leading to the observation of black metallic silver staining.20,48,49 Alizarin red staining is another technique used for the demonstration of calcium crystals.50 It binds directly to calcium ions and can be used to differentiate CaOx from CaP because CaOx can only be stained with Alizarin Red at a pH of 7 but not at pH 4.2, with the latter used in our study.51,52 We observed a strong correlation between these 2 nephrocalcinosis staining methods, suggesting that the mineral deposits were primarily composed of CaP. This observation is interesting because the majority of upper urinary tract uroliths in cats contain CaOx.53 It may be that precipitation of CaP is a prerequisite for nephrocalcinosis, nephrolithiasis, or both in cats, a process that resembles the formation of Randall's plaques in humans.8 Micro-CT scanning was performed on the FFPE kidney samples to determine whether a single von Kossa-stained slide was representative of nephrocalcinosis. We found a moderate correlation between micro- and macroscopic nephrocalcinosis, further supporting the techniques and quantitative methods used for classifying nephrocalcinosis severity in our study, but suggesting, as might be expected, that a single kidney tissue section gives an approximate estimate of the severity of macroscopic nephrocalcinosis assessed by 3D imaging.

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Our retrospective study had some limitations. Nephrocalcinosis was identified from kidney samples collected at necropsy, and it is unknown at what point renal calcification developed in these cats. Nephrocalcinosis can have developed before CKD diagnosis. Only cats with a necropsy examination performed were included in the study. Therefore, selection bias may have occurred even though necropsy examinations were offered to all clients when their cats were euthanized at our clinics. A wide variety of maintenance diets were fed before the transition to a PRD. Although the exact mineral concentrations in these diets were challenging to determine, it is reported that the median values of phosphorus content and Ca:P are 3 g/Mcal and 1.3, respectively, among 82 commercially available cat foods.25 Therefore, it is logical to assume that the cats that accepted a PRD after CKD diagnosis received lower dietary phosphate intake and higher dietary Ca:P than those that continued to be fed maintenance diets. Because the allocation of the PRD group was not randomized (all cats were offered a PRD as part of their management strategy for CKD in our study for ethical reasons), selection bias may have been introduced, even though no differences in clinicopathological variables were identified among 3 groups at baseline. Therefore, PRD effects on CKD progression and mortality should be interpreted with caution because the unknown reason why the cats did not eat the PRD may be linked to their poorer survival. Additionally, a variety of PRDs, with differences in phosphorus content and Ca:P, were offered throughout the study period and it cannot be inferred that the results from our study can be generalized to all commercially available PRD because although the general properties of clinical renal diets are similar, variations in diet formulation exist. A prospective longitudinal study is required to further investigate the causal effect of dietary phosphate restriction on nephrocalcinosis. Intriguingly, the extent of macroscopic calcification could be evaluated using micro-CT scanning in our study. However, the volume of FFPE kidney tissue could have been overestimated because of interference of the paraffin wax. Although measurements of multiple tissue surface areas at regular depth intervals were obtained to provide the best possible estimate of tissue volume, the VN: KT ratio potentially could have been underestimated. However, this possibility is deemed to have a relatively minor impact on the correlation between micro- and macroscopic nephrocalcinosis reported. Finally, nephrocalcinosis has been associated with hyperparathyroidism in human patients.29 This association is most likely attributable to the increased plasma and urine concentrations of calcium, urinary excretion of phosphate, and renal reabsorption of calcium stimulated by PTH, leading to renal calcification.54,55 Fibroblast growth factor-23 also has been suggested to be involved in nephrocalcinosis.56 Unfortunately, data on FGF23, PTH, and urinary electrolytes were not available in our study; hence, their involvement in nephrocalcinosis could not be investigated. This limitation prohibits further evaluation of the potential contribution of hypercalciuria or hyperphosphaturia to nephrocalcinosis. Calcium status in the cats in our study was assessed by tCa because the biologically active iCa measurement was not available in most cats. Additional studies, including plasma FGF23, PTH, iCa, and urinary calcium and phosphate measurements, are warranted to characterize whether these factors play a more important role in nephrocalcinosis.

Collectively, we showed that higher plasma tCa concentration at CKD diagnosis and dietary phosphate restriction are independent risk factors for nephrocalcinosis although causality cannot be determined. Extraosseous calcification is a multifaceted and complex process that is actively regulated by various inducers and inhibitors.57,58 The role of endogenous calcification inhibitors, such as magnesium, fetuin-A, and pyrophosphate, on nephrocalcinosis remains to be explored in CKD cats. Furthermore, in cats, nephrocalcinosis did not appear to be associated with the rapidity of disease progression and risk of all-cause mortality in our study. Nonetheless, ingestion of ≥50% PRD may decrease the risk of all-cause mortality and prolong survival in CKD cats. These contradictory observations require further study. In cats, CKD is a heterogeneous syndrome with highly variable progression rates most likely driven by multiple factors. Our results implicate derangements in phosphate homeostasis in contributing to the rapid progression of CKD even though nephrocalcinosis was not evident in these cats. Future prospective studies assessing the development and progression of nephrocalcinosis in cats with azotemic CKD are warranted and may lead to a new framework for diagnostic and therapeutic approaches in the management of CKD-MBD in cats.

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Pak-Kan Tang1 | Rosanne E. Jepson2 | Yu-Mei Chang3 | Rebecca F. Geddes2 | Mark Hopkinson1 | Jonathan Elliott1

1 Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, United Kingdom

2 Department of Clinical Science and Services, Royal Veterinary College, University of London, London, United Kingdom

3 Research Support Office, Royal Veterinary College, University of London, London, United Kingdom