Pascale Smets

Urinary Markers in Healthy Young and Aged Dogs and Dogs with Chronic Kidney Disease

BACKGROUND Blood urea nitrogen and serum creatinine concentrations only detect a decrease of > 75% of renal functional mass. Therefore, there is a need for markers that allow early detection and localization of renal damage. HYPOTHESIS Urinary albumin (uALB), C-reactive protein (uCRP), retinol binding protein (uRBP), and N-acetyl-beta-D-glucosaminidase (uNAG) concentrations are increased in dogs with chronic kidney disease (CKD) compared with healthy controls and in healthy older dogs compared with young dogs. ANIMALS Ten dogs with CKD, 10 healthy young dogs (age 1-3 years), and 10 healthy older dogs (age > 7 years) without clinically relevant abnormalities on physical examination, hematology, biochemistry, and urinalysis. METHODS Urinary markers were determined using an ELISA (uALB, uCRP, and uRBP) or a colorimetric test (uNAG). Results were related to urinary creatinine (c). The fixed effects model or the Wilcoxon rank sum test were used to compare the different groups of dogs. RESULTS uALB/c, uRBP/c, and uNAG/c were significantly higher in CKD dogs than in healthy dogs. No significant difference was found for uCRP, which was not detectable in the healthy dogs and only in 3 of the CKD dogs. Between the healthy young and older dogs, no significant difference was detected for any of the markers. CONCLUSION The urinary markers uALB/c, uRBP/c, and uNAG/c were significantly increased in dogs with CKD compared with healthy controls. Additional studies are needed to evaluate the ability of these markers to detect renal disease before the onset of azotemia.

Validation of immunoassays for the candidate renal markers C-reactive protein, immunoglobulin G, thromboxane B2 and retinol binding protein in canine urine

The study of early markers for glomerular and tubular dysfunction in dogs with renal diseases holds promise to gain new insights in the pathogenesis of canine nephropathies. However, the validation of such markers in canine urine is largely lacking. Therefore, immunoassays for the quantification of a set of four urinary markers, C-reactive protein (CRP), immunoglobulin G (IgG), thromboxane B(2) (TXB(2)) and retinol binding protein (RBP), were validated by determining their sensitivity, reproducibility, precision and accuracy in a large patient group. The results show that the immunoassays are appropriate for analysis of urinary CRP, IgG, TXB(2) and RBP in dogs. Furthermore, the significant differences in urinary concentrations of the selected glomerular and tubular markers between healthy (H) dogs and dogs with several types of nephropathies (R) support their future application in both clinical settings and research models.

Evaluation of Kidney Injury in Dogs with Pyometra Based on Proteinuria, Renal Histomorphology, and Urinary Biomarkers

BACKGROUND Proteinuria is a feature of pyometra-associated renal dysfunction, but its prevalence and clinical relevance are not well characterized. OBJECTIVES To define which subset of dogs with pyometra has clinically relevant kidney injury by quantification of proteinuria; light, immunofluorescence, and electron microscopic examination of kidney biopsy specimens; and measurement of urinary biomarkers. ANIMALS Forty-seven dogs with pyometra. Ten clinically healthy intact bitches of comparable age. METHODS Prospective study. Routine clinicopathological variables including urinary protein to creatinine ratio (UPC) were analyzed. Validated assays were used to quantify urinary biomarkers for glomerular (urinary albumin, urinary immunoglobulin G, urinary C-reactive protein, urinary thromboxane B(2)) and tubular function (urinary retinol-binding protein, urinary N-acetyl-β-d-glucosaminidase). Kidney biopsy specimens from 10 dogs with pyometra and dipstick urine protein concentrations of 2+ or 3+ were collected during ovariohysterectomy. Urinalysis was repeated within 3 weeks after surgery in 9 of the 10 dogs. RESULTS UPC (median, range) was significantly higher in dogs with pyometra (0.48, 0.05-8.69) compared with healthy bitches (0.08, 0.02-0.16) (P < .01). Twenty-two of 47 dogs with pyometra had UPC>0.5, 12 had UPC>1.0, and 7 had UPC>2.0. Glomerulosclerosis and tubulointerstitial nephritis were common kidney biopsy findings in proteinuric dogs with pyometra. Dogs with glomerulosclerosis (5/10), either global or focal and segmental, had UPC>1.0 at ovariohysterectomy and afterward. Dogs with structural glomerular and tubular changes mostly had urinary biomarker to creatinine ratios above the 75th percentile. CONCLUSION Dogs with pyometra and UPC>1.0 or high ratios of urinary biomarkers appear likely to have clinically relevant renal histologic lesions and require monitoring after ovariohysterectomy. Future studies should evaluate the role of pyometra-associated pathogenic mechanisms in causing or exacerbating focal and segmental glomerulosclerosis in dogs.

Urinary Biomarkers for Acute Kidney Injury in Dogs

Routinely, kidney dysfunction and decreased glomerular filtration rate (GFR) are diagnosed by the evaluation of changes in the serum creatinine (SCr) and blood urea nitrogen (BUN) concentrations. However, neither of these tests is sensitive or specific enough for the early diagnosis of impaired kidney function because they are both affected by other renal and nonrenal factors. Furthermore, kidney injury can be present in the absence of kidney dysfunction. Renal reserve enables normal GFR even when nephrons are damaged. Renal biomarkers, especially those present in urine, may be useful for the study of both acute and chronic nephropathies. The aim of this review is to describe the current status of urinary biomarkers as diagnostic tools for kidney injury in dogs with particular focus on acute kidney injury (AKI). The International Renal Interest Society (IRIS) canine AKI grading system and the implementation of urinary biomarkers in this system also are discussed. The discovery of novel urinary biomarkers has emerged from hypotheses about the pathophysiology of kidney injury, but few proteomic urine screening approaches have been described in dogs. Lack of standardization of biomarker assays further complicates the comparison of novel canine urinary biomarker validation results among studies. Future research should focus on novel biomarkers of renal origin and evaluate promising biomarkers in different clinical conditions. Validation of selected urinary biomarkers in the diagnosis of canine kidney diseases must include dogs with both renal and nonrenal diseases to evaluate their sensitivity, specificity as well as their negative and positive predictive values.

Escherichia coli Pyometra Induces Transient Glomerular and Tubular Dysfunction in Dogs

BACKGROUND Pyometra in dogs has been associated with renal injury. HYPOTHESIS Examine pyometra-related nephropathy by evaluating novel renal biomarkers. ANIMALS Twenty-five dogs with Escherichia coli pyometra. Fourteen clinically healthy bitches of comparable age. METHODS Prospective study. Urinary biomarkers determined by immunoassays (uIgG, uCRP, uAlb, uRBP, uTXB2) or colorimetric test (uNAG) with results normalized to urine creatinine concentration. Nonparametric Mann-Whitney U-test and Wilcoxons signed-rank test used to compare healthy dogs and dogs with pyometra, and dogs with pyometra at initial and follow-up examination. RESULTS Urinary biomarkers (median, range) significantly increased in dogs with pyometra (uIgG/Cr: 169.7 mg/g, 4.8-1052.9; uCRP/Cr: 0.260 mg/g, 0.006-3.030; uAlb/Cr: 89.5 mg/g, 8.8-832.7; uRBP/Cr: 1.66 mg/g, 0.05-21.44; uNAG/Cr: 5.8 U/g, 1.6-27.7; uTXB2 /Cr: 15.3 μg/g, 3.2-139.6) compared with healthy bitches (uIgG/Cr: 3.4 mg/g, 0.6-8.9; uCRP/Cr: below detection limit; uAlb/Cr: 17.5 mg/g, 1.3-166.3; uRBP/Cr: 0.13 mg/g, 0.02-0.44; uNAG/Cr: 2.4 U/g, 1.4-7.4; uTXB2 /Cr: 2.4 μg/g, 1.2-4.7) (P<.001). Six months after ovariohysterectomy, urinary biomarkers in pyometra group (uIgG/Cr: 4.7 mg/g, 1.5-99.8; uCRP/Cr: below detection limit; uAlb/Cr: 13.9 mg/g, 2.1-471.2; uRBP/Cr: 0.05 mg/g, 0.02-0.32; uNAG/Cr: 1.6 U/g, 0.9-3.3; uTXB2 /Cr: 3.3 μg/g, 1.0-6.9) were significantly lower than before surgery (P<.01), and not significantly different to those of healthy dogs (P>.05). CONCLUSION AND CLINICAL IMPORTANCE Pyometra-related renal dysfunction affects the nephron both at glomerular and proximal tubular level and is a transient process in most dogs with E. coli pyometra.

Retinol-Binding Protein in Serum and Urine of Hyperthyroid Cats before and after Treatment with Radioiodine

BACKGROUND Retinol-binding protein (RBP) is suggested as a clinically useful marker of renal function in cats. HYPOTHESIS Serum and urinary RBP concentrations in hyperthyroid (HT) cats differ from those in healthy (H) cats; radioiodine ((131)I) treatment influences serum and urinary RBP concentrations in HT cats. ANIMALS Ten HT and 8 H cats. METHODS RBP concentration was evaluated in feline serum and urine samples from a prospective study. RESULTS There was a significant (P= .003) difference in the urinary RBP/creatinine (uRBP/c) ratios of H (-) and untreated HT (1.4 + or - 1.5 x 10(-2) microg/mg) cats. Serum total thyroxine concentration (1.8 + or - 1.9 microg/dL, 24 weeks) and uRBP/c (0.6 + or - 1.0 x 10(-2) microg/mg, 24 weeks) decreased significantly (P < .001) in HT cats at all time points after treatment with (131)I, and these variables were significantly correlated with one another (r= 0.42, P= .007). Serum RBP concentrations from HT cats (199 + or - 86 microg/L) did not differ significantly (P= .98) from those of H cats (174 + or - 60) and did not change after treatment with (131)I (182 + or - 124 microg/L, P= .80). CONCLUSION AND CLINICAL IMPORTANCE The presence of urinary RBP in HT cats is a potential marker of tubular dysfunction that is correlated to thyroid status, although it is independent of circulating RBP concentrations. The decreased uRBP/c combined with the absence of changes in serum RBP after treatment suggests that the suspected tubular dysfunction was partly reversible with treatment of (131)I.

Assessment of renal dysfunction using urinary markers in canine babesiosis caused by Babesia rossi

Renal damage is deemed a common, yet poorly documented, complication in canine babesiosis. Serum urea and creatinine are insensitive and non-specific markers of early renal dysfunction and their measurements are influenced by hemolysis caused by babesiosis. Therefore, the aim of this study was to use urinary markers to assess the localization and degree of renal dysfunction in dogs with Babesia rossi infection. Urinary immunoglobulin G (uIgG) and urinary C-reactive protein (uCRP) were measured as markers for glomerular dysfunction, while urinary retinol-binding protein (uRBP) was used as a marker for tubular dysfunction. Eighteen dogs presenting with uncomplicated babesiosis were included and compared with eight clinically healthy dogs. Previously validated commercial ELISA kits were used for the measurement of uIgG, uCRP, and uRBP. Results were related to urinary creatinine concentrations (c). Dogs with babesiosis had significantly higher concentrations of all three measured urinary markers compared to healthy dogs. Except for urinary protein/c ratio (UPC), routine urinary and serum markers for renal function (urine specific gravity (USG), serum urea and creatinine (sCr)) were not significantly different between dogs with babesiosis and healthy dogs. All three urinary markers were positively correlated with each other and with UPC. The data supports the presence of both glomerular and tubular dysfunction in dogs suffering from uncomplicated B. rossi infection. Urinary markers were superior to USG, serum urea and creatinine concentrations for the early detection of renal dysfunction in dogs with babesiosis.

Effect of Thyroxine Supplementation on Glomerular Filtration Rate in Hypothyroid Dogs

BACKGROUND Glomerular filtration rate (GFR) is decreased in humans with hypothyroidism, but information about kidney function in dogs with hypothyroidism is lacking. HYPOTHESIS Hypothyroidism influences GFR in dogs. The objective of this study was to assess GFR in hypothyroid dogs before implementation of thyroxine supplementation and after re-establishing euthyroidism. ANIMALS Fourteen hypothyroid dogs without abnormalities on renal ultrasound examination or urinalysis. METHODS Blood pressure and GFR (measured by exogenous creatinine clearance) were measured before treatment (T0, n=14) and at 1 month (T1, n=14) and at 6 months (T6, n=11) after beginning levothyroxine supplementation therapy (20 microg/kg/d, PO). The response to therapy was monitored at T1 by measuring serum total thyroxine and thyroid stimulating hormone concentrations. If needed, levothyroxine dosage was adjusted and reassessed after 1 month. Statistical analysis was performed using a general linear model. Results are expressed as mean+/-standard deviation. RESULTS At T0, the average age of dogs in the study group was 6.3+/-1.4 years. Their average body weight decreased from 35+/-18 kg at T0 to 27+/-14 kg at T6 (P<.05). All dogs remained normotensive throughout the study. GFR increased significantly with levothyroxine supplementation; the corresponding results were 1.6+/-0.4 mL/min/kg at T0, 2.1+/-0.4 at T1, and 2.0+/-0.4 at T6 (P<.01). CONCLUSION GFR was <2 mL/min/kg in untreated hypothyroid dogs. Re-establishment of a euthyroid state increased GFR significantly.

Cushing's syndrome, glucocorticoids and the kidney

Glucocorticoids (GCs) affect renal development and function in fetal and mature kidneys both indirectly, by influencing the cardiovascular system, and directly, by their effects on glomerular and tubular function. Excess GCs due to endogenous GC overproduction in Cushings syndrome or exogenous GC administration plays a pivotal role in hypertension and causes increased cardiac output, total peripheral resistance and renal blood flow. Glucocorticoids increase renal vascular resistance (RVR) in some species and experimental settings and decrease RVR in others. Short term administration of adrenocorticotrophic hormone or GCs causes an increased glomerular filtration rate (GFR) in humans, rats, sheep and dogs. Interestingly, chronic exposure may cause a decreased GFR in combination with a higher cardiovascular risk in human patients with Cushings syndrome. Glomerular dysfunction leads to proteinuria and albuminuria in canine and human Cushings patients, and some cases also show histological evidence of glomerulosclerosis. Tubular dysfunction is reflected by an impaired urinary concentrating ability and disturbed electrolyte handling, which can potentially result in increased sodium reabsorption, hypercalciuria and urolithiasis. Conversely, chronic kidney disease can also alter GC metabolism. More research needs to be performed to further evaluate the renal consequences of Cushings syndrome because of its implications for therapeutic aspects as well as the general well-being of the patient. Because there is a high incidence of Cushings syndrome in canines, which is similar to the syndrome in humans, dogs are an interesting animal model to investigate the link between hypercortisolism and renal function.

Long‐Term Follow‐Up of Renal Function in Dogs after Treatment for ACTH‐Dependent Hyperadrenocorticism

BACKGROUND Systemic hypertension and proteinuria are frequent complications in dogs with Cushings syndrome and do not always resolve after treatment of hypercortisolism. Therefore, dogs with Cushings syndrome may be at risk for renal dysfunction before and after treatment. HYPOTHESIS/OBJECTIVES To assess renal function in dogs with ACTH-dependent hyperadrenocorticism (ADHAC) before and after treatment. ANIMALS A total of 19 dogs with ADHAC and 12 control dogs. METHODS Renal function was assessed before and at 1, 3, 6, and 12 months after treatment. Twelve dogs were treated with trilostane and 7 dogs by transsphenoidal hypophysectomy. Routine renal markers were measured and urinary albumin (uALB), immunoglobulin G (uIgG), and retinol-binding protein (uRBP) were assessed by ELISA. Urinary N-acetyl-β-D-glucosaminidase (uNAG) was determined colorimetrically. All urinary markers were indexed to urinary creatinine concentration (c). Plasma clearance of creatinine (Cl(creat)), exo-iohexol (Cl(exo)), and endo-iohexol (Cl(endo)) was used to measure glomerular filtration rate (GFR). Data were analyzed using a general linear model. RESULTS Serum creatinine and urea concentrations increased post-treatment, but remained within reference ranges. Plasma Cl(creat) and Cl(endo) were significantly lower post-treatment, whereas Cl(exo) was not different. Urinary protein-to-creatinine ratio (UPC), uALB/c, uIgG/c, and uRBP/c were decreased post-treatment, but at 12 months 5/13 dogs remained proteinuric. Urinary NAG/c did not change significantly. CONCLUSIONS AND CLINICAL IMPORTANCE A decrease in GFR and persistent proteinuria post-treatment may warrant the clinicians attention. Future research including renal histopathology of dogs with persistent proteinuria or low GFR is needed to further assess renal outcome.