Bert Maddens

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.

Chitinase-like Proteins are Candidate Biomarkers for Sepsis-induced Acute Kidney Injury

Sepsis-induced acute kidney injury (AKI) is a frequent complication of critically ill patients and leads to high mortality rates. The specificity of currently available urinary biomarkers for AKI in the context of sepsis is questioned. This study aimed to discover urinary biomarkers for septic AKI by contemporary shotgun proteomics in a mouse model for sepsis and to validate these in individual urine samples of mice and human septic patients with and without AKI. At 48 h after uterine ligation and inoculation of Escherichia coli, aged mice (48 weeks) became septic. A subgroup developed AKI, defined by serum creatinine, blood urea nitrogen, and renal histology. Separate pools of urine from septic mice with and without AKI mice were collected during 12 h before and between 36–48 h after infection, and their proteome compositions were quantitatively compared. Candidate biomarkers were validated by Western blot analysis of urine, plasma, and renal tissue homogenates from individual mice, and a limited number of urine samples from human septic patients with and without AKI. Urinary neutrophil gelatinase-associated lipocalin, thioredoxin, gelsolin, chitinase 3-like protein 1 and -3 (CHI3L3) and acidic mammalian chitinase were the most distinctive candidate biomarkers selected for septic AKI. Both neutrophil gelatinase-associated lipocalin and thioredoxin were detected in urine of septic mice and increased with severity of AKI. Acidic mammalian chitinase was only present in urine of septic mice with AKI. Both urinary chitinase 3-like protein 1 and -3 were only detected in septic mice with severe AKI. The human homologue chitinase 3-like protein 1 was found to be more excreted in urine from septic patients with AKI than without. In summary, urinary chitinase 3-like protein 1 and -3 and acidic mammalian chitinase discriminated sepsis from sepsis-induced AKI in mice. Further studies of human chitinase proteins are likely to lead to additional insights in septic AKI.

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.

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.

Hypercortisolism affects glomerular and tubular function in dogs.

Renal function was assessed in 25 dogs with Cushings syndrome and in 12 healthy controls. Routine renal parameters and glomerular filtration rate (GFR) were measured and urinary biomarkers such as urinary albumin (uALB), urinary immunoglobulin G (uIgG), and urinary retinol-binding protein (uRBP) were assessed by ELISA. Urinary N-acetyl-β-D-glucosaminidase activity (uNAG) was determined colorimetrically. All urinary markers were indexed to urinary creatinine concentration (c). Plasma exo- (Cl(exo)) and endo-iohexol (Cl(endo)) clearance were used to measure GFR. Based on a Mann-Whitney U test, urea and Cl(exo) did not differ, sCr was significantly lower, and UPC, uALB/c, uIgG/c, uRBP/c, uNAG/c and Cl(endo) were higher in the dogs with Cushings syndrome when compared with controls. The findings indicate that glomerular and tubular function are both altered in dogs with Cushings syndrome. Further longitudinal studies will be required to elucidate the pathogenesis of the changes in GFR.

Effect of Sampling Method and Storage Conditions on Albumin, Retinol-Binding Protein, And N-Acetyl-β-D-Glucosaminidase Concentrations in Canine Urine Samples

Urinary markers for renal dysfunction are gaining interest, but effects of sampling method, storage conditions, and urinary tract inflammation or infection on these markers are unclear. Therefore, the objectives of the current study were to determine the difference in urinary albumin (uALB), urinary retinol-binding protein (uRBP), and urinary N-acetyl-β-D-glucosaminidase (uNAG) concentrations in cystocentesis and voided samples and to investigate concentration changes after storage at −20°C and at −80°C. Effects of a protease inhibitor were also assessed in samples stored at −80°C for 12 months. In a pilot experiment, influence of in vitro hematuria, pyuria, and bacteriuria on the urinary markers was evaluated. A mixed model was used to calculate mean differences and 95% confidence intervals. Urinary ALB, uNAG, and uRBP concentrations were similar in voided and cystocentesis samples. After storage for 4 months at −20°C, uALB concentration was not affected, and uRBP concentration showed a mild and clinically irrelevant decrease, whereas uNAG activity was significantly lower compared with fresh samples. After storage for 12 months at −80°C, uALB and uRBP concentrations did not differ from fresh samples, but uNAG activity was severely decreased. Protease inhibitor addition did not preserve uNAG activity. Experimental hematuria, pyuria, and bacteriuria did not seem to affect urinary markers, although further research is needed.

Severity of sepsis-induced acute kidney injury in a novel mouse model is age dependent.

Objective:Despite extensive research, the mortality rate of patients with sepsis-induced acute kidney injury (AKI) is unacceptably high, especially in the elderly. Current sepsis models have difficulties in reproducing AKI. This study aimed to develop a novel, clinically relevant mouse model for sepsis-induced AKI by uterine ligation and inoculation of bacteria. In addition, the age dependency of the severity of sepsis and sepsis-induced AKI was studied by validating this model in three different age categories. Design:Experimental animal investigation. Setting:University research laboratory. Subjects:Young (12–14 wks), aged (46–48 wks), and old (70–72 wks) C57BL/6 female mice were used as models for adolescent, adult premenopausal, and elderly postmenopausal women, respectively. Interventions:Uterine ligation and inoculation with 103 colony forming unit Escherichia coli or saline (sham) was performed; in vivo imaging with a luminescent Escherichia coli strain documented the course of infection. Measurements and Main Results:All mice had established Escherichia coli sepsis at 48 hrs postinfection, with higher mortality rate in old (43%) compared to aged (23%) or young (9%) mice. Infected mice had elevated serum or plasma cytokine, chemokine (tumor necrosis factor, interleukin-6, keratinocyte-derived chemokine, monocyte chemoattractant protein 1, and interleukin-10), and NOx− concentrations compared to sham mice. AKI was confirmed by renal histology. Serum creatinine concentrations at 48 hrs increased with age (mean ± SEM; controls 0.18 ± 0.03 mg/dL, young 0.28 ± 0.03 mg/dL, aged 0.38 ± 0.05 mg/dL, and old 0.44 ± 0.06 mg/dL). Conclusion:The uterine ligation and inoculation model for sepsis-induced AKI starts from a real infectious focus and shows an age-dependent severity of septic AKI that resembles AKI in humans.