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Keywords:

  • Chronic kidney disease;
  • Dog;
  • Endometritis;
  • Focal segmental glomerulosclerosis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

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 B2) 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.

Abbreviations:
ACE

angiotensin-converting enzyme

C3

complement factor 3

ESRD

end-stage renal disease

FSGS

focal segmental glomerulosclerosis (human)

PAS

periodic acid-Schiff

sCr

serum creatinine

sUN

serum urea nitrogen

uAlb

urinary albumin

uCRP

urinary C-reactive protein

uIgG

urinary immunoglobulin G

uNAG

urinary N-acetyl-β-d-glucosaminidase

UPC

urinary protein to creatinine ratio

uRBP

urinary retinol-binding protein

USG

urine specific gravity

UTI

urinary tract infection

uTXB2

urinary thromboxane B2

Pyometra is a common reproductive disease in female intact dogs.1 This chronic uterine inflammation often is accompanied by glomerular, or tubular dysfunction, or both.2,3 Clinically, dogs with pyometra benefit from prompt ovariohysterectomy.4 In follow-up studies from our group, resolution of renal dysfunction has been found in most dogs with pyometra several months after surgery.2,3,5 Nevertheless, renal failure after ovariohysterectomy has been reported in 2 of 25 (8%) dogs with Escherichia coli pyometra.3 In addition, 5 of 41 dogs with pyometra (12%) were reported to have persistent polyuria, polydipsia, and proteinuria after surgery. Two of these dogs (5%, n = 2/41) died from end-stage renal disease (ESRD) within 3 years.5 A similar postsurgical renal course in 1 proteinuric dog of 6 dogs with pyometra also was described in another study.6 Importantly, substantial proteinuria at ovariohysterectomy was present in the above specified dogs with pyometra that had renal failure.3,5,6 Therefore, renal dysfunction and more specifically proteinuria associated with pyometra merit the clinician's attention.

Previous renal histopathology studies agree on the variable extent of tubulointerstitial injury associated with pyometra, but no consensus exists on the type of glomerular lesions.5,7,8 For decades, underlying immune complex-mediated glomerulonephritis has been suspected.7,9–11 However, based on light, immunofluorescence, and electron microscopy, Stone et al8 found no evidence for glomerulonephritis and even concluded that pyometra-associated glomerular changes were similar to those of healthy control dogs of comparable age. A more recent study5 described only lesions consistent with glomerulosclerosis in dogs with pyometra, although similar lesions were detected in age-matched control dogs and renal immunofluorescence or ultrastructural examinations were not performed.

Complementary to urinary protein to creatinine ratio (UPC) and serum renal variables, urinary biomarkers can be useful for detecting and localizing functional abnormalities in the kidney. Recently validated assays12,13 provide the opportunity to study altered glomerular permselectivity by quantifying urinary albumin (uAlb), immunoglobulin G (uIgG), and C-reactive protein (uCRP). Tubular dysfunction is reflected by increased concentrations of urinary retinol-binding protein (uRBP) and N-acetyl-β-d-glucosaminidase (uNAG). In addition, intrarenal hemodynamics can be evaluated by urinary thromboxane B2 (uTXB2).

The aims of this study were (1) to study the prevalence of proteinuria in dogs with pyometra; (2) to describe light, immunofluorescence, and electron microscopy findings in the most severely proteinuric dogs; and (3) to evaluate the relationship between the kidney biopsy findings and urinary protein excretion as well as specific urinary biomarkers.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

Dogs

Forty-seven dogs with pyometra and without concurrent diseases and 10 clinically healthy intact female dogs of comparable age were included. All animals were privately owned and presented at the Blue Star Animal Hospital of Göteborg, Sweden (37 dogs with pyometra) or the Companion Animal Clinic of the Faculty of Veterinary Medicine, Ghent University, Belgium (10 dogs with pyometra and 10 healthy bitches) between April 2009 and January 2010. The owners gave informed consent to allow participation of their dogs in the study.

Physical examination, abdominal ultrasound examination, and routine hematologica and biochemicalb tests were performed on all dogs. Control dogs were considered healthy when no clinically relevant abnormalities were found on these examinations. Likewise, if concurrent diseases were detected in dogs with pyometra, this resulted in exclusion of these dogs from the study. Serologic testing and evaluation of blood pressure was not routinely performed. Blood coagulation profiles (prothrombin time, activated partial thromboplastin time) were performed in all dogs before surgery.c Urine was collected by cystocentesis on ultrasound examination (n = 11 dogs with pyometra and 4 healthy dogs) or at the start of the surgery (n = 36 dogs with pyometra and 6 healthy bitches). Urine specific gravity (USG) was measured with a refractometer. Urine was centrifuged (447 × g, 3 minutes) and routine dipstick analysis,d urine culture, and sediment analysis were performed. Urinary protein concentration was determined with a turbidimetric method with benzethonium chloride.e Urinary creatinine concentration was measured by the modified Jaffé reaction.14 The term pyometra in this study includes the disease complex characterized by history, clinical signs with or without vulvar discharge, uterine bacterial culture, and ultrasound examination with luminal content and uterine wall structure findings typical for pyometra.5,8,15 If the diagnosis of pyometra was equivocal, histological examination of the uterine wall was performed. Only dogs with classical presentation for pyometra or cystic endometrial hyperplasia and endometritis confirmed by histopathology were included.

Upon arrival at the clinic, all dogs with pyometra received fluid therapy adapted to their needs and most of the dogs received IV or SC antibiotic treatment (amoxicillin, n = 39/47; enrofloxacin, n = 5/47). Ovariohysterectomy was performed according to standard procedures after sedation with IV acepromazinef or diazepam,g and methadone.h Anesthesia was induced by IV propofoli and maintained by inhalation of isofluranej or sevoflurane.k Appropriate analgesia with opioids, nonsteroidal anti-inflammatory drugs, or both was provided postoperatively until discharge from the clinic. Antibiotic (amoxicillin or enrofloxacin) and analgesic (carprofenl or tramadolm) treatment was continued at home for 7 and 5 days, respectively.

If the dipstick urine protein result was 2+ or 3+ (on a scale from negative [<30 mg protein/dL urine] to 1+ [30 mg/dL], 2+ [100 mg/dL], and 3+ [500 mg/dL]), then the dog with pyometra was prospectively enrolled for kidney biopsy specimens during ovariohysterectomy. A total of 10 kidney biopsy specimens of dogs with pyometra were obtained.n Two cores of tissue were collected from the left kidney, divided in 3 parts in opposite directions and processed for light (10% buffered formalin), immunofluorescence (transported in Michel's medium and processed in OCT compound), and electron microscopy (3% glutaraldehyde).

Follow-up examination with urine sampling was performed 2 weeks to 3 months after ovariohysterectomy in 14 of the 47 dogs originally presented with pyometra, of which 9 were biopsied. If UPC was > 0.5 at the 1st follow-up examination, additional urine examinations were performed later. Follow-up samples were not available in 31 dogs because owners declined to participate when their dogs were doing well after surgery (including 1 biopsied dog that had UPC<0.2). Two dogs were excluded from the follow-up study because of mortality unrelated to kidney disease. Two dogs with pyometra from which kidney biopsy specimens were collected, were euthanized because the owners declined further treatment (1 dog developed neck pain and diabetes mellitus after ovariohysterectomy, and 1 dog developed severe uremia after ovariohysterectomy).

Light, Immunofluorescence, and Transmission Electron Microscopic Examination of Kidney Biopsy Specimens

For light microscopy, sections from each dog were stained with hematoxylin and eosin, periodic acid-Schiff (PAS; matrix deposition), Masson's trichrome (collagen) (3–4 μm thick), and periodic acid methenamine silver (basement membrane) (1 μm) according to standard procedures.16 Immunofluorescence labelling was performed on all kidney biopsy specimens utilizing sheep anti-dog IgG, goat anti-dog IgM, goat anti-dog IgA, goat anti-dog complement factor 3 (C3),o and rabbit anti-human κ light chain, rabbit anti-human λ light chain, and rabbit anti-human C1Q.p,17 The strength of the fluorescent signal was evaluated as negative (−), equivocal (±), 1+, 2+, or 3+, and the staining pattern was described. Ultrastructural examination was performed on 8 of 10 biopsied dogs with pyometra (samples collected from 2 dogs did not contain suitable glomeruli for evaluation). All sections were examined by board-certified veterinary pathologists (J.v.d.L., L.A.).

Urinary Biomarker Analysis

Remaining urine supernatant was aliquoted and stored at −20°C (Sweden) or −80°C (Belgium) for a maximum of 2 months before analysis, and no more than 1 freeze-thaw cycle per aliquot was allowed. Quantification of urinary biomarkers was performed on supernatant by immuno- (uAlb, uIgG, uCRP, uRBP, uTXB2)q,r,s,t,u and colorimetric assays (uNAG)v as validated previously by our group,12,13 with analysis of all samples in duplicate. Because of the use of spot urine samples, urinary biomarker concentrations were related to urinary creatinine and expressed as ratios.

Statistical Analysis

A commercial software package was used to analyze data.w The Mann-Whitney U-test was applied to compare UPC, USG, serum creatinine (sCr), serum urea nitrogen (sUN), and urinary biomarker ratios between dogs with pyometra and healthy dogs. Initial and follow-up UPC, USG, and urinary biomarker ratios were compared by the Wilcoxon signed-rank test for paired samples. Statistical significance was considered at P < .05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

Study Group Characteristics

Both groups of healthy dogs and dogs with pyometra included several breeds. The age of the group of healthy dogs (median age, 5.3 years; range, 0.7–12.5 years) and dogs with pyometra (median age, 7.3 years; range, 1.2–12.5 years) did not differ significantly (P = .228). History and clinical signs of dogs with pyometra included the presence of polyuria and polydipsia (n = 21/47), vomiting (n = 9/47), vulvar discharge (n = 33/47), fever (n = 9/47), and painful abdomen on palpation (n = 18/47). Uterine culture results were negative in 13 dogs with pyometra or indicated presence of E. coli (n = 30/47), Staphylococcus spp. (n = 2/47), or Streptococcus spp. (n = 2/47). Abdominal ultrasound examination in dogs with pyometra did not detect any abnormalities in organs other than the uterus or ovaries. Two dogs with pyometra had multiple cysts in their ovaries. Presence of a small amount of anechoic free abdominal fluid, indicative of peritonitis, was detected in 4 dogs with pyometra. These dogs did not have a 2+ or 3+ urine protein dipstick result. Leukocytosis (>16 × 109/L) with neutrophilia (>12 × 109/L) was observed in 28 dogs with pyometra. Five dogs with pyometra had hypoalbuminemia (<20 g/L) and 1 of these had hypoproteinemia (<55 g/L). Thirty dogs with pyometra had decreased PCV (<43%). Overall leukocyte (P = .179) and platelet counts (P = .208), and total plasma protein concentration (P = .191) were not significantly different between dogs with pyometra and healthy bitches. Plasma albumin concentration and PCV were significantly lower in the dogs with pyometra compared with the group of healthy dogs (P = .002 and 0.001, respectively).

sUN and sCr concentrations were not significantly different between dogs with pyometra (median, sUN 4.3 mmol/L; range, 2.2–49.2 mmol/L; median sCr, 91.3 μmol/L; range, 40.0–477.0 μmol/L) and healthy bitches (sUN, sCr; P = .709 and 0.096, respectively). However, 6 of 47 dogs with pyometra (13%) had increased concentrations of sCr (sCr > 125 μmol/L). Four of 47 dogs with pyometra (9%) had increased concentrations of sUN (sUN > 8.3 mmol/L), and 2 of these also had increased concentrations of sCr. Of these 8 dogs with increased concentrations of either sCr, sUN, or both, 4 also had USG<1.015, which persisted after surgery in 2 dogs. Urine culture and light microscopic examination of urine sediment showed evidence of urinary tract infection (UTI) in 5 of 47 dogs with pyometra, of which 2 were included in the kidney biopsy group. Pyuria was observed in these 5 dogs (>5 leukocytes per high power field), which was combined with hematuria in 1 dog. USG was significantly higher in the group of healthy dogs than in the dogs with pyometra (P < .01), but not compared with the nonproteinuric dogs with pyometra (P > .05) (Table 1). USG was significantly higher after ovariohysterectomy than before in the dogs available for follow-up urine samples (n = 14/47) (P < .05). Three of 14 dogs had USG<1.015 after surgery, and 2 of these also were proteinuric.

Table 1.  Urinary renal markers: results for healthy bitches (n = 10), dogs with pyometra (n = 47), and dogs with pyometra after ovariohysterectomy (follow-up) (n = 14) (expressed as median [range]).Thumbnail image of

Proteinuria

Six of the 10 dogs from which kidney biopsy specimens were collected had UPC>1.0 at ovariohysterectomy. Dogs with pyometra had significantly higher UPC compared with healthy dogs (P < .01) (Table 1). Twenty-two of 47 dogs with pyometra had UPC > 0.5 (47%), and 12 had UPC > 1.0 (26%, n = 12/47). Seven dogs with pyometra had UPC>2.0 (15%, n = 7/47) (Fig 1). Of the 8 dogs with increased concentrations of either sCr, sUN, or both, 2 had UPC<0.5, 5 had UPC between 0.5 and 1.0, and 1 had UPC of 8.69. The UPC values of the 5 dogs with a UTI were 0.05, 0.06, 0.60, 2.34, and 8.69.

image

Figure 1.  Concentrations of urinary protein and renal biomarkers in dogs with pyometra, normalized to urinary creatinine and expressed as ratios. Dogs with pyometra (n = 47) are visualized as individual dots, with indication of median values (full lines) and 75th percentile (dotted lines). Numbered dots refer to dogs with pyometra from which kidney biopsies were collected. UPC, urinary protein to creatinine ratio; Alb, urinary albumin to creatinine ratio; IgG, urinary immunoglobulin G to creatinine ratio; CRP, urinary C-reactive protein to creatinine ratio; TXB, urinary thromboxane B2 to creatinine ratio; RBP, urinary retinol-binding protein to creatinine ratio; NAG, urinary N-acetyl-β-d-glucosaminidase index.

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In dogs with pyometra, UPC decreased significantly after ovariohysterectomy (P < .05) (Table 1). Nine of 14 dogs had UPC<0.2 after surgery. Persistent proteinuria (UPC>1.0) occurred in 5 of the 6 biopsied dogs with initial UPC>1.0.

Kidney Biopsy Specimens Findings in Dogs with Pyometra

Table 2 provides detailed blood and urinalysis data of the 10 dogs with pyometra from which kidney biopsy specimens were collected (median age, 6.9 years; range, 2.0–8.3 years). Uterine culture results were negative (n = 5/10) or indicated presence of E. coli (n = 4/10) or Staphylococcus spp. (n = 1/10). Two dogs also had positive urine culture results (E. coli) and had UPC of 8.69 and 0.60. A 3+ urine protein dipstick was observed in 6 biopsied dogs. A summary of light microscopic kidney biopsy findings of these 10 dogs is presented in Table 3. The most common glomerular lesion was glomerulosclerosis (n = 5/10), which was segmental or global (Fig 2). Diagnosis of focal and segmental glomerulosclerosis was based on the presence of mesangial expansion and obliteration of capillary lumina with or without adhesions to Bowman's capsule affecting <50% of the glomerular tuft in <50% of glomeruli.18 Two dogs with glomerulosclerosis also had cystic atrophy of the glomerulus (1 and 5 affected glomeruli of 7 and 34 examined glomeruli, respectively). Low level glomerular (±) to 1+ positivity for IgM and C3 were detected in 2 of 4 dogs with focal and segmental glomerulosclerosis (in areas of sclerosis), in a proteinuric dog (in mesangium) and in a nonproteinuric dog with normal histology on light microscopic examination (in capillary wall). Only 1 dog had strong (3+) immunostaining, and diffuse global coarse granular capillary wall and mesangial staining for IgG and IgA was observed. Electron dense deposits were observed in subendothelial spaces of the examined kidney section of this dog. Ultrastructural evidence of effacement or fusion of foot processes (Fig 3) was found in 7 dogs with pyometra. In all but 1 dog, glomerular basement membranes were of normal thickness. Mild glomerular hypercellularity (in 18/23 examined glomeruli) and occasional neutrophils were present in glomerular capillary lumina of 1 dog (2/23 examined glomeruli) or infiltrated in the interstitium (1 other dog). Tubular atrophy was noted in 5 of 10 dogs with pyometra (Fig 2b), and interstitial inflammation with infiltration of lymphocytes and plasma cells was present in 7 of 10 dogs with pyometra (Fig 2c, Table 3). Chronic interstitial changes, represented by fibrosis, were found in 5 of 10 dogs (Fig 2b). No relevant histopathological abnormalities were detected on light microscopy in dogs with UPC<0.2 (2 dogs).

Table 2.  Selected clinical data of the dogs with pyometra from which a kidney biopsy was collected.
Urine Variable (Median (Range))Reference RangesHematology and Biochemistry Results (Median (Range))Reference Ranges
  1. USG, urine specific gravity; UPC, urine protein to creatinine ratio; UPCfup; UPC at follow-up visit 2 weeks (6 dogs) or 3 weeks (3 dogs) after ovariohysterectomy; sAlb, serum albumin; sUN, serum urea nitrogen; sCr, serum creatinine.

USG1.014 (1.006–1.048)1.015–1.045Leucocytes (109/L)11.8 (5.3–33.6)6.0–16.0
UPC1.37 (0.08–8.69)<0.5sAlb (g/L)28.0 (23.5–29.7)30.0–45.0
UPCfup1.01 (0.09–4.06)<0.5sUN (mmol/L)6.2 (3.4–49.2)3.3–8.3
   sCr (μmol/L)89.2 (46.0–477.0)<125
Table 3.  Summary of light microscopic kidney biopsy findings in 10 dogs with pyometra and urine dipstick protein 2+ or 3+ at ovariohysterectomy.
Renal Pathological Findings
  1. # dogs, number of dogs; # glom, number of affected, respectively, examined glomeruli; degree of tubular atrophy: 0 = no atrophy, + = focal, ++ = multifocal; degree of interstitial inflammation and/or interstitial fibrosis: 0 = no inflammation/fibrosis; + = inflammation/fibrosis that does not distort the tubulointerstitial architecture; ++ = inflammation/fibrosis that widely separates tubules; +++ = inflammation/fibrosis that replaces tubules.

Glomerular lesions
 GlomerulosclerosisGlobalFocal and segmental  
 # dogs24  
 # glom4/7, 13/2616/16, 12/34, 10/16, 13/26  
Tubular lesions
 Tubular atrophy0++++++
 # dogs5140
Interstitial lesions
 Inflammation0++++++
 # dogs3502
 Fibrosis0++++++
 # dogs5212
image

Figure 2.  Kidney section, dog, light microscopy, periodic acid-Schiff staining. (a) Segmental area of sclerosis (arrow) with adhesion near the origin of the proximal convoluted tubule; (b) glomerulosclerosis of approximately 40% of the tuft, with nodular hyalinosis (arrow) and thickening of Bowman's capsule. Interstitial fibrosis and tubular atrophy (asterisk); (c) global glomerulosclerosis (glomerular solidification) (arrow), interstitial infiltration of lymphocytes and plasma cells (asterisk) with tubular atrophy, and protein casts in tubules.

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image

Figure 3.  Kidney, glomerulus, dog, electron microscopy, 10,000 × . Capillary wall. Multifocal podocyte foot process effacement (arrow).

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Urinary Biomarkers

Dogs with pyometra had significantly increased ratios of uAlb/Cr, uIgG/Cr, uCRP/Cr, uRBP/Cr, uNAG/Cr, and uTXB2/Cr compared with healthy dogs (P < .01) (Table 1). With the exception of uCRP/Cr, uNAG/Cr, and uTXB2/Cr, urinary biomarker ratios in nonproteinuric (UPC < 0.5) dogs with pyometra were significantly lower than in dogs with pyometra and UPC>0.5 (P < .05, Table 1). Urinary biomarker ratios decreased significantly after ovariohysterectomy in the studied dogs (P < .05) (Table 1, supporting information Table S1). Most of the dogs with glomerulosclerosis or marked tubulointerstitial lesions tended to have UPC or urinary biomarker ratios above the 75th percentile of the total group of dogs with pyometra (Fig 1). The 2 dogs with UPC<0.2 had low ratios of urinary biomarkers (under median) and no relevant light microscopic lesions but strong glomerular immunostaining for IgG and IgA was observed in 1 of these 2 dogs.

Clinical Course of Biopsied Dogs with Proteinuria after Ovariohysterectomy

Based on the persistent proteinuria, treatment with an angiotensin-converting enzyme (ACE) inhibitorx was instituted after ovariohysterectomy in 4 dogs with pyometra. In 2 dogs, the UPC did not increase further, but because of their UPC remained above 2.0 after treatment with ACE inhibitor, additional treatment with lozartany was instituted. The UPC of another dog initially decreased from 3.05 to 1.69 two weeks after ovariohysterectomy, but fluctuated between 1.69 and 2.48 and then increased to 4.1 three months after surgery, at which time ACE inhibitor treatment was initiated. The dog that developed uremia postoperatively was euthanized 3 weeks after ovariohysterectomy. Theoretically, the uremia in this dog might have been the result of acute-on-chronic kidney disease. Having biopsy-documented evidence of the chronicity of the renal lesions in this dog was helpful with regard to prognosis.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

This study describes in detail a subset of dogs with pyometra and substantial proteinuria, and their related renal histomorphological changes by light, immunofluorescence, and electron microscopy. Proteinuria was present in approximately half of the dogs with pyometra, although it usually was mild and frequently transient. A few dogs remained severely proteinuric and required therapeutic intervention. In addition, glomerulosclerosis, tubular atrophy, and interstitial nephritis were common lesions in the dogs with pyometra that were biopsied in this study. Increased ratios of urinary biomarkers were associated with these structural lesions and provided further evidence of glomerular and tubular dysfunction.

Study group characteristics and blood and urine analysis results document that the included dogs with pyometra were similar to those of previous studies.2,3,5,8 The proportion of azotemic dogs (13%) was smaller than the proportion of dogs with pyometra and proteinuria (47%). Importantly, 26% of the dogs with pyometra had UPC>1.0, which has been described to be a factor in progression to ESRD in dogs with chronic kidney disease.19 Substantial chronic renal histological changes and persistent proteinuria were noted in 5 dogs with pyometra. One of these dogs was euthanized because of ESRD 3 weeks after ovariohysterectomy. Fifteen percent of the dogs with pyometra had UPC>2.0, which may indicate severely altered glomerular permselectivity.20 Only 4 dogs with a UPC>2.0 were included in the follow-up examination, and it cannot be concluded whether pyometra itself, a preexisting glomerular disease, or an exacerbation of existing renal disease by pyometra resulted in a severely altered glomerular permselectivity. Concurrent diseases such as leishmaniasis or hypertension might result in glomerular pathology and cannot be excluded as a cause of the observed lesions because serological testing and evaluation of blood pressure was not routinely performed in the studied dogs with pyometra. However, regardless of the exact underlying etiology of renal changes and proteinuria, our findings indicate that monitoring proteinuric dogs with pyometra after surgery is important to optimize their care. In this study, only 5 of 47 dogs with pyometra had detectable UTI, and they had varied UPC results. Urinary tract inflammation might be a potential source of increased UPC21,22 and cannot be ruled out as the predominant source in some of these samples. Renal proteinuria is difficult to assess in dogs with concurrent UTI. Although severe renal lesions were present in 1 of the 3 dogs with UTI and UPC>0.5, the increased UPC of the other 2 dogs could be the result of UTI. In addition, the effect of UTI on urinary biomarker concentrations needs to be further examined in future studies.23 Although the presence of upper UTI is less likely because of the absence of ultrasonographic findings typical for pyelonephritis,24 it cannot be definitively excluded in the dogs with UTI.

Urinary renal biomarkers followed the trend of UPC, and were evaluated for the first time in a larger population of dogs with pyometra, regardless of uterine culture results. Our findings indicate that the higher the UPC, the more severe are the renal histopathologic changes that can be expected. In addition, in the dogs biopsied and with relevant lesions of the glomeruli and tubulointerstitium, high ratios of urinary glomerular biomarkers, tubular biomarkers, or both were observed. Although this finding suggests an association between urinary biomarkers and renal structural lesions, the number of dogs was limited, and 1 dog had minimal histological lesions but increased UPC and urinary biomarkers. High urinary enzyme concentrations previously were found to reflect extensive proximal tubular lesions in dogs with pyometra.2 However, future work is needed to define the specificity of urinary biomarkers for localization of kidney injury in dogs with pyometra. In contrast to earlier results,3 approximately 40% of dogs with pyometra had uCRP concentrations below the detection limit, independent of the uterine culture results and magnitude of UPC. Because CRP is a high molecular weight protein, its appearance in urine might be the result of a severely altered glomerular permselectivity. Increased systemic CRP concentrations have been reported in dogs with pyometra.25 Whether variations in plasma concentrations of CRP influence urinary concentrations in dogs with pyometra requires further investigation.

Based on the literature and previous pilot studies, a kidney biopsy specimens was considered clinically indicated in dogs with pyometra and substantial proteinuria.2,5,26 In the current study, UPC results were not available at the time of surgery. Inclusion criteria for the kidney biopsy specimens could have been refined by obtaining UPC or combining a dipstick urine protein result of 3+ with a low USG (<1.015). This probably would have resulted in the selection of dogs with UPC>0.5. On the other hand, a recent study suggests that dogs with dipstick urine protein 2+ or 3+ are likely proteinuric regardless of their USG.27 A limitation of the study is that no healthy age-matched bitches nor nonproteinuric dogs with pyometra were included for kidney biopsy specimens (for ethical reasons). Because the degree of pyometra-associated kidney dysfunction can vary widely,3 the presence of the 3 dogs with lower UPC (0.08, 0.19, 0.51) provides valuable alternative renal histologic information. One half of the dogs with pyometra in our study were not proteinuric (UPC<0.5). Only 2 dogs with pyometra and UPC<0.2 were biopsied, and these showed minimal renal histologic changes. Although this might indicate that nonproteinuric dogs with pyometra also have less kidney injury, evaluation of a larger number of nonproteinuric dogs is needed to address proteinuria and histologic kidney changes associated with pyometra.

All biopsied dogs with persistent UPC>1.0 after ovariohysterectomy also had glomerulosclerotic lesions. Because glomerulosclerosis can be present before pyometra and persist after ovariohysterectomy, it is not clear if pyometra-associated pathogenic mechanisms cause or exacerbate glomerulosclerosis and proteinuria in dogs. In addition, focal and segmental glomerulosclerosis is clinically and pathologically not yet well defined in dogs. According to the initial data of the world small animal veterinary association renal standardization project,28 glomerulosclerosis occurs in middle-aged dogs and is more common in female than in male dogs. Dogs with focal and segmental glomerulosclerosis generally are nonazotemic or mildly azotemic, and show moderate proteinuria and infrequently have hypoalbuminemia. Our findings corroborate these features. The presence of glomerulosclerosis in healthy dogs has been described to increase with age.29 Furthermore, the occurrence of glomerulosclerosis has been reported in dogs with decreased functional renal mass after nephrectomy (reviewed in Brown et al30). Glomerulosclerosis also has been mentioned as part of leishmaniasis, albeit not as a primary aspect of the disease.31 Indeed, glomerulosclerosis often is the final stage of several glomerulopathies of variable etiology.30 Until kidney biopsy specimens of healthy bitches are evaluated, glomerulosclerosis cannot be excluded as an underlying preexisting renal disease in dogs with pyometra.

No evidence for an immune complex-mediated glomerulopathy was found in dogs with pyometra except for dog, because no relevant positivity was detected on immunofluorescence microscopic evaluation and no electron dense deposits were seen on ultrastructural examination. Occasional nonspecific trapping of IgM and C3 might occur in sclerotic regions of glomeruli in humans with focal segmental glomerulosclerosis (FSGS) and dogs with focal and segmental glomerulosclerosis.32,33 Renal histologic changes in dogs with pyometra have been characterized previously by older microscopic techniques.7–10 In addition, classification criteria for glomerular diseases in dogs were largely lacking at that time, thus not preventing interpretation of nonspecific trapping as immune-mediated glomerulonephritis. On the other hand, a variable degree of tubulointerstitial lymphoplasmacytic inflammation and fibrosis was found in the present study and is in agreement with previous reports.2,5,7,8 Although neutrophils have been described as the main interstitial inflammatory cell type in 2 of 19 dogs with pyometra in a previous study,5 this was not observed in any kidney biopsy specimens of our study.

Based on the persistent proteinuria in some dogs with pyometra, treatment with ACE inhibitors was started to prevent worsening of proteinuria. Because these dogs also had chronic kidney lesions, this therapy was instituted in hope of limiting possible progression to ESRD.34–36 Controlled data regarding the optimal treatment of focal and segmental glomerulosclerosis in dogs are not yet available. In human medicine, both ACE inhibitors and immunomodulators are used for the treatment of FSGS, but evidence-based guidelines also are lacking.37 Extensive follow-up studies are needed to evaluate the appropriate treatment in dogs with pyometra and glomerulosclerotic lesions.

In conclusion, proteinuria is an important aspect of pyometra-associated nephropathy. Kidney biopsy findings indicate tubulointerstitial nephritis in many of the dogs with pyometra and UPC>0.5, and a frequent association with glomerulosclerosis when UPC>1.0. Urinary biomarkers supported glomerular and tubular dysfunction in dogs with pyometra. Future studies should elucidate whether focal segmental glomerulosclerosis is a primary lesion of pyometra-associated nephropathy.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

a LaserCyte Hematology Analyzer, Idexx, Hoofddorp, the Netherlands

b Catalyst Dx Chemistry Analyzer, Idexx

c Coag Dx Analyzer, Idexx

d Combur stick, Roche Diagnostics, Burgess Hill, UK

e Urinary/CSF Protein 911 Analyzer, Roche Diagnostics

f Placivet, Codifar, Wommelgem, Belgium or Plegicil, Pherrovet, Malmö, Sweden

g Valium, Roche, Hertfordshire, UK

h Mephenon, Denolin, Brussels, Belgium or Metadon Recip, RecipAB, Solna, Sweden

i Propovet 10 mg/mL, Abbott Lab, Maidenhead, UK

j Isoflo, Abbott Lab

k Petrem, Piramal, Mulund, Mumbai, India

l Rimadyl, Pfizer A.H., London, UK

m Tradolan, Nordic Drugs, Limhamn, Sweden

n Bard Biopty-Cut device with 14 G (1.2 mm × 19 mm) biopsy needles, C.R. Bard Inc, Covington, GA

o Bethyl Laboratories Inc, Montgomery, TX

p Dako, Glostrup, Denmark

q Dog Albumin enzyme-linked immunosorbent assay (ELISA) kit, Immunology Consultants Laboratory, Newberg, OR

r Dog Immunoglobulin G ELISA kit, Immunology Consultants Laboratory

s Dog C-reactive protein ELISA kit, Immunology Consultants Laboratory

t Human Retinol-Binding Protein ELISA kit, Immunology Consultants Laboratory

u Thromboxane B2 competitive enzyme immunoassay, Assay Designs, Ann Arbor, MI

v β-N-acetylglucosaminidase assay kit, Sigma-Aldrich, St Louis, MO

w SPSS 15, SPSS Inc, Chicago, IL

x Fortekor, Novartis, Basel, Switzerland

y Cozaar, MSD-Merck & Co Inc, White House Station, NJ

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

This research was funded by the Bijzonder Onderzoeksfonds of the University of Ghent (BOF, grant to B. Maddens). The authors are grateful to Sofia Nardai Persson for her excellent technical assistance and appreciate the cooperation of all dog owners.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
  9. Supporting Information

Table S1. UPC and urinary biomarker ratios of dogs with pyometra from which a kidney biopsy was collected, at ovariohysterectomy and at follow-up visit 2 weeks after ovariohysterectomy (fup).

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