Resolution of a Proteinuric Nephropathy Associated with Babesia gibsoni Infection in a Dog

Introduction

Glomerular disorders are challenging for the veterinarian, because, although many causes have been described, the identification of an underlying cause is not commonly found in the clinical setting. The following case demonstrated a suspected role of a traditionally nonclinical patient with Babesia gibsoni in the participation of antigenic stimulation and glomerular immune complex deposition, leading to the development of proteinuric nephropathy. In this case, the procurement of renal biopsies was extremely valuable in understanding the pathogenesis of the insult, rationale for specific therapy, and histopathologic severity of the lesions, which helped to predict prognosis and response to therapy. Animals with proteinuric nephropathies can have a poor long-term prognosis, although the expected survival is largely unknown.

The clinical implications of this case report included identification of the suspected role of B gibsoni in etiology of proteinuric nephropathy and response to treatment, the importance of expanding infectious disease searches in similar cases, and the utility of renal biopsies in providing valuable information in similar cases. To the authors' knowledge, only one other case report in the veterinary literature has described protein-losing nephropathy associated with B gibsoni. By advancing awareness of this atypical presentation of the disease, more cases may be diagnosed and more effectively treated.

Case Report

A 4 yr old male castrated Labrador retriever was referred to the Internal Medicine service with a 2.5 wk history of inappetance, lethargy, weight loss, polyuria/polydipsia, and small bowel diarrhea. Test results performed 14 days earlier at the local veterinarian revealed azotemia (blood urea nitrogen [BUN] 66 mg/dL, reference range 6–25 mg/dL; creatinine 2.9 mg/dL, reference range 0.5–1.6 mg/dL), hypoalbuminemia (1.5 g/L, reference range 2.7–4.4 g/L), hyperphosphatemia (7.9 mg/dL, reference range 2.5–6.0 mg/dL), proteinuria (3+via dipstick, verified by 3% sulfosalicylic acid, reference range negative), and mild pyuria (4–10 white blood count [WBC]/high-powered field [HPF], reference range 0–3 WBC/HPF, voided sample) with isosthenuric urine (urine specific gravity [USG] 1.016). Clinical signs persisted despite treatment with enalapril^a (15 mg per os [PO] q 24 hr) and metronidazole^b (500 mg PO q 24 h). No recent travel history outside of Pennsylvania and New Jersey, tick exposure, or intoxications were reported. Three weeks before illness, the dog was vaccinated for Leptospirosis and Bordetellosis^c. Previous medical treatment included year-round heartworm^d and flea/tick^e preventatives, as well as phenobarbital^f and thyroxine^g for previously diagnosed epilepsy and hypothyroidism.

Physical examination showed the dog was thin (body condition score 2.5/5), weighing 33.1 kg (6 kg loss over 1 mo), with stable vital parameters and no cardiopulmonary abnormalities. The abdomen was tense on palpation. No peripheral lymphadenopathy was appreciated. Muscle atrophy was present along the hind limbs, spine, and head. Fundic examination was unremarkable, and the dog was normotensive (systolic pressure 160 mm Hg).

A serum biochemical profile at admission showed worsening azotemia (BUN 146 mg/dL, creatinine 3.2 mg/dL), hypoalbuminemia (1.4 g/dL), and hyperphosphatemia (8.0 mg/dL). In addition, increased alkaline phosphatase activity (220 U/L, reference range 20–155 U/L) and hypercholesterolemia (458 mg/dL, reference range 128–317 mg/dL) were present. Urinalysis (cystocentesis) showed a USG of 1.020, persistent proteinuria (3+), and the presence of coarse granular casts. A complete blood count was within normal limits. Colloid oncotic pressure was low at 10.1 mm Hg (reference range 15.3–26.3 mm Hg).

Further diagnostics confirmed proteinuria (urine protein/creatinine [UPC] 11.5, reference range <0.5). A urine culture was negative. Serologic test results for heartworm antigen and antibodies against Ehrlichia canis, Borrelia burgdorferi, Anaplasma phagocytophilum^h, Rickettsia rickettsiiⁱ, and Bartonella^j spp. (via Western blot) were negative. Leptospirosis titers were negative for Leptospira canicola, L hardjo, L pomona, and L bratislava serovars; titers for serovars L grippotyphosa and L icterohemorrhagiae were mildly increased at 1:200 (the dog was vaccinated 5 wk earlier). All serology was performed at least 2 wk after initial clinical signs were reported.

Thoracic radiographs showed no significant findings. Abdominal ultrasound revealed both kidneys to be in the upper range of normal size with normal echostructure. Areas of mesentery were slightly hyperechoic in the area surrounding the pancreas, with normal pancreatic architecture.

The dog was diagnosed with proteinuric nephropathy of unknown etiology. Treatment included intravenous fluid therapy (Plasmalyte^k at 2 mL/kg/hr and Hetastarch^l at 1 mL/kg/hr, both discontinued within 24 hr), ampicillin^m (22 mg/kg IV q 8 hr), doxycyclineⁿ (10 mg/kg IV q 12 hr), famotidine^o (0.5 mg/kg IV q 12 hr), enalapril (0.4 mg/kg PO q 12 hr), and moderate protein restricted diet (Hill's D/D dry diet^p). Maintenance phenobarbital (1.5 grain PO q 12 hr) was continued and thyroid supplementation was discontinued.

Due to clinical improvement, the dog was discharged after a 3 day hospitalization with oral doxycycline^q (1 mo total), enalapril, famotidine, Hill's D/D, and amoxicillin^r (20 mg/kg PO q 12 hr for 2 wk). Biochemical abnormalities were relatively unchanged (BUN 97 mg/dL, creatinine 2.7 mg/dL, phosphorus 6.3 mg/dL, albumin 1.5 g/dL) when he returned 3 days later for percutaneous needle biopsies of kidney obtained with ultrasonographic guidance under general anesthesia. A coagulation profile was not performed due to documentation of normal platelet count and blood pressure and no clinical evidence of coagulopathy. Portions of the samples obtained were separately preserved appropriately in 10% formalin solution, 3% glutaraldehyde solution, and Michel's Transport Medium^s for histopathologic evaluation, transmission electron microscopic (TEM) evaluation, and immunostaining, respectively, and transported overnight on ice to the Texas Veterinary Renal Pathology Service, Texas A&M University, for further processing and examination. After the biopsy procedure, administration of antithrombotic low-dose aspirin (0.5 mg/kg PO q 24 hr) was started.

Light microscopic changes were present in the glomerular and tubulointerstitial compartments (Figure 1). This photomicrograph shows light microscopic changes present in the glomerular and tubulointerstitial compartments. The glomerulus exhibits mild hypercellularity and a segmentally increased mesangial matrix (arrow). The interstitium was variably and mildly expanded by edematous collagenous connective tissue containing widely scattered inflammatory cells (periodic acid-Schiff stain; 20×). The 23 glomeruli in the sections examined were mildly to moderately hypercellular with parietal and visceral epithelial cell hypertrophy, increased endothelial prominence, and mild mesangial hypercellularity. Small numbers of neutrophils were occasionally present within glomerular capillary lumina. The mesangial matrix was variably and segmentally increased in prominence with rare extension into peripheral capillary loops. Glomerular tufts were occasionally focally adherent to minimally thickened Bowman's capsules. The proximal tubular epithelial cells were moderately swollen with a subset of tubular profiles exhibiting diffuse cytoplasmic microvesiculation. The interstitium was multifocally and mildly expanded by dense to loose (edematous) collagenous connective tissue containing widely scattered and mild infiltrates of mixed mononuclear and fewer neutrophilic inflammatory cells. Few tubules contained intraluminal accumulations of homogenous to granular eosinophilic material. Glomerular arterioles were often dilated with one arteriole exhibiting a focal area of medial fibrinoid change. Small numbers of individual necrotic and/or apoptotic cells were randomly scattered within glomeruli and tubular epithelia.

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Electron microscopy revealed mild to moderate glomerular, tubular, and interstitial changes. The three glomeruli examined showed multifocal to confluent areas with visceral epithelial cell foot process fusion. The mesangium had multifocal areas with increased matrix and cells. Electron-dense deposits (irregular, 0.1–2.5 μm) were scattered within the substrate (Figure 2). This electron micrograph shows electron-dense deposits within the mesangium (arrows; 5,000×).

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There were multifocal areas with mesangial cell interpositioning with the filtration membrane. Focal areas with mesangial cell interpositioning showed subendothelial to intramembranous deposits (Figure 3). This electron micrograph shows mesangial cell interpositioning of the filtration membrane (arrows; 15,000×). The visceral epithelial, mesangial, and tubular epithelial cells had an increase in cytoplasmic lipid droplets and residual bodies. The interstitium had focal areas with increased collagen and edema. Scattered lymphocytes and macrophages were present.

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Immunostaining of cryosections from all samples submitted for this evaluation was performed, but informative results were not obtained due to lack of glomeruli within the samples.

The changes in the sample were consistent with primary immune-mediated glomerular disease characterized by immune complex glomerulonephritis with immune deposits located mainly in the mesangium. Overall, the changes were considered mild to moderate in severity.

Because of concern for ongoing immune-complex deposition, an immunosuppressive protocol of SoluMedrol^t (5 mg/kg IV q 24 hr ×2 doses) and cyclophosphamide^u (200 mg/m² IV) was initiated to potentially reduce ongoing glomerular damage. At the same time, an infectious disease search was expanded to include Babesia spp. whole blood polymerase chain reaction (PCR), results of which were positive for B gibsoni (Asian genotype)^v.

Because of these PCR results, immunosuppressive therapy was stopped and the dog was given azithromycin^w (10 mg/kg PO q 24 hr for 10 days) and atovaquone^x (13.3 mg/kg PO q 8 hr for 10 days) for treatment of B gibsoni. In total, the dog received one treatment of 320 mg of SoluMedrol and 202 mg of cyclophosphamide.

One week after starting antibabesial therapy, the dog was doing well clinically. A complete blood count was unremarkable other than a mild normochromic, normocytic anemia (hematocrit 33.5%, reference range 36–60%). The dog's azotemia had resolved, but hypoalbuminemia (1.5 g/L), hypercholesterolemia (369 mg/dL), and increased alkaline phosphatase activity (724 U/L) remained.

Two weeks after azithromycin and atovaquone therapy was finished, the dog's azotemia resolved, and both hypoalbuminemia (1.9 g/dL) and proteinuria (UPC 2.95) were of lesser magnitudes than previously noted. Anemia was still present (hematocrit 30%). Due to palatability, the diet was changed to commercial moderately protein-restricted renal diet^y.

Approximately 3 mo after therapy was finished, the dog had no clinical signs of disease. At recheck, he was no longer azotemic, had normal serum albumin, and was no longer significantly proteinuric (UPC 0.41). His hematocrit was within normal limits (42%). Another Babesia spp. PCR was performed and was negative^w. Based on his status, enalapril and aspirin were discontinued.

Six months after therapy, diagnostics performed at the local veterinarian showed that the dog was not azotemic or anemic, and continued to exhibit negligible proteinuria (UPC 0.5) without medications. Phenobarbital and famotidine administration was continued throughout this time.

Approximately 18 mo after therapy, diagnostics performed at the local veterinarian showed that the dog continued to be nonazotemic with a normal hematocrit and serum albumin.

Discussion

Glomerular disorders are a frequent cause of renal disease in dogs. Causes of canine glomerular damage are numerous and include infectious diseases, chronic inflammatory conditions, neoplasia, and certain inherited disorders.¹ Infectious diseases previously reported to cause glomerular disease in dogs include Ehrlichiosis, Brucellosis, Borreliosis, Bartonellosis, Dirofilariasis, Blastomycosis, Babesiosis, Hepatozoonosis, Leishmaniasis, Rocky Mountain Spotted Fever, and Infectious Canine Hepatitis.¹ Further categorization of canine glomerular diseases based on their histopathological appearance or underlying cause may enhance understanding of disease progression, prognosis, and in some cases, therapy.

Membranoproliferative glomerulonephritis (MPGN), possibly the most common histopathologic type of acquired glomerular disease in dogs, is often induced when infectious diseases cause immune complexes to become deposited in the glomerular mesangia and on the subendothelial aspect of the glomerular basement membranes, leading to activation of cytokines and inflammatory mediators, leukocyte influx, mesangial expansion, and ultimately to loss of normal filtration ability.² Treatments for proteinuric nephropathies in dogs mainly are symptomatic and supportive (angiotensin-converting enzyme inhibitors, antihypertensives, ultra-low-dose aspirin, diet modification), with specific therapy administered only if a suspected underlying cause (e.g., an infectious agent) is identified. In the human literature, many glomerular diseases, including variants of MPGN, have been treated with immunosuppressive drugs in addition to symptomatic therapy with the goal of reducing both circulating immune complex load and glomerular inflammation.^3–5 However, similar therapeutic protocols have not been established as standard care for glomerular diseases in companion animals.²

B gibsoni and B canis are the two predominant species capable of natural canine Babesiosis infection worldwide.⁶ The protozoal organisms are transmitted through the bites of infected ticks or orally via fights or bites with infected dogs; in the eastern United States, most infections occur in American Staffordshire and American pit bull terriers or dogs involved in fighting with them (B gibsoni) or in greyhounds (B canis).⁶ The common clinical presentations of Babesiosis include signs secondary to hemolytic anemia and multiple organ dysfunction— weakness, pallor, fever, splenomegaly, and anorexia. Clinicopathologic findings are typically consistent with regenerative anemia and thrombocytopenia, usually with evidence of intra- or extravascular hemolysis. Diagnosis can be made by identification of organisms within infected red blood cells, positive serology, or confirmation of Babesia spp. DNA from infected tissue or blood.⁶ Because of increased specificity and sensitivity, nucleic acid dectection via PCR may be a more advantageous test for not only speciating Babesia spp., but for detecting subclinical infections.⁶ Treatment of B canis (a large Babesia sp.) includes the use of imidocarb diproprionate; however, this drug has not been demonstrated to clear B gibsoni (a small Babesia sp.) from infected dogs. The efficacy of combined atovaquone/azithromycin as therapy for treatment of B gibsoni has been demonstrated.⁷

This case report identified a dog with a proteinuric nephropathy and concurrent evidence of B gibsoni infection whose clinical and clinicopathologic abnormalities resolved after treatment targeted at reducing the presence of both antigen and antibody sources potentially responsible for glomerular immune complex deposition. Renal biopsy showed histologic changes consistent with MPGN and ultrastructural evidence of glomerular immune complex deposition. Immunostaining of kidney for immunoglobulins (G, M, A) and complement (C3) is yet another way to identify deposits of immune reactants in the glomeruli. Unfortunately in this case, the tissue samples available for immunostaining did not contain sufficient glomeruli suitable for evaluation. Evaluation of biopsy specimens under low-magnitude microscopy to identify glomeruli before submission might have increased the likelihood of appropriate samples for immunostaining. However, the clinical diagnosis and treatment decisions were made with reasonable confidence based on the TEM changes and clinicopathologic evidence of glomerular disease.

Elution of specific Babesia antigen–antibody complexes from glomeruli may provide further evidence of immune-mediated injury in future cases but is not readily available currently. Immunohistochemistry is not currently performed on renal biopsies for Babesia because it is an erythrocyte-associated pathogen and unlikely to be found in renal cells. Similarly, results of PCR of renal tissue itself may be difficult to interpret due to the confounding presence of potentially infected erythrocytes in submitted samples, in addition to the fact that any DNA amplified from renal parenchyma would not discriminate between glomerular and tubulointerstitial compartments.

As a disease that is primarily characterized by interstitial nephritis, and resultant mild proteinuria consistent with tubular damage, Leptospirosis was considered a less likely cause in this patient.⁸ However, due to potential public health concerns with a zoonotic agent, serologic testing was submitted. Although other studies demonstrated a glomerular range of moderate to severe proteinuria with dogs diagnosed with Leptospirosis, this presentation subset was less common and might reflect glomerular lesions associated with chronic disease.⁸

Vaccinations for Leptospirosis and Bordetellosis were given 3 wk before the onset of illness. Postvaccinal glomerulonephritis has not been documented in dogs but is theoretically possible because circulating antigen–antibody complexes may rise. Elution studies to check for Bordetella or Leptospira spp. specific antigen–antibody complexes in glomeruli are unavailable. Whole cell Leptospira bacterins have been associated with high allergenicity/anaphylaxis, but increased acute adverse events were not found to be associated with Bordetella bacterin or purified subunit Leptospira bacterin compared with other vaccines. ^9–11 Also, because postvaccinal Leptospirosis titers were borderline or low, postvaccinal (Leptospira) glomerulonephritis was deemed unlikely.

Although PCR positive for Babesia, the dog in this report did not show the classic presentation for this disease (i.e., significant anemia), but a positive PCR test suggested evidence of a subclinical infection. He had no history of dog fights or previous transfusions and was given year-round flea and tick preventatives. However, after appropriate therapy, the dog became PCR negative, suggesting that, even if subclinical, treatment might have reduced an antigenic source in the body that was contributing to glomerular immune complex deposition.

It is unclear whether resolution of the dog's glomerular disease was due partly or entirely to immunosuppressive therapy, atovaquone/azithromycin administration, or both. Either therapy could have acted to reduce circulating immune complex deposition in the glomeruli, allowing adequate time for tissue repair. Human immunosuppressive protocols are often given over several weeks and, in this case, an abbreviated dosage was administered.⁵ Therefore, the role of immunosuppression might have been minor relative to the full course of antiprotozoal therapy that was given. In addition, no other underlying cause (infectious or otherwise) for the MPGN was found, but it is possible that unidentified agents or factors responsive to therapy might have contributed to the immune complex glomerulonephritis in this case.

Conclusion

This report documented a case of a proteinuric nephropathy in a dog PCR positive for B gibsoni who had resolution of clinicopathologic abnormalities after treatment with both immunosuppression and antiprotozoal agents. Although an unexpected presentation for Babesiosis, the authors suspected that chronic carrier status of this pathogen provided an antigenic source for immune complex formation and deposition in glomeruli; azotemia and proteinuria resolved after successful treatment of Babesiosis. To the authors’ knowledge, only one other case report in the literature has described proteinuric nephropathy associated with B gibsoni (confirmed via PCR); in that case, the dog was severely anemic and no renal biopsies were performed. Treatment involved prednisolone and buparvaquone, and proteinuria and anemia improved (but did not resolve) after therapy; however, that dog remained PCR positive.¹² Recent literature also reported a number of azotemic and proteinuric dogs positive for Babesia microti-like organisms (a strain of B gibsoni) via PCR, which provided the further suggestion that Babesial organisms might play a role in the development of canine proteinuric nephropathy.¹³ Because the potential for variants of B gibsoni resistant to atovaquone and azithromycin have recently been reported, the dog in this report will have whole blood PCR tests repeated in the future to confirm status.¹⁴

This case underscored the importance of identifying potential infectious etiologies in cases of MPGN in hopes of identifying a source of immune stimulation so that specific therapeutics might be selected. Specifically, diagnostics for Babesiosis might be recommended for dogs with protein-losing nephropathy even in cases in which infection might be subclinical or the dog is not typically a breed at risk. This case's clinical presentation was similar to the proteinuric nephropathy sometimes seen with Lyme disease (Lyme nephropathy) in both clinicopathologic and renal biopsy findings.^15,16 Had Babesiosis not been tested for or treated, or if the dog concurrently had serologic evidence of exposure to Borrelia burgdorferi, he might have been given a poorer prognosis traditionally ascribed to that population and further therapeutics might not have been pursued. In addition, this case demonstrated the diagnostic benefit of renal biopsy in confirming glomerular pathology, assessing subtype of glomerular disease, and documenting disease progression. When feasible, renal biopsy of affected dogs might provide keener insight into the most effective treatment of disease or estimate response to therapy.