Drug-Induced Minimal Change Nephropathy in a Dog

A 7-year-old, spayed female Giant Schnauzer was referred to the veterinary internal medicine service of The University at Georgia because of severe proteinuria. The dog had a 5-year clinical history of recurring otitis externa and allergic dermatitis, which were diagnosed as atopy 7 weeks before initial examination. At this time (Day −48), the dog was enrolled into a multicenter, randomized, double-blinded clinical trial evaluating the efficacy of masitinib mesylatea for the treatment of atopy. Masitinib is a selective and potent inhibitor of c-KIT-dependent cell proliferation as well as PDGFR-dependent cell proliferation in vitro. Furthermore, masitinib inhibits to a lesser extent FGFR3.1 Another potentially important target of masitinib is LYN, which is a key component of the transduction pathway leading to IgE-induced degranulation.2 A physical examination (PE), CBC, serum biochemistry profile (chem), and urinalysis (UA) were performed 2 hours before enrollment in the clinical trial. Results of the blood work were within reference ranges except for an increased mean platelet volume (MPV) of 11.2 fL (normal: 6.1–10.1 fL) and total protein (TP) of 7.4 g/dL (normal: 5.2–7.3 g/dL). Blood urea nitrogen (BUN) and creatinine concentrations were within the reference range at 11 mg/dL (normal: 10–30 mg/dL) and 1.4 mg/dL (normal: 0.5–1.5 mg/dL), respectively. UA revealed a low urine specific gravity (USG) of 1.015, pH was 7.5, and protein was negative on dipstick testing.b,c The study protocol included a PE and UA every 2 weeks and PE, CBC, chem, and UA every 4 weeks. Treatment with masitinib was initiated at a dose of 12 mg/kg PO q24h. The owner was also told to feed Hill's d/d Potato & Salmon dietd exclusively, to clean her dog's ears twice weekly with Epi-Otic Ear Cleanser,e and to bathe the dog once weekly with Malaseb shampoo.f Two weeks later (Day −35), freshly voided urine was obtained and analyzed by dipstick and refractometry. The USG was again 1.015, pH was 7, and trace protein was present on dipstick testing. Five weeks after initiation of therapy (Day −13), the dog presented late for her recheck appointment, which included a PE, CBC, chem, and UA. The dog's dermatologic signs were still present but the owner reported no other abnormalities. However, the PE showed a secondary bacterial pyoderma on the front feet and in both ears, and weight loss of 5 kg. Blood work revealed the following abnormalities: leukopenia (3.5 × 103 leukocytes/μL, normal: 7.12–20.21 × 103/μL), neutropenia (1.2 × 103 mature neutrophils/μL, normal: 3.91–13.7 × 103/μL), and lymphopenia (1.6 × 103 lymphocytes/μL, normal: 1.88–6.22 × 103/μL). The dog was hypoalbuminemic with a serum albumin concentration (Alb) of 1.7 g/dL (normal: 2.6–3.8 g/dL) but had an increased serum globulin concentration of 5.0 g/dL (normal: 1.8–3.1 g/dL). Other abnormalities included an increased blood glucose (124 mg/dL, normal: 61–114 mg/dL) and decreased phosphate (3.5 mg/dL, normal: 3.8–9.8 mg/dL). Cephalexing was prescribed at a dose of 25 mg/kg PO every 12 hours for 3 weeks for treatment of pyoderma. A UA completed 2 days later revealed a USG of 1.015, a pH of 7.5, and 3+ protein (300 mg/dL). The owner was advised to check for proteinuria via dipstick every other day. The proteinuria persisted at 3+ to 4+ (ie, 300 to >2,000 mg/dL) as assessed by urine dipstick for the next 7 days. Two days after the 1st documentation of proteinuria (Day −9), administration of masitinib was discontinued, and 4 days later (Day −5), cephalexin was discontinued. A follow-up examination 13 days after the initial diagnosis of proteinuria (Day −1) revealed that there was a mild nonregenerative, normocytic, normochromic anemia with a hematocrit (Hct) of 35.5% (normal: 37–55%) and a hemoglobin concentration (Hgb) of 11.5 g/dL (normal: 12–18 g/dL). The hypoalbuminemia had worsened, with Alb decreasing from 1.7 g/dL on Day −13 to 0.8 g/dL on Day −1. The dog remained mildly hyperglobulinemic (3.3 g/dL). The UA (cystocentesis urine) showed a USG of 1.007, pH of 7, and 3+ protein (confirmed by Robert's reagent testing) and 1+ blood (50 erythrocytes/μL) on urine dipstick. Microscopic examination of the urine sediment revealed 2 to 4 erythrocytes/hpf. The urine protein-creatinine ratio (UPC) was 8.2 (normal: <0.3). Based on the worsening of the proteinuria and hypoalbuminemia, the dog was hospitalized for further evaluation. On presentation (Day 0) to the Internal Medicine Service, the owner mentioned a several-day history of polydipsia and polyuria. Other presenting complaints included mild signs of lethargy and a reduced appetite of approximately 1 week duration. Upon PE, the Giant Schnauzer still had the previously noted skin abnormalities, as well as slightly enlarged mandibular and popliteal lymph nodes. Systolic blood pressure was normal at 120 mmHg. The dog's current weight was 33 kg, a decrease of 4.5 kg compared with enrollment in the clinical trial (Day −48). No other abnormalities were identified. During the 1st day of hospitalization, the following abnormalities were noted: a persistent, normocytic, normochromic anemia with an Hct of 36.3% and an Hgb of 12.1 g/dL. The dog was hypoalbuminemic and hypoproteinemic, with an Alb of 1.4 and a TP of 4.1 mg/dL. Colloid osmotic pressure was decreased at 11.8 mmHg (normal: 19.95–22.05 mmHg). The USG was 1.008, the pH was 7.5, and the urine dipstickc showed 3+ protein (confirmed by Robert's reagent testing). The urine sediment was unremarkable. Antithrombin activity was low with 74% activity (normal: 108–146%). Aerobic urine culture showed no growth after 48 hours, and Gram staining of the submitted urine did not show any organisms. Coagulation profile, indirect Coombs', and testing for antinuclear antibodies were all unremarkable. Serologic testing for Rickettsia rickettsii, Ehrlichia canis, Ehrlichia chaffeensis, Borrelia burgdorferi, Anaplasma spp., and Dirofilaria immitis was negative. Thoracic and abdominal radiographs revealed mild right-sided microhepatica and mild spondylosis deformans within the caudal thoracic and cranial lumbar spine. Abdominal ultrasound showed a single, small well-defined hypoechoic splenic nodule but was otherwise unremarkable. Additionally, fine needle aspirates of the mandibular and popliteal lymph nodes were performed, which revealed mild lymphoid hyperplasia. A presumptive diagnosis of subacute glomerulopathy was made based on the persistent proteinuria and hypoalbuminemia. On Day 2 of hospitalization, 0.5 × 0.5 × 0.5 cm wedge biopsies were obtained from both left and right kidneys via laparotomy. Some small pieces of renal cortex were placed in 10% buffered formalin, while others were placed in glutaraldehyde for fixation and processing for light and electron microscopic evaluation, respectively. Postsurgical treatment included a constant rate infusion of isotonic, polyionic crystalloidsh IV at a rate of 3.4 mL/kg/h for 7 hours, then 1 mL/kg/h for 5 hours. Buprenorphinei at a dose of 0.01 mg/kg was given IV every 6 hours for postsurgical analgesia and 0.5 mg/kg benazeprilj PO once a day as a glomeruloprotective agent. Aspirink at a dose of 0.5 mg/kg was also administered PO once a day as an antithrombotic, while histopathology of the kidney biopsies was pending. The crystalloids were discontinued after 24 hours and a concentrated fatty acid dietary supplement, 1 Derm Caps ESl capsule PO daily, was added to provide Ω-3 fatty acids. The dog recovered well from surgery and was discharged on Day 3 after hospitalization. At that time, buprenorphine was discontinued and tramadolm tablets were given to the owner to administer for pain control as needed over the next 2 days (up to 2 mg/kg PO TID). Sections of formalin-fixed, paraffin-embedded kidney were sectioned at 3 μm and stained with hematoxylin/eosin, periodic acid-Shiff hematoxylin, and Masson's trichrome stain. Histopathologic changes within the kidney biopsy were minimal. Glomeruli were of normal cellularity, and thickening of the glomerular capillary loops was not evident. Within many glomeruli, a few podocytes were enlarged with increased cytoplasmic volume and slightly enlarged nuclei (Fig 1); rare parietal epithelial cells lining Bowman's capsule were similarly hypertrophied. Deposits were not observed with Masson's trichrome stain, and basement membrane changes were not evident when stained with periodic acid-Schiff hematoxylin. A few renal corpuscles had mild thickening or splitting of the basement membrane of Bowman's capsule. The proximal tubular epithelium was slightly swollen with eosinophilic and granular cytoplasm (hyaline droplet change) and hyaline casts were present in a small number of distal tubules, indicative of proteinuria. Photomicrograph (light microscopy) of kidney wedge biopsy illustrating the appearance of the majority of glomeruli from a dog with minimal change nephropathy. Glomeruli often had a few podocytes with slightly enlarged nuclei and increased amounts of cytoplasm (see 3 podocytes at arrow), but no mesangial changes or thickening of basement membranes. Hematoxylin and eosin stain. Bar = 100 μm. Based on the histopathological findings, most likely diagnoses were early membranous glomerulopathy or minimal change nephropathy (MCN). Transmission electron microscopy was used to characterize the kidney pathology. Ultrastructurally, changes were restricted to the glomerular visceral epithelial cells (podocytes) and consisted of markedly diffuse foot process effacement and villous transformation (2, 3). The cytoplasm of the podocytes was swollen and occasional vacuoles containing debris were identified. The glomerular basement membrane (GBM) and endothelium were normal in appearance, and immune deposits were not observed. A diagnosis of MCN was made based on these ultrastructural findings. Ultrastructural glomerular changes are characterized by diffuse podocyte foot process effacement (long arrows) and podocyte villous transformation. The glomerular basement membrane (asterisk) and the capillary endothelial cells (arrowheads) are normal. VS, vascular space containing an erythrocyte. Transmission electron microscopy. Bar = 1 μm. Ultrastructurally, podocytes (long arrow) form an almost continuous sheet over the glomerular basement membrane (asterisk) and there are microvillous projections (short arrow). The endothelium is normal (arrowhead). Occasional vacuoles containing debris are observed in podocytes (end of long arrow). Transmission electron microscopy. Bar = 200 nm. Three days after discharge (Day 6), follow-up bloodwork and UA were performed (Fig 4). Hct and Hgb were mildly decreased at 34.4% and 11.7 g/dL. The platelet count was increased (627 × 103/μL, normal: 170–400 × 103/μL), and there was a monocytosis (1,192 × 103 monocytes/μL; normal: 0–840 × 103/μL). The dog was hypoalbumemic (1.5 g/dL) and hyperglobulinemic (3.7 g/dL). The urine was dilute (SG of 1.012) with 3 fat droplets/hpf and no detectable protein on urine dipstick. UPC was within the reference range (0.1). Laboratory values before (Day −48 to Day −1), during (Day 0 to Day 3), and after hospitalization (Day 4 to Day 47). Masitinib treatment occurred from Day −48 until Day −9. Two weeks later (Day 19), the dog was presented for a follow-up examination. The owner reported that the dog had an improved appetite and attitude. Additionally, the owner had observed a decrease in drinking and frequency of urination. The dog had gained 1.4 kg of body weight. On clinical examination, the skin problems had worsened characterized by an increased pododermatitis. No other significant findings were noted. Blood and urine samples were obtained to check on the previously noted abnormalities. Hct (44.4%) and Hgb (14.4 g/dL) were within normal reference ranges. Clinicopathologic abnormalities at this time included an elevated MPV of 11.1 fL (normal: 6.1–10.1 fL), hypoglycemia (60 mg/dL), hyperkalemia (5.2 mmol/L; normal: 3.9–5.0 mmol/L), and hypercholesterolemia (285 mg/dL; normal: 126–264 mg/dL). The hypoproteinemia and hypoalbuminemia had resolved: TP and Alb had increased to normal concentrations of 6.3 and 2.9 g/dL, respectively. The USG was 1.019 with a pH of 8.5 and unremarkable sediment. Protein was absent on urine dipstick and the UPC was within the normal reference range (0.08). Benazepril, aspirin, and Derm Caps ES were discontinued at that time. Although the dog continued to be seen for skin-related problems on a monthly basis, the severe proteinuria resolved without reoccurrence and neither clinical signs nor laboratory abnormalities suggestive of urinary protein loss have been observed for the following 6 months. The dog's body weight increased to 36 kg. MCN has been rarely described in the dog, which might be because of low prevalence, and/or failure of detection when tissues are only evaluated at the light microscopic level. Transient MCN has been described in dogs experimentally infected with E. canis.3 Infected dogs developed proteinuria with foot process effacement and without ultrastructural evidence of immune complex deposition. This proteinuria resolved spontaneously 10 weeks after E. canis inoculation. The dog described in this report was serologically negative for anti-E. canis antibodies. A single case of idiopathic MCN has been reported in a 4-year-old, female Collie with nephrotic syndrome.4 The dog was euthanized shortly after presentation and a diagnosis of MCN was made based on histologic and ultrastructural findings in renal tissue obtained at necropsy. The Giant Schnauzer in this case report recovered quickly after masitinib was discontinued. It is unlikely that the proteinuria developed because of other medication administered at that time. This dog continued to receive oral cephalosporins for treatment of pyoderma without developing proteinuria again. In vitro, masitinib mesylate is a tyrosine kinase inhibitor that potently and selectively inhibits the c-KIT WT receptor (half inhibitory concentration [IC50] of 150 nM), and its juxtamembrane mutation (IC50 of 5 nM).1 Masitinib is currently undergoing clinical trial investigations of numerous indications, including treatment for gastrointestinal stromal tumors in humans and mast cell tumors in canines and humans. Both types of tumors are characterized by expression of a gain-of-function mutation in c-KIT.5,6 In 12 dogs (7.5% of the study population) renal disorders occurred after treatment with masitinib for mast cell tumors, compared with 2 dogs (4.9%) in the placebo group.6 These dogs were diagnosed with renal failure, glomerulonephritis, nephrotic syndrome, and/or proteinuria. Of these 12 masitinib-treated dogs, 6 recovered, 3 had ongoing abnormalities, and 3 were euthanized. Dogs that developed increased BUN and creatinine values already had high normal to mildly increased values at the beginning of the study. The decreased renal function was thought to be caused by a direct inhibitory effect on the function of the renal tubules, which have been shown to express c-KIT.7 The dog described in this report also had a high normal serum creatinine concentration and a low specific gravity both at the beginning of treatment with masitinib and later. Regarding to the IRIS staging system, these values already indicate mild renal tubular disease (early stage II). Nonetheless, in this case, masitinib did not cause a progression of renal tubular failure (serum creatinine concentration and USG did not worsen for 6 months). Instead, the dog developed a severe proteinuria caused by glomerular disease. Masitinib also potently inhibits the PDGF receptor (PDGFRα, IC50 of 300 nM and PDGFRβ, IC50 of 50 nM).1 Cells within the glomeruli in primates do express PDGF receptors8; it is thus hypothesized that canine glomerular cells also express PDGF receptors, and that in this case the decreased podocyte function was because of PDGF receptor inhibition. However, we could not exclude that other kinases, which might be inhibited by masitinib, could play a role in podocyte integrity. This report describes the clinical signs, laboratory and histopathologic abnormalities, and clinical course of drug-induced MCN in a dog. Clinical signs at the time of presentation, 48 days after institution of masitinib therapy for treatment of a dermatologic condition, were mild. This case demonstrates the limitation of using light microscopy alone in characterizing glomerular diseases. A complete pathologic evaluation of the glomerulus in proteinuric renal disease should include light, electron, and immunofluorescent microscopy techniques. Diagnosis of MCN in this dog was dependent on the ultrastructural evaluation of glomeruli, which revealed diffuse effacement of podocyte foot processes as well as podocyte villous transformation. No changes in the GBM were identified, and immune deposits were not evident. While not done in this case, immunofluorescent microscopy could also be used to further verify the lack of immunoreactants in cases of MCN. This dog recovered quickly and without relapse after discontinuation of masitinib administration. Based on this report, dogs receiving masitinib mesylate should be carefully monitored for proteinuria. From our broader observation of this condition in other dogs and humans currently enrolled in clinical trials, the low frequency of this adverse event suggests that a genetic defect in podocyte functions might be evidenced by masitinib treatment. Development of significant and persistent renal proteinuria after initiation of masitinib therapy justifies prompt discontinuation of masitinib, and mandates ongoing surveillance of proteinuria in the dog. a Masitinib mesylate, AB1010, AB Science, S.A., Paris, France b Reichert VET 360, Reichert Analytic Instruments, Reichert Inc, Depew, NY c Multistix, Bayer HealthCare, Elkhart, IN d Hill's d/d Potato & Salmon Formula Canine, Hill's Pet Nutrition, Inc, Topeka, KS e Epi-Otic Ear Cleanser, Virbac, Forth Worth, TX f Malaseb, DVM Pharmaceuticals, Teva Animal Health Inc, Saint Joseph, MO g Cephalexin, Aurobindo Pharma USA Inc, Cranbury, NJ h Lactated Ringer's, Hospira Inc, Lake Forest, IL i Buprenorphine Hydrochloride Injection, Ben Venue Laboratories Inc, Bedford, OH j Benazepril Hydrochloride, Apotex Inc, Toronto, Ontario, Canada k Bayer Genuine Aspirin, Bayer HealthCare, Morristown, NJ l Derm Caps ES, DVM Pharmaceuticals Inc, Miami, FL m Tramadol Hydrochloride, Apotex Inc The skillful technical assistance of Mrs Mary Ard, Section of Electron Microscopy, The University of Georgia and the critical review of the manuscript by Mr Colin Mansfield, PhD, AB Science, S.A., are gratefully acknowledged.

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