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OBJECTIVE To characterize the signalment, clinical signs, clinical pathological and histologic findings, and outcome in 8 related Bracchi Italiani with proteinuric kidney disease. ANIMALS 8 client-owned Bracchi Italiani. PROCEDURES Health records submitted to the Bracco Italiano Health Foundation and the Bracco Italiano Club of America between 2012 and 2019 were reviewed for dogs with evidence of nephropathy for which histologic diagnoses were obtained. Pedigree, signalment, clinical signs, diagnostic test results (including microscopic examination of kidney tissue samples collected ante- or postmortem), and outcome were acquired. Results were presented as descriptive statistics. RESULTS The most common clinical sign in affected dogs was inappetence. All dogs were proteinuric, and 4 dogs were azotemic. Seven dogs developed clinical signs of kidney disease and were euthanized a median of 75 days postdiagnosis. Six dogs had glomerular amyloidosis, and 1 dog each had nephrosclerosis and nonamyloidotic fibrillar glomerulopathy. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the clinical presentation may vary in affected dogs, and proteinuria in young or middle-aged Bracchi Italiani should raise the concern for hereditary nephropathy. Prognosis is likely poor once clinical signs are noted.

Objectives In humans, renal aging is associated with an increased frequency of glomerulosclerosis, interstitial fibrosis, inflammation and tubular atrophy. The purpose of this study was to describe the frequency of renal histopathologic lesions in cats without kidney disease. Methods A cross-sectional study of archival kidney tissue from 74 cats without kidney disease (serum creatinine 1.035) was carried out: 0–4 years (young, n = 18); 5–9 years (mature, n = 16); 10–14 years (senior, n = 34), 15+ years (geriatric, n = 6). Glomerulosclerosis, tubular atrophy, interstitial inflammation and fibrosis, and the presence or absence of lipid in the interstitium and tubules were scored by a pathologist masked to clinical data. Statistical analyses were performed as appropriate. Results Geriatric cats had significantly more glomerulosclerosis than mature ( P = 0.01) and young cats ( P = 0.004). Senior cats had significantly more glomerulosclerosis than young cats ( P = 0.006). Glomerulosclerosis was weakly positively correlated with age ( r = 0.48; P Conclusions and relevance Evidence of renal aging exists in cats. These changes imply that the aging kidney may be more susceptible to injury and impaired healing.

Equine ingesta-associated choledocholithiasis is a rare cause of morbidity and mortality. We describe here the clinical, gross, histologic, and microbiologic features of this condition in 2 horses and compare the features to 2 previous cases. Case 1 was a 4-y-old Thoroughbred mare with colic. Case 2 was an 18-y-old American Paint Horse mare with colic, chronic weight loss, and inappropriate mentation. Both had elevated biochemical markers of hepatocellular injury and cholestasis and were euthanized given a poor prognosis. Case 1 had a well-formed 5-cm choledocholith surrounding a piece of hay, and had chronic neutrophilic cholangiohepatitis, bridging fibrosis, and extrahepatic obstruction. Case 2 had an ill-formed choledocholith with occasional hay fragments, wood stick, and twigs, and had regionally extensive hepatocellular necrosis with mild neutrophilic cholangiohepatitis and bridging fibrosis. Enterococcus casseliflavus and Escherichia coli were isolated in both cases; Clostridium spp. were also isolated from case 2. All 4 reported cases had increased activity of cholestatic enzymes, hyperbilirubinemia, portal inflammation, and bridging fibrosis. Colic, pyrexia, leukocytosis with neutrophilia, and elevated hepatocellular enzyme activity were documented in 3 cases. Foreign material in all 4 cases was plant origin (choledochophytolithiasis), including hay ( n = 2), sticks/twigs ( n = 2), and grass awns ( n = 1). Ingesta-associated choledocholithiasis may be considered as a cause of colic, pyrexia, and elevated cholestatic biomarkers in horses.

German Shorthaired Pointer (GSHP) dogs with a UNC93B1 gene mutation develop exfoliative cutaneous lupus erythematosus (ECLE) and kidney disease resembling lupus nephritis in humans. The objective of this study was to characterize the kidney disease by light microscopy, immunofluorescence, and electron microscopy in a population of GSHP dogs with ECLE. Medical records were reviewed, and light microscopy of kidneys from 7 GSHP dogs with a previous histologic diagnosis of ECLE was performed. Immunofluorescence of fresh-frozen kidney from 1 dog and transmission electron microscopy of kidney from that dog and 2 additional dogs were performed. Five of 7 dogs had proteinuria diagnosed by urinalysis or urine protein-to-creatinine ratio. Two of 7 dogs were intermittently hypoalbuminemic, and none were azotemic. Histologic findings included early (2 dogs) to late (5 dogs) membranous glomerulonephropathy characterized by mild-to-severe glomerular capillary loop thickening and tubular proteinosis. In all 7 cases, trichrome staining revealed red granular immune deposits on the subepithelial surface of the glomerular basement membrane. Immunofluorescence revealed strong granular labeling for immunoglobulins and complement protein C3. Electron microscopy demonstrated subepithelial electron-dense immune deposits encircled by the remodeled glomerular basement membrane. These findings are diagnostic of immune-complex membranous glomerulonephropathy and are similar to class V lupus in humans. This cohort of GSHP dogs with ECLE developed immune-complex membranous glomerulonephropathy, which we hypothesize is a manifestation of systemic lupus erythematosus. GSHP dogs with ECLE should undergo clinical evaluation of renal function for early identification and treatment.

Background Proteinuria is an independent risk factor for morbidity and mortality in dogs. An association between proteinuria and gallbladder mucocele formation in dogs is unknown. Objective Determine if gallbladder mucocele formation or clinicopathologic comorbidities are associated with proteinuria. Animals Twenty-five dogs with mucocele formation and 25 breed and age-matched control dogs from a prior study. Methods Retrospective case control study. Proteinuria defined by calculated urine dipstick protein concentration (mg/mL) to urine specific gravity (USG) ratio. Clinicopathologic findings, postcosyntropin cortisol concentration, thyroid function profile, and illness severity score were recorded. Results Median urine dipstick protein concentration to USG ratio and number of dogs having a ratio ≥1.5 were significantly higher for dogs with mucocele formation compared to control dogs. Proteinuria was not significantly associated with CBC or serum biochemistry profile abnormalities but increased in relation to severity of illness. Conclusions and clinical importance Gallbladder mucocele formation is significantly associated with proteinuria in dogs. Diagnosis and treatment of proteinuria in dogs with mucocele formation might minimize long term kidney morbidity in these patients.

Objective To describe 2 dogs with acute kidney injury secondary to type III hypersensitivity reaction to 25% human serum albumin (HSA). Case series summary Two dogs were presented with evidence of septic peritonitis. The dogs were hospitalized following definitive surgical correction of a jejunal laceration following routine ovariohysterectomy, and removal of a jejunal foreign body. In the postoperative period, both dogs developed hypoalbuminemia and received 25% HSA. At the time of initial discharge, both dogs were doing well clinically and had normal renal parameters. Eleven and 18 days after HSA infusion, respectively, both dogs were re-presented with clinical signs of inappetence, vomiting, and lameness that progressed to urticaria, peripheral and angioedema, and petechiae, consistent with a delayed type III hypersensitivity reaction. Treatment for the type III hypersensitivity reaction to HSA included administration of diphenhydramine and glucocorticoids. Despite partial resolution of edema and joint swelling, both dogs developed progressive azotemia together with hypoalbuminemia and proteinuria. One dog developed an anuric acute kidney injury (AKI). Both dogs were humanely euthanized. Histopathology of the kidneys of both dogs was consistent with immune complex deposition and vasculitis. New or unique information Severe type III hypersensitivity reactions have been documented in healthy dogs and clinical patients following the administration of HSA. This report describes the first documented delayed type III hypersensitivity reaction in 2 dogs with septic peritonitis that resulted in AKI, glomerulonephritis, and oligo- to anuria in clinical patients following administration of 25% HSA.

Background Focal segmental glomerulosclerosis (FSGS) is a common cause of nonimmune complex glomerulopathy and the prognosis and clinicopathologic findings associated with this condition have not been described in dogs. Objective To characterize the presentation and identify clinical factors associated with the survival of dogs with FSGS. Animals Seventy‐seven dogs diagnosed with FSGS based on evaluation of renal biopsy samples submitted to the International Veterinary Renal Pathology Service. Methods Retrospective review of medical records of dogs biopsied for evaluation of proteinuria between January 2015 and May 2017. Results The incidence of FSGS among all dogs biopsied for proteinuria was 26%. Significantly more females (48; 62.3%) than males (29; 37.7%) were affected (P = .04). At the time of biopsy, median serum creatinine concentration (SCr) was 1.2 mg/dL (range, 0.3‐8.7), median serum albumin concentration (Alb) was 2.8 g/dL (range, 1.1‐4.6), median systolic blood pressure was 153.5 mm Hg (range, 95‐260), and median urine protein : creatinine ratio was 5.9 (range, 1.4‐22). Median survival time after biopsy was 258 days (range, 26‐1003) for dogs that died from all causes (n = 32). Factors that were associated with a shorter survival time included SCr ≥ 2.1 mg/dL (P

Objectives The aim of this study was to describe the causes, clinicopathologic features and outcomes of feline protein-losing nephropathy (proteinuria secondary to glomerular disease [PLN]). Methods Kidney biopsy/necropsy samples from proteinuric cats submitted to the International Veterinary Renal Pathology Service were retrospectively reviewed. Diagnoses based on histopathology were categorized by primary disease compartment. Clinicopathologic variables at diagnosis, development of hypoalbuminemia, anemia, hypertension, azotemia and effusion/edema, and survival were compared between cats with immune-complex glomerulonephritis (ICGN) and other causes of PLN. Results Fifty-eight percent (n = 31/53) of proteinuric cats had ICGN and 74% (n = 31/42) of cats with PLN had ICGN. Cats with glomerular diseases other than ICGN had a higher median urine protein:creatinine ratio than ICGN cats (14.5 vs 6.5; P Conclusions and relevance Taken together, these data suggest that clinical suspicion for glomerular proteinuria should increase in young, male cats with higher degrees of proteinuria, and immune-mediated disease is common. Further studies are needed to determine the effect of immunosuppression on morbidity and mortality in cats with ICGN.

Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death. Our screen and validation studies reveal an essential role of Cdkl5-kinase in RTEC cell death. In mouse models, genetic or pharmacological Cdkl5 inhibition mitigates nephrotoxic and ischemia-associated AKI. We propose that Cdkl5 is a stress-responsive kinase that promotes renal injury in part through phosphorylation-dependent suppression of pro-survival transcription regulator Sox9. These findings reveal a surprising non-neuronal function of Cdkl5, identify a pathogenic Cdkl5-Sox9 axis in epithelial cell-death, and support CDKL5 antagonism as a therapeutic approach for AKI.

A 4-year-old male neutered domestic shorthair cat was presented to The Ohio State University College of Veterinary Medicine for a 2-month history of severe weight loss, lethargy, anemia, and bilaterally hyperechoic kidneys with loss of corticomedullary distinction as reported by the referring veterinarian. Relevant initial laboratory results included severe non-regenerative normocytic hypochromic anemia, increased blood urea nitrogen, minimally concentrated urine, proteinuria, and an increased urine protein:creatinine ratio. Cytologic evaluation of a bone marrow aspirate revealed a markedly hypocellular marrow with abundant mucinous material. Gelatinous marrow transformation (GMT) was confirmed histologically by the presence of mucinous material in the bone marrow that stained positive for Alcian blue but negative for periodic acid-Schiff. The cat died despite repeated blood transfusions and supportive care. Gelatinous marrow transformation, immune complex-mediated membranoproliferative glomerulonephritis, and gastrointestinal hemorrhage were observed on autopsy and histology. It is likely that the development of GMT was secondary to chronic kidney disease (CKD) and that CKD, GMT, and gastrointestinal hemorrhage contributed to the cat's non-regenerative anemia.

Background: Angiosarcomas are a broad category of vascular origin neoplasms that are poorly characterized in veterinary species. Lymphangiosarcoma (LAS) is an uncommon type of angiosarcoma reported in humans and canines arising from lymphatic endothelium. LAS can be differentiated from other angiosarcomas in dogs based on expression of Prospero-related homeobox gene-1 (PROX-1) or lymphatic vessel endothelial receptor-1 (LYVE-1). Composite hemangioendothelioma (CHE) is a rare angiosarcoma subtype described in people and characterized by a variable biologic behavior and infrequent metastasis. This variant of angiosarcoma histologically combines features of retiform hemangioendothelioma and epithelioid hemangioendothelioma. Information regarding the cytologic and histopathologic appearance and clinical course of dogs with vascular tumors that exhibit features of CHE are unknown. Here, we report a case of pleomorphic LAS with features of CHE arising in a dog and treated with surgery and adjuvant chemotherapy.Case presentation: A 10-year-old intact male Labrador retriever presented with an approximately 6-cm-diameter cutaneous mass caudal to the left elbow that was progressively growing over 1.5 years. On physical examination, palpable extensions were identified coursing proximally over the triceps with concurrent loco-regional peripheral lymphadenopathy. Fine needle aspirates (FNA) and cytologic assessment of the cutaneous mass, left prescapular, and accessory axillary lymph nodes reported that this appeared to be a metastatic epithelial neoplasm, although a mixed carcinoma or collision tumor could not be excluded. An incisional biopsy of the mass was submitted for histopathology and was consistent with a well-differentiated angiosarcoma with features of CHE. The neoplasm expressed vimentin, CD31, von Willebrand factor (vWf), and PROX-1, supporting the diagnosis of LAS. Complete staging was performed, and no additional metastatic lesions were identified. Left forelimb amputation and lymph node removal were performed. Based on the diagnosis of metastatic LAS, doxorubicin chemotherapy was administered. 7 months post-amputation, the tumor recurred at the amputation site without evidence of metastatic disease.Conclusion: This report describes a malignant, locally aggressive lymphatic origin vascular tumor in a dog, with features consistent with descriptions of CHE in humans. Cytologic features in this case were discordant with its true mesenchymal etiology, obfuscating diagnosis. The morphologic features of the mesenchymal neoplastic population and immunohistochemistry (IHC) labeling ultimately supported a diagnosis of pleomorphic LAS with features of CHE.

BackgroundGlomerular disease, often manifesting as nephrotic syndrome (NS) with high proteinuria, can be refractory to standard treatment and is typically associated with hypoalbuminemia, hypercholesterolemia and hypercoagulopathy. We hypothesized that the nuclear receptor PPARγ can be selectively modulated using a novel partial agonist, GQ-16, to gain therapeutic advantage over traditional PPARγ agonists (e.g. thiazolidinediones) for the treatment of glomerular disease.MethodsNephropathy was induced with puromycin amino-nucleoside (PAN) in Wistar rats and treated with Pioglitazone (Pio) or GQ-16. Plasma, serum, and urine chemistries were performed, and kidneys, glomeruli, liver, and white adipose tissue (WAT) were harvested. Lipid accumulation and adipogenic gene expression were measured in adipocytes.ResultsPAN-induced proteinuria was significantly reduced with Pio to 64% of PAN-value. It was reduced robustly with GQ-16 to 81% of PAN, which was comparable to controls. While both GQ-16 and Pio restored glomerular Nphs1 and hepatic Pcsk9 expression and reduced hypercholesterolemia, GQ-16 also restored glomerular Nrf2, and reduced hypoalbuminemia and hypercoagulopathy. Furthermore, RNA-seq analysis identified both common and distinct restored glomerular genes downstream of Pio and GQ-16. Pio but not GQ-16 significantly induced aP2 (fatty acid binding protein) in adipocytes and in WAT. Pio induced more lipid accumulation than GQ-16 in differentiated adipocytes. Both, Pio and GQ-16 induced insulin sensitizing adipokines in WAT with varying degrees.ConclusionsSelective modulation of PPARγ by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria and NS associated co-morbidities, while reducing the adipogenic side-effects conferred by traditional PPARγ full agonists.Translational StatementThe authors have previously reported that type-II diabetes drugs, thiazolidinediones (PPARγ agonists), also provide beneficial effects in reducing podocyte and glomerular injury. However, these drugs are associated with adverse effects such as weight gain, and their effects on glomerular disease-associated features are largely unexplored. Their current findings demonstrate that PPARγ can be selectively modulated by its partial agonist, GQ-16, which reduces proteinuria and improves nephrotic syndrome (NS) with reduced side-effects typically conferred by thiazolidinediones. These findings not only deepen our molecular understanding of the role of PPARγ in glomerular disease and underscore the potential for partial agonists of PPARγ, such as GQ-16 as a treatment modality for NS, but also lend the possibility of its potential benefits in diabetic nephropathy.

Cytomegalovirus (CMV) infection is associated with renal allograft failure by unknown mechanisms. In a murine renal transplant model, murine CMV (MCMV) induces intragraft infiltration of Th17 cells co-expressing Th1 cytokines, IFN-γ and TNF-α, but only a minority of intragraft Th17 cells are specific for MCMV antigens. Instead, MCMV promotes viral antigen-independent Th17 cell recruitment via CCL20-CCR6 and CXCL10-CXCR3 interactions. Th17 cells correlate directly with Th1 cell frequencies and inversely with Tregs in MCMV infected grafts. Pharmacologic inhibition of IL-17A reduces intragraft Th17 cells and neutrophils, increases Tregs, and reduces total but not MCMV-specific Th1 cells. Among a clinical renal transplant cohort with acute rejection, patients with CMV DNAemia had significantly higher serum IL-17A quantities compared to those without CMV DNAemia. Together, these findings indicate that CMV infection upregulates Th17 cell activity during acute rejection and suggests that inhibition of IL-17A may ameliorate CMV-associated allograft injury without impairing antiviral Th1 cells.

To document changes in urinary biomarker concentration and conventional diagnostic tests of acute kidney injury (AKI) following hypotension and fluid resuscitation in anaesthetized dogs.Experimental, repeated measures, prospective study.A group of six male adult Greyhound dogs.Following general anaesthesia, severe hypotension was induced by phlebotomy, maintaining mean arterial blood pressure (MAP)40 mmHg for 60 minutes, followed by resuscitation with intravenous gelatine solution to maintain MAP60 mmHg for 3 hours. Following euthanasia, renal tissue was examined by light microscopy (LM) and transmission electron microscopy (TEM). Urinary and serum concentrations of neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CysC), and gamma-glutamyl transpeptidase (GGT), serum creatinine and urine output were measured at baseline and hourly until euthanasia. Data are presented as mean and 95% confidence interval and analysed using repeated measures analysis of variance with Dunnett's adjustment, p0.05.Structural damage to proximal renal tubular cells was evident on LM and TEM. Urinary biomarker concentrations were significantly elevated from baseline, peaking 2 hours after haemorrhage at 19.8 (15.1-25.9) ng mLUrinary NGAL, CysC and GGT concentrations, and UPC were consistently elevated within 1 hour of severe hypotension, suggesting that proximal renal tubules are damaged in the earliest stage of ischaemia-reperfusion AKI. Measurement of urinary biomarkers may allow early diagnosis of AKI in anaesthetized dogs. Urinary GGT concentration and UPC are particularly useful as they can be measured on standard biochemistry analysers.

Resveratrol has been shown to preserve organ function and improve survival in hemorrhagic shock rat models. This study investigated whether seven days of oral resveratrol could improve hemodynamic response to hemorrhage and confer benefits on risk of acute kidney injury (AKI) without inducing coagulopathy in a canine model. Twelve greyhound dogs were randomly allocated to receive oral resveratrol (1000 mg/day) or placebo for seven days prior to inducing hemorrhage until a targeted mean blood pressure of ≤40 mmHg was achieved. AKI biomarkers and coagulation parameters were measured before, immediately following, and two hours after hemorrhage. Dogs were euthanized, and renal tissues were examined at the end of the experiment. All investigators were blinded to the treatment allocation. A linear mixed model was used to assess effect of resveratrol on AKI biomarkers and coagulation parameters while adjusting for volume of blood loss. A significant larger volume of blood loss was required to achieve the hypotension target in the resveratrol group compared to placebo group (median 64 vs. 55 mL/kg respectively, p = 0.041). Although histological evidence of AKI was evident in all dogs, the renal tubular injury scores were not significantly different between the two groups, neither were the AKI biomarkers. Baseline (pre-hemorrhage) maximum clot firmness on the Rotational Thromboelastometry (ROTEM®) was stronger in the resveratrol group than the placebo group (median 54 vs. 43 mm respectively, p = 0.009). In summary, seven days of oral resveratrol did not appear to induce increased bleeding risk and could improve greyhound dogs’ blood pressure tolerance to severe hemorrhage. Renal protective effect of resveratrol was, however, not observed.

Objectives To determine the proportion of dogs diagnosed with immune-complex glomerulonephritis in a large cohort of UK dogs with clinical suspicion of glomerular disease in which renal histopathology, including routine light microscopy, transmission electron microscopy and immunofluorescence, had been performed. The second objective was to describe treatment and long-term clinical outcome of dogs diagnosed with immune-complex glomerulonephritis. Materials and methods Sixty-two UK dogs that underwent renal biopsies for investigation of suspected glomerulopathy (urine protein-to-creatinine ratio persistently >0.5) were included in this retrospective multicentre study. Signalment, clinico-pathological abnormalities, histopathological diagnosis, treatment following diagnosis and survival were recorded. Results Seventeen (27%) of the dogs with suspected glomerular disease were diagnosed with immune-complex glomerulonephritis and nine (53%) of these were still alive at the study end point, with a median follow-up of 366 days (range 52 to 1299). Six dogs diagnosed with immune-complex glomerulonephritis were treated with mycophenolate. Four received mycophenolate alone for immunosuppression and two received mycophenolate and chlorambucil; all these six dogs were alive at data collection [median follow-up time 712.5 days (range 73 to 1299)]. Seven dogs diagnosed with immune-complex glomerulonephritis did not receive immunosuppressive treatment; only one of these dogs was alive at study end point [median survival time 302 days (range 52 to 723)]. Clinical significance Immune-complex glomerulonephritis may be less common in the UK than previously reported in North America and mainland Europe, reducing the likelihood of treatment modification following renal biopsy. Mycophenolate was the most commonly used immunosuppressant for cases of immune-complex glomerulonephritis.

With the advent and increased accessibility of deep neural networks (DNNs), complex properties of histologic images can be rigorously and reproducibly quantified. We used DNN-based transfer learning to analyze histologic images of periodic acid-Schiff–stained renal sections from a cohort of mice with different genotypes. We demonstrate that DNN-based machine learning has strong generalization performance on multiple histologic image processing tasks. The neural network extracted quantitative image features and used them as classifiers to look for differences between mice of different genotypes. Excellent performance was observed at segmenting glomeruli from non-glomerular structure and subsequently predicting the genotype of the animal on the basis of glomerular quantitative image features. The DNN-based genotype classifications highly correlate with mesangial matrix expansion scored by a pathologist (R.E.C.), which differed in these animals. In addition, by analyzing non-glomeruli images, the neural network identified novel histologic features that differed by genotype, including the presence of vacuoles, nuclear count, and proximal tubule brush border integrity, which was validated with immunohistologic staining. These features were not identified in systematic pathologic examination. Our study demonstrates the power of DNNs to extract biologically relevant phenotypes and serve as a platform for discovering novel phenotypes. These results highlight the synergistic possibilities for pathologists and DNNs to radically scale up our ability to generate novel mechanistic hypotheses in disease.

Biofilm formation enhances bacteria’s ability to colonize unique niches while protecting themselves from environmental stressors.Escherichia colithat colonize the urinary tract can protect themselves from the harsh bladder environment by forming biofilms. These biofilms promote persistence that can lead to chronic and recurrent urinary tract infections (UTI). While biofilm formation is frequently studied among urinaryE.coli, its association with other pathogenic mechanisms and adaptations in certain host populations remains poorly understood. Here we utilized whole genome sequencing and retrospective medical record analysis to investigate associations between the population structure, phenotypic resistance, resistome, virulome, and patient demographic and clinical findings of 104 unique urinaryE.coliand their capacity to form biofilms. We show that population structure including multilocus sequence typing and Clermont phylogrouping had no association with biofilm capacity. Among clinical factors, exposure to multiple antibiotics within that past 30 days and a clinical history of recurrent UTIs were positively associated with biofilm formation. In contrast, phenotypic antimicrobial reduced susceptibility and corresponding acquired resistance genes were negatively associated with biofilm formation. While biofilm formation was associated with increased virulence genes within the cumulative virulome, individual virulence genes did not influence biofilm capacity. We identified unique virulotypes among different strata of biofilm formation and associated the presence of thetosA/R-ibeAgene combination with moderate to strong biofilm formation. Our findings suggest thatE.colicausing UTI in dogs utilize a heterogenous mixture of virulence genes to reach a biofilm phenotype, some of which may promote robust biofilm capacity. Antimicrobial use may select for two populations, non-biofilm formers that maintain an arsenal of antimicrobial resistance genes to nullify treatment and a second that forms durable biofilms to avoid therapeutic insults.

A multitude of disease and therapy related factors drive the frequent development of kidney disorders in cancer patients. Along with chemotherapy, the newer targeted therapeutics can also cause kidney dysfunction through on and off-target mechanisms. Interestingly, among the small molecule inhibitors approved for the treatment of cancers that harbor BRAF-kinase activating mutations, vemurafenib can trigger tubular damage and acute kidney injury. BRAF is a proto-oncogene involved in cell growth. To investigate the underlying mechanisms, we developed cell culture and mouse models of vemurafenib kidney toxicity. At clinically relevant concentrations vemurafenib induces cell-death in transformed and primary mouse and human kidney tubular epithelial cells. In mice, two weeks of daily vemurafenib treatment causes moderate acute kidney injury with histopathological characteristics of kidney tubular epithelial cells injury. Importantly, kidney tubular epithelial cell-specific BRAF gene deletion did not influence kidney function under normal conditions or alter the severity of vemurafenib-associated kidney impairment. Instead, we found that inhibition of ferrochelatase, an enzyme involved in heme biosynthesis contributes to vemurafenib kidney toxicity. Ferrochelatase overexpression protected kidney tubular epithelial cells and conversely ferrochelatase knockdown increased the sensitivity to vemurafenib-induced kidney toxicity. Thus, our studies suggest that vemurafenib-associated kidney tubular epithelial cell dysfunction and kidney toxicity is BRAF-independent and caused, in part, by off-target ferrochelatase inhibition.

Background: Glomerular disease, often manifesting as nephrotic syndrome (NS) with high proteinuria, can be refractory to standard treatment and is typically associated with hypoalbuminemia, hypercholesterolemia and hypercoagulopathy. We hypothesized that the nuclear receptor PPARγ can be selectively modulated using a novel partial agonist, GQ-16, to gain therapeutic advantage over traditional PPARγ agonists (e.g. thiazolidinediones) for the treatment of glomerular disease. Methods: Nephropathy was induced with puromycin amino-nucleoside (PAN) in Wistar rats and treated with Pioglitazone (Pio) or GQ-16. Plasma, serum, and urine chemistries were performed, and kidneys, glomeruli, liver, and white adipose tissue (WAT) were harvested. Lipid accumulation and adipogenic gene expression were measured in adipocytes. Results: PAN-induced proteinuria was significantly reduced with Pio to 64% of PAN-value. It was reduced robustly with GQ-16 to 81% of PAN, which was comparable to controls. While both GQ-16 and Pio restored glomerular Nphs1 and hepatic Pcsk9 expression and reduced hypercholesterolemia, GQ-16 also restored glomerular Nrf2, and reduced hypoalbuminemia and hypercoagulopathy. Furthermore, RNA-seq analysis identified both common and distinct restored glomerular genes downstream of Pio and GQ-16. Pio but not GQ-16 significantly induced aP2 (fatty acid binding protein) in adipocytes and in WAT. Pio induced more lipid accumulation than GQ-16 in differentiated adipocytes. Both, Pio and GQ-16 induced insulin sensitizing adipokines in WAT with varying degrees. Conclusions: Selective modulation of PPARγ by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria and NS associated co-morbidities, while reducing the adipogenic side-effects conferred by traditional PPARγ full agonists.​

BACKGROUND Human cytomegalovirus (CMV) infection is associated with renal allograft dysfunction and loss, particularly in combination with acute rejection. Emerging literature suggests that non-HLA antibodies may contribute to antibody-mediated rejection, but pathogen-induced antibodies have not been investigated in this context. This study examines the presence of CMV-induced antibodies in murine CMV (MCMV)-infected renal allografts during acute rejection. METHODS Intragraft immunoglobulin G (IgG) and complement C3 immunostaining were compared among allogeneic MCMV D-/R-, D+/R-, and D+/R+ renal transplants. Intragraft antibody deposition was examined in B cell-deficient recipients treated with MCMV immune sera. Antibody binding and complement-dependent cytotoxicity (CDC) of D-/R- and D+/R+ sera against infected renal tubular epithelial cells (TECs) were measured in vitro. IgG immunostaining was performed in D+/R+ allografts and native kidneys and in D+/R- allografts treated with ganciclovir to inhibit viral replication. RESULTS D+/R- and D+/R+ transplants had more abundant IgG and C3 deposition compared with D-/R- recipients. Greater IgG deposition was associated with more severe allograft injury in B cell-deficient recipients treated with MCMV immune sera compared with nonimmune sera. D+/R+ sera induced greater CDC of infected TECs compared with D-/R- sera. Native kidneys had lower IgG deposition compared with allografts, despite similar organ viral loads. Ganciclovir-treated allografts had reduced IgG deposition compared with untreated allografts. CONCLUSIONS In this murine model, complement-fixing antibodies can deposit into MCMV-infected renal allografts, are associated with allograft damage, and can induce CDC of MCMV-infected renal TECs. The allogeneic response and viral replication may also contribute to intragraft antibody deposition.

Acute kidney injury (AKI) is a common clinical syndrome associated with adverse short- and long-term sequelae. Renal tubular epithelial cell (RTEC) dysfunction and cell death are among the key pathological features of AKI. Diverse systemic and localized stress conditions such as sepsis, rhabdomyolysis, cardiac surgery, and nephrotoxic drugs can trigger RTEC dysfunction. Through an unbiased RNA inhibition screen, we recently identified cyclin-dependent kinase-like 5 (Cdkl5), also known as serine/threonine kinase-9, as a critical regulator of RTEC dysfunction associated with nephrotoxic and ischemia-associated AKI. In the present study, we examined the role of Cdkl5 in rhabdomyolysis-associated AKI. Using activation-specific antibodies and kinase assays, we found that Cdkl5 is activated in RTECs early during the development of rhabdomyolysis-associated AKI. Furthermore, we found that RTEC-specific Cdkl5 gene ablation mitigates rhabdomyolysis-associated renal impairment. In addition, the small-molecule kinase inhibitor AST-487 alleviated rhabdomyolysis-associated AKI in a Cdkl5-dependent manner. Mechanistically, we demonstrated that Cdkl5 phosphorylates the transcriptional regulator sex-determining region Y box 9 (Sox9) and suppresses its protective function under stress conditions. On the basis of these results, we propose that, by suppressing the protective Sox9-directed transcriptional program, Cdkl5 contributes to rhabdomyolysis-associated renal impairment. All together, the present study identified Cdkl5 as a critical stress-induced kinase that drives RTEC dysfunction and kidney injury linked with distinct etiologies.

Several protein biomarkers have been shown to be useful for the early diagnosis of acute kidney injury (AKI) in animals and people. Multiplex assays for measurement of a panel of renal biomarkers in canine samples have recently become available. This study compared the use of two such assays, versus previously validated ELISAs, to measure five biomarkers in canine samples during ischaemia-reperfusion (IR) AKI. Blood and urine was collected from six male anaesthetised greyhounds that underwent 1-h of renal ischaemia (severe hypotension induced by acute haemorrhage) and 2-h of reperfusion (intravenous fluid resuscitation). Histology confirmed presence of acute tubular injury at 2 h of reperfusion. Concentrations of clusterin, cystatin C, kidney-injury molecule 1 (KIM-1), monocyte chemoattractant protein 1, and neutrophil gelatinase-associated lipocalin (NGAL) at baseline and following IR, measured by two different multiplex assays and previously-validated single analyte immunoassays, were compared. Only NGAL was significantly elevated following IR with all assays investigated. Whether concentrations of the other four biomarkers were significantly increased following IR depended on the assay used. Concentrations of cystatin C and KIM-1 measured with the multiplex assays were of a vast magnitude lower than those measured with the corresponding single analyte ELISAs. We conclude that further validation is required before these assays can reliably be used to measure AKI biomarkers in canine samples.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background Urine protein loss is common in dogs with chronic kidney disease (CKD). Currently available noninvasive means of evaluating CKD in dogs cannot accurately predict the severity of glomerular and tubulointerstitial damage. Electrophoretic analysis of urine proteins can indicate the compromised renal compartment (glomerular vs tubular), but extensive evaluation of protein banding pattern associations with histologic damage severity has not been performed in dogs. Objectives We aimed to evaluate electrophoretic banding patterns as indicators of the presence and severity of glomerular and tubulointerstitial damage in dogs with naturally occurring, predominantly proteinuric CKD. Methods We performed a retrospective study using urine and renal tissue from 207 dogs with CKD. Urine protein banding patterns were correlated with histologic severity of renal damage. Sensitivity and specificity of banding patterns for the detection of glomerular and tubulointerstitial damage were determined. Results Banding patterns were 97% sensitive and 100% specific for the detection of glomerular damage and 90% sensitive and 100% specific for the detection of tubulointerstitial damage. Correlations between composite banding patterns and the severity of renal damage were strong, while glomerular banding patterns correlated moderately with glomerular damage severity, and tubular gel scores correlated weakly to moderately with the severity of tubulointerstitial damage. Conclusions and clinical importance Urine protein banding patterns are useful for the detection of glomerular and tubulointerstitial damage in dogs with proteinuric CKD.

Comprehensive renal biopsy evaluation of canine glomerular disease uses immunofluorescence (IF) labeling of fresh frozen tissue to detect immune complexes that are confirmed with transmission electron microscopy. This methodology requires the veterinarian to harvest additional tissue samples, whereas sections for immunohistochemistry (IHC) could be performed on paraffin sections. If adequate IHC labeling of formalin-fixed, paraffin-embedded tissue was possible, the additional tissue samples would be unnecessary. We compared the specificity and sensitivity of IHC to IF for diagnosis of immune complex–mediated glomerulonephritis (ICGN). Commercial anti-canine IHC and IF antibodies targeting the lambda light chain component of immunoglobulins were evaluated, using previously diagnosed cases of ICGN and cases without immune complexes (non-ICGN). Because the pattern of IF labeling is crucial for accurate interpretation, sections were evaluated by a trained nephropathologist and a novice to assess the impact of experience in the diagnosis of ICGN. Unfortunately, our attempts to develop an IHC protocol that could improve the workflow for clinicians and laboratory personnel were unsuccessful; the IHC protocol did not demonstrate staining patterns that could be detected reliably by either evaluator. Moreover, the IHC antibody demonstrated abundant nonspecific staining in non-ICGN cases, and 60% of true ICGN cases were misdiagnosed as non-ICGN. We did not achieve a reliable IHC protocol for the anti-lambda light chain antibody and, therefore, IF for lambda light chain remains the method of choice for ICGN detection.

Serum canine α1-proteinase inhibitor (cα1-PI) concentrations were evaluated in dogs with pancreatitis (n=24), exocrine pancreatic insufficiency (EPI; n=29), chronic hepatitis (CH; n=11) or proteinuric chronic kidney disease (CKD-P; n=61) to determine whether systemic proteinase/proteinase-inhibitor balance is altered in these conditions. Dogs with CKD-P had significantly lower cα1-PI concentrations than dogs with pancreatitis, EPI or CH; 16% of dogs with CKD-P had serum cα1-PI concentrations below the reference interval. Serum and urine cα1-PI concentrations were inversely correlated in dogs with CKD-P, but not in dogs with CH. This suggests that renal loss of cα1-PI contributes to decreased serum concentrations in dogs with CKD-P, while hepatic cα1-PI synthesis with CH either is not compromised or is counterbalanced by extrahepatic production.

Thymoma‐associated nephropathies have been reported in people but not in dogs. In this report, we describe a dog with thymoma and concurrent renal amyloidosis. A 7‐year‐old castrated male Weimaraner was presented for progressive anorexia, lethargy, and tachypnea. The dog was diagnosed with azotemia, marked proteinuria, and a thymoma that was surgically removed. Postoperatively, the dog developed a large left ventricular thrombus and was euthanized. Necropsy confirmed the presence of a left ventricular thrombus and histopathology revealed renal amyloidosis. We speculate that the renal amyloidosis occurred secondary to the thymoma, with amyloidosis in turn leading to nephrotic syndrome, hypercoagulability, and ventricular thrombosis. This case illustrates the potential for thymoma‐associated nephropathies to occur in dogs and that dogs suspected to have thymoma should have a urinalysis and urine protein creatinine ratio performed as part of the pre‐surgical database.

Focal segmental glomerulosclerosis (FSGS) recently has been recognized as a common cause of proteinuria in dogs in general, and in Miniature Schnauzer dogs in particular. This study describes the morphologic features present in the kidneys of 8 related proteinuric Miniature Schnauzer dogs. The FSGS, characterized by solidification of portions of the capillary tuft, affected 32% to 49% of examined glomeruli in these dogs. Synechiae, often accompanied by hyalinosis, were present in 13% to 54% of glomeruli and were more prevalent in older dogs. Seven of 8 dogs had arteriolar hyalinosis. Ultrastructurally, all dogs had evidence of a podocytopathy in the absence of electron-dense deposits, glomerular basement membrane splitting, or fibrils. All dogs had multifocal to extensive podocyte foot process effacement. Other podocyte changes included microvillous transformation, the presence of vacuoles or protein resorption droplets, cytoplasmic electron-dense aggregates, and occasional binucleation. Variable amounts of intraglomerular lipid were present in all dogs. All dogs were proteinuric, with measured values for the urine protein-to-creatinine ratio ranging from 1.2 to 6.5. Azotemia was mild to absent and dogs were euthanatized at 5.1 to 14 years of age, in all cases due to nonrenal diseases. The underlying cause of FSGS in these Miniature Schnauzer dogs has yet to be determined, but contributors likely include genetic podocytopathy, lipid abnormalities, and glomerular hypertension.

Background The European Veterinary Renal Pathology Service (EVRPS) is the first Web-based registry for canine renal biopsy specimens in Europe. Hypothesis/Objectives The aim was to verify whether differences exist between the clinical and laboratory presentation of dogs with nephropathy according to renal pathological findings, as defined by light and electron microscopy of renal biopsy specimens submitted to EVRPS. Animals Renal biopsy specimens of dogs were collected from the archive of the service (n = 254). Cases were included if both light and electron microscopy were available (n = 162). Methods Renal biopsy specimens were classified based on the morphological diagnoses. Thereafter, they were grouped into 3 disease categories, including immune-complex-mediated glomerulonephritis (ICGN), non-immune-complex-mediated GN (non-ICGN), and renal lesions not otherwise specified (RL-NOS). Differences among morphological diagnoses and among disease categories were investigated for clinical and laboratory variables. Results Serum albumin concentration was lower in dogs with ICGN than in those with non-ICGN (P = 0.006) or RL-NOS (P = 0.000), and the urine protein-to-creatinine ratio (UPC) was significantly higher in ICGN than in the other 2 disease categories. Regarding morphological diagnoses, albumin was significantly lower in amyloidosis (AMY) and membranous (MGN), membranoproliferative (MPGN) or mixed glomerulonephritis (MixGN) than in minimal change disease, primary (FSGS I) or secondary (FSGS II) focal and segmental glomerulosclerosis and juvenile nephropathies (JN). The UPC was higher in MPGN than in FSGS I and FSGS II. Conclusions and clinical importance Dogs with ICGN, in particular MPGN, had higher protein loss than those with non-ICGN or RL-NOS, leading to more severe hypoalbuminemia. Clinical and laboratory differentiation among dogs with the different morphological diagnoses and among dogs with different disease categories was difficult due to overlapping results.

Foamy viruses (FVs) are globally prevalent retroviruses that establish apparently apathogenic lifelong infections. Feline FV (FFV) has been isolated from domestic cats with concurrent diseases, including urinary syndromes. We experimentally infected five cats with FFV to study viral kinetics and tropism, peripheral blood mononuclear cell (PBMC) phenotype, urinary parameters, and histopathology. A persistent infection of primarily lymphoid tropism was detected with no evidence of immunological or hematologic perturbations. One cat with a significant negative correlation between lymphocytes and PBMC proviral load displayed an expanded FFV tissue tropism. Significantly increased blood urea nitrogen and ultrastructural kidney changes were noted in all experimentally infected cats, though chemistry parameters were not outside of normal ranges. Histopathological changes were observed in the brain, large intestine, and other tissues. In order to determine if there is an association of FFV with Chronic Kidney Disease, we additionally screened 125 Australian pet cats with and without CKD for FFV infection and found that FFV is highly prevalent in older cats, particularly in males with CKD, though this difference was not statistically significant compared to controls. Acute FFV infection was clinically silent, and while some measures indicated mild changes, there was no overt association of FFV infection with renal disease.

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The purpose of this work is to investigate whether imaging sequences of flash-replenishment contrast enhanced ultrasound (CEUS) of the kidney result in chronic or acute bioeffects. Kidneys of female Fischer 344 rats were imaged using the flash-replenishment technique. Animals were separated into four groups (N = 31). Imaging was conducted with a 4C1 probe, driven by an Acuson Sequoia system with Definity microbubbles as the ultrasound contrast agent. During the flash phase of the imaging sequence, one kidney in each animal was exposed to either a mechanical index (MI) of 1.0 or 1.9. For each MI, half of the animals were sacrificed shortly after imaging (4 h) or after 2 weeks. A blinded veterinary nephropathologist reviewed the histopathology of both the imaged and control (non-imaged) kidney. Blood urea nitrogen (BUN) was measured for each animal prior to imaging and at the time of necropsy. Histopathology assessments in both the 1.0 and 1.9 MI groups revealed no signs of hemorrhage at either the 4-h or 2-week time point. BUN showed minor but statistically significant elevations in both the 1.0 and 1.9 MI groups, but no significant difference was present at the 2-week time point in the 1.0 MI group. All BUN levels (at both time points) remained in the normal range. In conclusion, CEUS with flash-replenishment imaging sequences did not result in kidney bioeffects observable with histology at early or late time points. Increases in BUN levels were observed after imaging, but were minimized when using a moderate MI (1.0).

Many recent advances in the evaluation of dogs with kidney disease have improved our diagnostic algorithms and have impacted our therapeutic strategies. Non-invasive techniques, such as urinary and serologic biomarker evaluation, can help a clinician diagnose and treat a patient that cannot undergo a renal biopsy for clinical or financial reasons. Some biomarkers might help localize the affected structure (glomerulus vs. tubule) and indicate the type or severity of injury present. Although more research is needed, studies indicate that some biomarkers (e.g. urine protein to creatinine ratio and urinary immunoglobulins) can be useful in predicting adverse outcomes. Importantly, the sensitivity and specificity of biomarkers for renal injury should be established and clinicians need to understand the limitations of these assays. If a renal biopsy is performed, then it should be evaluated by a specialty diagnostic service with expertise in nephropathology. A panel of special stains, immunofluorescence for the detection of immunoglobulins and complement factors, and transmission electron microscopy can be routinely employed in cases of glomerular disease. These advanced diagnostics can be used to detect immune deposits in order to definitively diagnose immune complex mediated glomerular disease. Integrating the results of biomarker assays and comprehensive renal biopsy evaluation, the clinician can make informed therapeutic decisions, such as whether or not to immunosuppress a patient.

Background Urine protein loss is common in dogs with chronic kidney disease (CKD). Hypothesis/Objectives To evaluate new biomarkers of glomerular and tubulointerstitial (TI) damage compared with histology and as survival indicators in dogs with naturally occurring, proteinuric CKD. Animals One hunderd and eighty dogs with naturally occurring kidney disease. Methods Retrospective study using urine, serum, and renal biopsies from dogs with kidney disease, 91% of which had proteinuric CKD. Biomarkers were evaluated and correlated with pathologic renal damage, and significant associations, sensitivities, and specificities of biomarkers for renal disease type were determined. Results Fractional excretions of immunogloblin M (IgM_FE) and immunoglobulin G (IgG_FE) correlated most strongly with glomerular damage based on light microscopy (r = 0.58 and 0.56, respectively; P < .01). Serum creatinine (SCr) correlated most strongly with TI damage (r = 0.70, P < .01). Urine IgM/creatinine and urine NAG/creatinine had the highest sensitivity (75%) and specificity (78%) for detection of immune complex-mediated glomerulonephritis. Although individually most biomarkers were significantly associated with decreased survival time (P < .05), in a multivariate analysis, SCr, IgM_FE, and glomerular damage based on transmission electron microscopy (TEM) were the only biomarkers significantly associated with survival time (SCr: P = .001; IgM_FE: P = .008; TEM: P = .017). Conclusions and Clinical Importance Novel urine biomarkers and FEs are useful for detection of glomerular and TI damage in dogs with proteinuric CKD and might predict specific disease types and survival.

Aberrant cell cycle activation is a hallmark of carcinogenesis. Recently three cell cycle targeting cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have been approved for the treatment of metastatic breast cancer. CDK4/6 inhibitors suppress proliferation through inhibition of CDK4/6-dependent retinoblastoma-1 (Rb1) phosphorylation and inactivation, a key regulatory step in G1-to-S-phase transition. Importantly, aberrant cell cycle activation is also linked with several non-oncological diseases including acute kidney injury (AKI). AKI is a common disorder caused by toxic, inflammatory, and ischemic damage to renal tubular epithelial cells (RTECs). Interestingly, AKI triggered by the anti-cancer drug cisplatin can be mitigated by ribociclib, a CDK4/6 inhibitor, through mechanisms that remain unclear. Employing in vivo cell cycle analysis and functional Rb1 knock-down, here, we have examined the cellular and pharmacological basis of the renal protective effects of ribociclib during cisplatin nephrotoxicity. Remarkably, siRNA-mediated Rb1 silencing or RTEC-specific Rb1 gene ablation did not alter the severity of cisplatin-associated AKI; however, it completely abrogated the protective effects conferred by ribociclib administration. Furthermore, we find that cisplatin treatment evokes CDK4/6 activation and Rb1 phosphorylation in the normally quiescent RTECs, however, this is not followed by S-phase entry likely due to DNA-damage induced G1 arrest. The cytoprotective effects of ribociclib are thus not a result of suppression of S-phase entry but are likely dependent on the maintenance of Rb1 in a hypo-phosphorylated and functionally active form under stress conditions. These findings delineate the role of Rb1 in AKI and illustrate the pharmacological basis of the renal protective effects of CDK4/6 inhibitors.