Evaluation of darbepoetin therapy on platelet count and function in healthy cats and cats with surgically induced chronic kidney disease.

OBJECTIVE: To evaluate the effects of darbepoetin on platelet population and reactivity in healthy cats (HCs) and azotemic cats with remnant kidney (RK) model-induced chronic kidney disease. ANIMALS: 12 purpose-bred domestic shorthair cats (n = 6 HCs and n = 6 RK). METHODS: In this pilot study, all cats received darbepoetin (1 µg/kg, SC) on days 0, 7, and 14. Blood was sampled at baseline and on days 3, 10, 15, 17, 20, and 21. At each time point, a CBC was performed, platelet aggregometry was assessed by impedance and optical methods, and platelet P-selectin (CD62P) was quantified before and after thrombin stimulation. Additionally, reticulated platelets were quantified using both thiazole orange staining and proprietary analysis by the CBC analyzer. For RK cats, systemic blood pressure (BP) was serially measured. RESULTS: No adverse effects of darbepoetin were seen. There was no statistically significant change in platelet count between or within groups at any time point. Hematocrit increased significantly over time in the RK but not the HC group. RBC reticulocyte numbers in both groups increased over time. Reticulated platelet percentage did not increase in either group. Differences in platelet reactivity within or between groups were not seen in the aggregometry or flow cytometric assessments. In RK cats, indirect BP did not significantly change during the study. CLINICAL RELEVANCE: This preliminary investigation did not find evidence that darbepoetin administration impacted platelet number, reactivity, nor reticulated platelet count. Anemic RK cats experienced increased hematocrit and RBC reticulocytes as expected with darbepoetin therapy.

Bioinspired and Photo-Clickable Thiol-Ene Bioinks for the Extrusion Bioprinting of Mechanically Tunable 3D Skin Models.

Bioinks play a fundamental role in skin bioprinting, dictating the printing fidelity, cell response, and function of bioprinted 3D constructs. However, the range of bioinks that support skin cells' function and aid in the bioprinting of 3D skin equivalents with tailorable properties and customized shapes is still limited. In this study, we describe a bioinspired design strategy for bioengineering double crosslinked pectin-based bioinks that recapitulate the mechanical properties and the presentation of cell-adhesive ligands and protease-sensitive domains of the dermal extracellular matrix, supporting the bioprinting of bilayer 3D skin models. Methacrylate-modified pectin was used as a base biomaterial enabling hydrogel formation via either chain-growth or step-growth photopolymerization and providing independent control over bioink rheology, as well as the mechanical and biochemical cues of cell environment. By tuning the concentrations of crosslinker and polymer in bioink formulation, dermal constructs were bioprinted with a physiologically relevant range of stiffnesses that resulted in strikingly site-specific differences in the morphology and spreading of dermal fibroblasts. We also demonstrated that the developed thiol-ene photo-clickable bioinks allow for the bioprinting of skin models of varying shapes that support dermis and epidermis reconstruction. Overall, the engineered bioinks expand the range of printable biomaterials for the extrusion bioprinting of 3D cell-laden hydrogels and provide a versatile platform to study the impact of material cues on cell fate, offering potential for in vitro skin modeling.

Uroliths composed of antiviral compound GS-441524 in 2 cats undergoing treatment for feline infectious peritonitis.

Feline infectious peritonitis (FIP) historically has been a fatal disease in cats. Recent unlicensed use of antiviral medication has been shown to markedly improve survival of this infection. An 8-month-old female spayed domestic short-haired cat undergoing treatment for presumptive FIP with the antiviral nucleoside analog GS-441524 developed acute progressive azotemia. Abdominal ultrasound examination identified multifocal urolithiasis including renal, ureteral, and cystic calculi. Unilateral ureteral obstruction progressed to suspected bilateral ureteral obstruction and subcutaneous ureteral bypass (SUB) was performed along with urolith removal and submission for analysis. A 2-year-old male neutered domestic medium-haired cat undergoing treatment for confirmed FIP with GS-441524 developed dysuria (weak urine stream, urinary incontinence, and difficulty expressing the urinary bladder). This cat also was diagnosed sonographically with multifocal urolithiasis requiring temporary tube cystostomy after cystotomy and urolith removal. In both cases, initial urolith analysis showed unidentified material. Additional testing confirmed the calculi in both cats to be 98% consistent with GS-441524. Additional clinical studies are required to determine best screening practices for cats presented for urolithiasis during treatment with GS-441524.

Effect of gabapentin on ambulatory, direct, systemic arterial blood pressure in apparently healthy cats in the at-home and in-clinic environments.

OBJECTIVES: Situational increases in blood pressure (BP) frequently confound the accurate diagnosis of pathological systemic hypertension in cats. The objective of this study was to investigate the effect of gabapentin on direct, ambulatory systolic arterial BP (SBP) in cats in at-home and in-clinic environments. METHODS: Six adult purpose-bred cats with surgically implanted femoral artery telemetric BP-sensing catheters were administered 100 mg of gabapentin or a placebo orally in two randomized, masked, crossover study phases. In the first, direct BP was measured continuously in undisturbed cats for 24 h before (at-home baseline) and 4 h after administration of study drug. The mean SBP after administration of the drug was compared between treatments. In the second study period, cats were administered gabapentin or placebo 90 mins before transport to a clinic, where direct BP was measured continuously during a simulated veterinary visit that included an indirect BP measurement session. Changes in mean direct SBP relative to the 24-h at-home pre-treatment period were calculated for each of one waiting room and two examination-room periods, and compared between treatments. Concurrent in-clinic direct and indirect SBP measurements were compared within-cat. Data were compared using linear mixed models. RESULTS: Direct SBP data from one cat were excluded due to implant failure. There were no differences in at-home or in-clinic SBP between treatment groups, with large inter-individual variability. Cats in both treatment groups experienced in-clinic increases in direct SBP relative to at-home baseline (range 11-50 and 10-52 mmHg in placebo- and gabapentin-treated cats, respectively). Across all visits, direct SBP was 15.6 mmHg higher than indirect SBP (P <0.001). No effects of treatment on difference between direct and indirect SBP were identified. CONCLUSIONS AND RELEVANCE: Significant effects of gabapentin on direct SBP were not identified, though a type II error is possible. Situational increases cannot be excluded in gabapentin-treated cats with high SBP.

Acute kidney injury is common in dogs with septic peritonitis and is associated with increased mortality.

OBJECTIVE: To identify the frequency of and risk factors for acute kidney injury (AKI) in dogs undergoing abdominal surgery for septic peritonitis, and to evaluate outcome and kidney-related risk factors for survival to discharge in those dogs. ANIMALS: 77 dogs that underwent abdominal surgery for septic peritonitis. METHODS: Medical records of dogs that underwent surgery for septic peritonitis from 2012 through 2022 were reviewed. Data regarding signalment, clinical and biochemical findings at presentation, blood creatinine concentration throughout hospitalization, surgery characteristics, postoperative monitoring, and outcome were collected. Dogs were classified based on occurrence of AKI and whether they presented with or developed AKI in-hospital. Perioperative risk factors were evaluated, and outcomes were compared with univariable logistic regression. RESULTS: 31 dogs (40.3%) had AKI diagnosed; 18/77 (23.4%) dogs presented with AKI, 11 (61.1%) of which had it postoperatively, and 13/77 (16.9%) dogs developed AKI postoperatively. Significant factors for presenting with AKI included increasing baseline respiratory rate (OR 2.5 for every 10 beats per minute higher), decreasing systolic blood pressure (OR 0.8 for every 10 mm Hg higher), and increasing body condition score (OR 2.2 for every score greater). No significant factors for developing AKI postoperatively were identified after multiple comparisons adjustment. Sixteen dogs (20.8%) did not survive to discharge; 12 (75.0%) had AKI and 4 (25.0%) did not. Dogs with AKI had decreased odds of survival to discharge (OR 0.2). CLINICAL RELEVANCE: AKI was common in dogs with septic peritonitis and was a significant risk factor for survival to discharge. Clinical surveillance of AKI is critical in this population.

A remnant kidney surgery model in cats induces acute and chronic outcomes that mimic spontaneous chronic kidney disease.

OBJECTIVE: To report acute and chronic outcomes of cats with chronic kidney disease (CKD) induced by a remnant kidney model. ANIMALS: 32 purpose-bred cats (n = 15 female, n = 17 male). PROCEDURES: Cats underwent a 2-stage reduction in renal mass through partial arterial ligation of 1 kidney (day 28) and delayed contralateral nephrectomy (day 0), targeting an 11/12th functional nephrectomy. Acute (days -28 - 29) survival and renal function parameters were compared over time, and the latter were evaluated as predictors for acute mortality. Chronic (days 30 to >1,100) survival, renal function, and morphology were described. RESULTS: Acutely, renal function deteriorated in all cats (mean ± SD baseline and day 28 serum creatinine mean concentration, 1.13 ± 0.23 mg/dL and 3.03 ± 1.20 mg/dL, respectively; P < .001; and GFR, 3.22 mL/min/kg ± 0.12 and 1.21 mL/min/kg ± 0.08, respectively; P < .001). Seven (22%) cats were euthanized after because of clinical signs of uremia after contralateral nephrectomy. Prenephrectomy renal function tests were not significant indicators for survival during this acute phase. Twenty-five cats entered the chronic phase. Ten cats were euthanized at a median of 163 days from nephrectomy because of progressive renal dysfunction. Median survival times were significantly different when stratified by acute kidney injury grade at day 29. Cats in the chronic phase had clinical courses similar to cats with naturally occurring CKD, and most (13/15) were in CKD stage 2. CLINICAL RELEVANCE: The remnant kidney model is effective at reducing kidney function to an extent that mimics important characteristics of spontaneous CKD in cats.

Cross-sectional characterization of renal function in cats with caudal stomatitis.

OBJECTIVES: The objective of the study was to compare renal functional biomarkers in cats and in caudal stomatitis (CS) and in age-matched control cats. METHODS: A cross-sectional, case-control study was conducted on 44 client-owned cats with CS that were prospectively enrolled and evaluated for a Comprehensive Oral Health Assessment and Treatment at one of four institutions. Renal function was assessed with measurement of serum creatinine, urea nitrogen, serum symmetric dimethylarginine, urinalysis, urine protein:creatinine ratio and urine protein electrophoresis. Affected gingiva was biopsied to confirm the diagnosis of stomatitis. Renal biochemical analyses from the experimental group were compared with those of 44 age-matched controls without CS enrolled prospectively or retrospectively after presenting to the primary institution for routine healthcare. Control cats were included if they were clinically stable, their chronic illnesses were well managed and minimal dental disease was present on examination. Renal biomarkers were compared between groups using a t-test or the Mann-Whitney U-test. Frequency of azotemia, proteinuria and the clinical diagnosis of renal disease were compared using Fisher's exact test. RESULTS: Relative to the control group, cats in the CS group had significantly lower serum creatinine (P <0.001) and albumin concentrations (P <0.001), urine specific gravity (P = 0.024) and hematocrit (P = 0.003), and higher serum phosphorus (P <0.001), potassium (P <0.001) and globulin concentrations (P <0.001), white blood cell count (P <0.001) and urine protein:creatinine ratio (P = 0.009). There were no significant differences in serum symmetric dimethylarginine or urea nitrogen concentrations. No clinically significant findings were noted on urine protein electrophoresis. There were no significant differences in the frequency of azotemia, proteinuria or renal disease categories between the two groups. CONCLUSIONS AND RELEVANCE: The present study does not demonstrate a significant difference in the frequency of kidney disease between cats with and without CS. Longitudinal evaluation is warranted to investigate the relationship between renal disease and CS.

Evaluation of renal values during treatment for heartworm disease in 27 client-owned dogs.

BACKGROUND: Canine heartworm disease (CHD) caused by Dirofilaria immitis remains a common preventable disease with increasing incidence in some parts of the USA. The treatment guidelines of the American Heartworm Society (AHS) currently recommend monthly macrocyclic lactone administration, 28 days of doxycycline given orally every 12 h and three injections of melarsomine dihydrochloride (1 injection on day 2 of treatment followed 30 days later by 2 injections 24 h apart). Minocycline has also been utilized when doxycycline is unavailable. The systemic effects of CHD, which particularly impact cardiac and renal function, have been described, with infected dogs often experiencing renal damage characterized by an increase in serum concentrations of renal biomarkers. Although the AHS treatment protocol for CHD has been shown to be safe and effective in most cases, the potential for complications remains. No study as of yet has evaluated changes in symmetric dimethylarginine (SDMA), a sensitive marker of renal function, during treatment for CHD. The purpose of the present study was to evaluate renal function in dogs by measuring serum creatinine and SDMA concentrations during the adulticide treatment period. METHODS: Serum creatinine and SDMA concentrations were measured in 27 client-owned dogs affected by CHD at the following time points: prior to starting doxycycline or minocycline therapy (baseline), during doxycycline or minocycline therapy (interim), at the time of the first dose of melarsomine (first dose), at the time of the second dose of melarsomine (second dose) and at the dog's follow-up visit after treatment, occurring between 1 and 6 months after completion of therapy (post-treatment). Concentrations of creatinine and SDMA were compared between time points using a mixed effects linear model. RESULTS: Mean SDMA concentrations following the second dose of melarsomine were significantly lower (-1.80 ug/dL, t-test, df = 99.067, t = -2.694, P-Value = 0.00829) than baseline concentrations. There were no other statistically significant differences in the concentration of either biomarker between the baseline and the other time points in CHD dogs undergoing treatment. CONCLUSIONS: The results suggest that the current AHS protocol may not have a substantial impact on renal function.

MicroRNAs are differentially expressed in the serum and renal tissues of cats with experimentally induced chronic kidney disease: a preliminary study.

OBJECTIVE: To identify differentially expressed microRNA in the serum and renal tissues of cats with experimentally induced chronic kidney disease (CKD). SAMPLE: Banked renal tissues and serum from 4 cats. PROCEDURES: Cats previously underwent 90-minute unilateral ischemia with delayed contralateral nephrectomy 3 months after ischemia. Tissues were collected from the contralateral kidney at the time of nephrectomy and from the ischemic kidney 6 months after nephrectomy (study end). Serum was collected prior to ischemia (baseline serum) and at study end (end point serum). Total RNA was isolated from tissues and serum, and microRNA sequencing was performed with differential expression analysis between the contralateral and ischemic kidney and baseline and end point serum. RESULTS: 20 microRNAs were differentially expressed between ischemic and contralateral kidneys, and 52 microRNAs were differentially expressed between end point and baseline serum. Five microRNAs were mutually differentially expressed between ischemic and contralateral kidneys and baseline and end point serum, with 4 (mir-21, mir-146, mir-199, and mir-235) having increased expression in both the ischemic kidney and end point serum and 1 (mir-382) having increased expression in the ischemic kidney and decreased expression in end point serum. Predicted target search for these microRNA revealed multiple genes previously shown to be involved in the pathogenesis of feline CKD, including hypoxia-inducible factor-1α, transforming growth factor-ß, hepatocyte growth factor, fibronectin, and vascular endothelial growth factor A. CLINICAL RELEVANCE: MicroRNAs were differentially expressed after CKD induction in this preliminary study. Regulation of renal fibrosis in feline CKD may occur through microRNA regulation of mRNAs of pro- and anti-fibrotic genes.

Characterization of the intrarenal renin-angiotensin system in cats with naturally occurring chronic kidney disease.

BACKGROUND: The role of the renin-angiotensin-aldosterone system in cats with chronic kidney disease (CKD) is incompletely understood. OBJECTIVE: To characterize components of the intrarenal renin-angiotensin system (RAS) in cats with CKD. ANIMALS: Eleven cats with naturally occurring CKD (CKD group) and 8 healthy control cats. METHODS: Renal tissue samples were evaluated by reverse-transcription polymerase chain reaction for renin, angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin II type 1 receptor transcript levels, and by liquid chromatography-mass spectrometry for quantification of angiotensin I, II, III, and IV concentrations. Linear mixed models were used to compare gene transcript levels and concentrations of angiotensin peptides between groups. RESULTS: Cats of the CKD group were significantly older (P < .001) and more likely to be neutered (P = .007) than healthy control cats. Kidneys from cats with CKD had significantly higher transcript levels of angiotensinogen (P < .001) and lower transcript levels of ACE (P < .001) than those from control cats. Renal angiotensin I concentrations were increased in CKD compared with control kidneys (P = .001). No other significant differences in renal transcript levels or angiotensin peptide concentrations were noted between groups. CONCLUSION AND CLINICAL IMPORTANCE: The intrarenal RAS might be activated in cats with CKD. Small sample size and differences in age, neuter status, and dietary sodium intake between groups might have limited the ability to identify a significant difference in concentration of renal angiotensin II.

Analysis of genes associated with proinflammatory and profibrotic pathways upregulated in ischemia-induced chronic kidney disease in cats.

OBJECTIVE: To use RNA sequencing (RNAseq) to characterize renal transcriptional activities of genes associated with proinflammatory and profibrotic pathways in ischemia-induced chronic kidney disease (CKD) in cats. SAMPLES: Banked renal tissues from 6 cats with experimentally induced CKD (renal ischemia [RI] group) and 9 healthy cats (control group). PROCEDURES: Transcriptome analysis with RNAseq, followed by gene ontology and cluster analyses, were performed on banked tissue samples of the right kidneys (control kidneys) from cats in the control group and of both kidneys from cats in the RI group, in which unilateral (right) RI had been induced 6 months before the cats were euthanized and the ischemic kidneys (IKs) and contralateral nonischemic kidneys (CNIKs) were harvested. Results for the IKs, CNIKs, and control kidneys were compared to identify potential differentially expressed genes and overrepresented proinflammatory and profibrotic pathways. RESULTS: Genes from the gene ontology pathways of collagen binding (eg, transforming growth factor-ß1), metalloendopeptidase activity (eg, metalloproteinase [MMP]-7, MMP-9, MMP-11, MMP-13, MMP-16, MMP-23B, and MMP-28), chemokine activity, and T-cell migration were overrepresented as upregulated in tissue samples of the IKs versus control kidneys. Genes associated with the extracellular matrix (eg, TIMP-1, fibulin-1, secreted phosphoprotein-1, matrix Gla protein, and connective tissue growth factor) were upregulated in tissue samples from both the IKs and CNIKs, compared with tissues from the control kidneys. CONCLUSIONS AND CLINICAL RELEVANCE: Unilateral ischemic injury differentially altered gene expression in both kidneys, compared with control kidneys. Fibulin-1, secreted phosphoprotein-1, and matrix Gla protein may be candidate biomarkers of active kidney injury in cats.

Engineering Modular Half-Antibody Conjugated Nanoparticles for Targeting CD44v6-Expressing Cancer Cells.

Gastric cancer (GC) remains a major cause of death worldwide mainly because of the late detection in advanced stage. Recently, we proposed CD44v6 as a relevant marker for early detection of GC, opening new avenues for GC-targeted theranostics. Here, we designed a modular nanoscale system that selectively targets CD44v6-expressing GC cells by the site-oriented conjugation of a new-engineered CD44v6 half-antibody fragment to maleimide-modified polystyrene nanoparticles (PNPs) via an efficient bioorthogonal thiol-Michael addition click chemistry. PNPs with optimal particle size (200 nm) for crossing a developed biomimetic CD44v6-associated GC stromal model were further modified with a heterobifunctional maleimide crosslinker and click conjugated to the novel CD44v6 half-antibody fragment, obtained by chemical reduction of full antibody, without affecting its bioactivity. Collectively, our results confirmed the specific targeting ability of CD44v6-PNPs to CD44v6-expressing cells (1.65-fold higher than controls), highlighting the potential of CD44v6 half-antibody conjugated nanoparticles as promising and clinically relevant tools for the early diagnosis and therapy of GC. Additionally, the rational design of our nanoscale system may be explored for the development of several other nanotechnology-based disease-targeted approaches.

Bioprinting a Multifunctional Bioink to Engineer Clickable 3D Cellular Niches with Tunable Matrix Microenvironmental Cues.

The properties of the surrounding cell environment are major determinants of cell response in 3D. However, the ability to unravel how these cues dictate the biological function in bioprinted constructs is limited by the lack of extracellular matrix (ECM)-mimetic bioinks with fully controllable properties. In this study, a multifunctional bioink that uniquely combines the independent control over the biochemical and biophysical cues that regulate cell fate with the bioorthogonal nature of thiol-norbornene photoclick chemistry is designed for the extrusion bioprinting of bioinspired 3D cellular niches with tunable properties. The bioink rheology is controlled by ionic gelation, being dependent on both the type and content of divalent ions (calcium and barium), while the mechanical and biochemical properties of hydrogels are tailored via a post printing thiol-ene reaction. Bioprinted cell-adhesive and protease-degradable hydrogels modulate cell proliferation and ECM deposition in a matrix-stiffness dependent manner over 14 days of culture regardless of cell spreading, demonstrating the ability to probe the effect of matrix cues on cell response. This bioink can be used as a versatile platform where building blocks can be rationally combined for the bioprinting of functional cell- and tissue-specific constructs with controlled cellular behavior.

Efficacy of telmisartan for the treatment of persistent renal proteinuria in dogs: A double-masked, randomized clinical trial.

BACKGROUND: Information regarding efficacy of the angiotensin II receptor blocker, telmisartan, for treatment of proteinuria in dogs is limited. OBJECTIVE: To evaluate the antiproteinuric efficacy of telmisartan, as compared to enalapril, in dogs with chronic kidney disease and persistent, renal proteinuria. ANIMALS: Thirty-nine client-owned dogs with chronic kidney disease and urinary protein-to-creatinine ratio (UPC) > 0.5 (if azotemic) or ≥ 1.0 (if nonazotemic). METHODS: In this prospective, randomized, double-masked clinical trial, dogs were block randomized, according to presence or absence of azotemia and systemic arterial hypertension, to receive telmisartan (1.0 mg/kg PO q24h), or enalapril (0.5 mg/kg PO q12h), and followed for 120 days. Up-titration of study drug dosage on days 30 and 60, and addition of the other study drug at day 90, were performed if UPC > 0.5 was noted at these visits. Percentage change in UPC relative to baseline was calculated for all time points. Data are presented as median (range). RESULTS: Thirty-nine (20 telmisartan-treated, 19 enalapril-treated) dogs were included. At day 30, percentage change in UPC was greater for telmisartan-treated (-65% [-95% to 104%]) vs enalapril-treated (-35% [-74% to 87%]) dogs (P = .002). Among dogs persistently proteinuric at earlier visits, telmisartan remained superior to enalapril at days 60 (P = .02) and 90 (P = .02). No difference in percentage change in UPC between study groups was observed at day 120, when combination therapy was allowed. Combination therapy resulted in relevant azotemia in 4/13 (31%) dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Telmisartan might be a suitable first-line therapy for dogs with renal proteinuria.

Evaluation of profibrotic gene transcription in renal tissues from cats with naturally occurring chronic kidney disease.

BACKGROUND: Increased gene transcription of hypoxia-induced mediators of fibrosis in renal tissue has been identified in experimentally induced, ischemic chronic kidney disease (CKD). OBJECTIVE: To characterize hypoxia-induced profibrotic pathways in naturally occurring CKD in cats. ANIMALS: Twelve client-owned cats with CKD and 8 healthy control cats. METHODS: In this prospective, cross-sectional study, bilateral renal tissue samples were assessed histologically for inflammation, tubular atrophy, and fibrosis, and by reverse transcription-quantitative PCR for characterization of transcript levels of hypoxia-inducible factor-1α (HIF1A), matrix metalloproteinases-2 (MMP2), -7 (MMP7), and -9 (MMP9), tissue inhibitor of metalloproteinase-1 (TIMP1), transforming growth factor-ß1 (TGFB1), and vascular endothelial growth factor-A (VEGFA). Linear mixed models were used to compare gene transcription between diseased and healthy kidneys, and to examine the association between transcript levels and serum creatinine concentration for all cats, and between transcript levels and histologic scores of diseased kidneys. RESULTS: Kidneys from cats with CKD had significantly higher transcript levels of HIF1A, MMP2, MMP7, MMP9, TIMP1, and TGFB1 (all P < .001), and lower levels of VEGFA (P = .006) than those from control cats. Transcript levels of MMP7 (P = .05) and TIMP1 (P = .005) were positively associated with serum creatinine in cats with CKD, but not in control cats. In diseased kidneys, transcript levels of MMP2 (P = .002), MMP7 (P = .02), and TIMP1 (P = .02) were positively, whereas those of VEGFA (P = .003) were negatively, associated with histologic score severity. CONCLUSION AND CLINICAL SIGNIFICANCE: Evaluation of the expression of the corresponding proteins in larger populations could identify therapeutic targets and/or biomarkers of tubulointerstitial fibrosis in cats.

Profibrotic gene transcription in renal tissues from cats with ischemia-induced chronic kidney disease.

OBJECTIVE: To characterize transcription of profibrotic mediators in renal tissues of cats with ischemia-induced chronic kidney disease (CKD). SAMPLE: Banked renal tissues from 6 cats with experimentally induced CKD (RI group) and 8 healthy control cats. PROCEDURES: For cats of the RI group, both kidneys were harvested 6 months after ischemia was induced for 90 minutes in 1 kidney. For control cats, the right kidney was evaluated. All kidney specimens were histologically examined for fibrosis, inflammation, and tubular atrophy. Renal tissue homogenates underwent reverse transcription quantitative PCR assay evaluation to characterize gene transcription of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor-ß1, and vascular endothelial growth factor A. Gene transcription and histologic lesions were compared among ischemic and contralateral kidneys of the RI group and control kidneys. RESULTS: Ischemic kidneys had greater transcript levels of MMP-7, MMP-9, and transforming growth factor-ß1 relative to control kidneys and of MMP-2 relative to contralateral kidneys. Transcription of TIMP-1 was upregulated and that of vascular endothelial growth factor A was downregulated in ischemic and contralateral kidneys relative to control kidneys. Transcription of HIF-1α did not differ among kidney groups. For ischemic kidneys, there were strong positive correlations between transcription of HIF-1α, MMP-2, MMP-7, and TIMP-1 and severity of fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Transcription of genes involved in profibrotic pathways remained altered in both kidneys 6 months after transient renal ischemia. This suggested that a single unilateral renal insult can have lasting effects on both kidneys.

Relationships among urinary protein-to-creatinine ratio, urine specific gravity, and bacteriuria in canine urine samples.

BACKGROUND: Proteinuria is a marker of chronic kidney disease in dogs and a risk factor for increased morbidity and death. Predictive models using the results of readily available screening tests could foster early recognition. OBJECTIVE: To determine whether urine specific gravity (USG) and semiquantitative category of dipstick protein can be used to predict urinary protein-to-creatinine ratio (UP : C) and to examine the effect of urine culture results on UP : C in dogs. ANIMALS: Three hundred ninety-four dogs (482 visits) presented to a university Community Practice Clinic or Veterinary Teaching Hospital between January 2011 and November 2015. METHODS: Retrospective study. Medical records were searched to identify dogs for which urinalysis, UP : C measurement, and urine culture testing were performed during a single hospital visit. Urine specific gravity, UP : C, dipstick protein concentration, and findings of urine sediment analysis and urine culture were recorded. Regression or Spearman correlation analysis was used to test for relationships between UP : C and USG within dipstick categories and between UP : C and bacterial colony-forming units per milliliter, respectively. Cohen's kappa test was used to evaluate agreement between urine culture and UP : C testing. RESULTS: There were significant (P < .05) weak negative correlations (R2 range, 0.14-0.37) between UP : C and USG for all nonnegative urine protein dipstick categories. The presence of a positive urine culture did not agree with the presence of abnormal UP : C (κ = -0.06). CONCLUSIONS AND CLINICAL IMPORTANCE: Within dipstick protein categories, UP : C cannot be accurately predicted from USG. Repeating UP : C measurement after resolution of urinary tract infection is advisable.

Bioengineering a novel 3D in vitro model of gastric mucosa for stomach permeability studies.

The field of stomach-directed therapeutics and diagnosis is still hampered by the lack of reliable in vitro models that closely mimic the gastric mucosa where gastric cancer cells are generally confined. Here we propose a rapid, complex, and innovative 3D in vitro model of the gastric mucosa, by extending a conventional gastric monolayer model to an inner stratum of the mucosa - the lamina propria. The developed model comprises normal stomach fibroblasts embedded in a 3D RGD-modified alginate hydrogel prepared on the basolateral side of a Transwell® insert, mimicking the extracellular matrix and cellular component of the lamina propria, onto which a moderately differentiated adenocarcinoma stomach cell line (MKN74) was seeded, reproducing the physiological conditions of the gastric barrier. The integrity and functionality of the in vitro model was evaluated through permeability studies of FITC-dextran and 200 nm fluorescent polystyrene nanoparticles at gastric conditions. Nanoparticle transport was pH-dependent and strongly impacted by the biomimetic lamina propria, highlighting that a gastric extracellular matrix (ECM)-like microenvironment should be integrated in an in vitro permeability model to be adopted as a reliable evaluation tool of innovative therapeutics and diagnosis of gastric diseases. STATEMENT OF SIGNIFICANCE: Current in vitro models of the gastric mucosa are limited to simplistic 2D cell culture systems, which ignore the dimensionality of the stomach wall and make it difficult to reliably test new therapeutic approaches to gastric pathologies. By combining stomach fibroblasts embedded within a 3D RGD-modified alginate hydrogel and epithelial gastric cancer cells in a Transwell® system, we established a new biomimetic model of the stomach mucosa. Epithelial cells recreate the gastric epithelium, while the cell-laden 3D hydrogel recapitulates both the cellular composition and dimensionality of the extracellular matrix of gastric lamina propria. This cellularized 3D model stands as a promising evaluation platform to assist the development of new strategies for the treatment and diagnosis of gastric diseases.

CD44v6 increases gastric cancer malignant phenotype by modulating adipose stromal cell-mediated ECM remodeling.

CD44, an abundantly expressed adhesion molecule, and its alternative splice variants have been associated with tumorigenesis and metastasis. In the context of gastric cancer (GC), de novo expression of CD44 variant 6 (CD44v6) is found in more than 60% of GCs, but its role in the pathogenesis and progression of this type of cancer remains unclear. Using a combination of media conditioning experiments and decellularized extracellular matrices (ECMs), this study investigates the hypothesis that CD44v6 overexpression enhances tumor cell malignant behavior by modulating stromal cell-mediated ECM remodeling. Our findings indicate that soluble factors secreted by CD44v6 expressing GC cells particularly increase proliferation and myofibroblastic differentiation of adipose stromal cells (ASCs). These changes in ASC phenotype mediate the deposition of fibrotic/desmoplastic ECM that, in turn, stimulates GC proliferation and inhibits GC clustering. Pharmacological inhibition of matrix metalloproteinase (MMP) activity in tumor cells abrogated matrix-induced changes in tumor cell malignant behavior. Additionally, studies in mice confirmed the pathological relevance of CD44v6 expression and consequential changes in ECM remodeling to gastric tumorigenesis in vivo. Collectively, these results indicate a direct link between CD44v6, ECM remodeling, and GC malignant behavior opening new insights into potential CD44v6-targeted therapies.

Abdominal ultrasonographic findings in acromegalic cats.

OBJECTIVES: Acromegaly is increasingly recognized as a cause of insulin resistance in cats with diabetes mellitus (DM). The objective of this study was to determine if ultrasonographic changes in selected abdominal organs of acromegalic cats could be used to raise the index of suspicion for this condition. METHODS: In this retrospective case-control study, medical records of cats presenting to North Carolina State University or Colorado State University from January 2002 to October 2012 were reviewed. Cats were included in the acromegaly group if they had insulin-resistant DM with increased serum insulin-like growth factor (IGF-1) concentrations and had an abdominal ultrasound examination performed with report available. A control group included age-matched cats that had abdominal ultrasound examination performed for investigation of disease unlikely to involve the kidneys, adrenal glands, pancreas or liver. RESULTS: Twenty-four cats were included in each group. IGF-1 concentrations in the acromegaly group ranged from >148 to 638 nmol/l. When compared with age-matched controls, cats with acromegaly demonstrated significantly increased median left and right kidney length, significantly increased median left and right adrenal gland thickness, and significantly increased median pancreatic thickness. Hepatomegaly and bilateral adrenomegaly were reported in 63% and 53% of acromegalic cats, respectively, and in none of the controls. Pancreatic abnormalities were described in 88% of the acromegalic cats and 8% of the controls. CONCLUSIONS AND RELEVANCE: These findings indicate that compared with non-acromegalic cats, age-matched acromegalic patients have measurably larger kidneys, adrenal glands and pancreas. Diagnostic testing for acromegaly should be considered in poorly regulated diabetic cats exhibiting organomegaly on abdominal ultrasound examination.