Safety and efficacy of intravenous infusion of allogeneic cryopreserved mesenchymal stem cells for treatment of chronic kidney disease in cats: results of three sequential pilot studies

INTRODUCTION: Administration of mesenchymal stem cells (MSCs) has been shown to improve renal function in rodent models of chronic kidney disease (CKD), in part by reducing intrarenal inflammation and suppressing fibrosis. CKD in cats is characterized by tubulointerstitial inflammation and fibrosis, and thus treatment with MSCs might improve renal function and urinary markers of inflammation in this disease. Therefore, a series of pilot studies was conducted to assess the safety and efficacy of intravenous administration of allogeneic adipose-derived MSCs (aMSCs) in cats with naturally occurring CKD. METHODS: Cats enrolled in these studies received an intravenous infusion of allogeneic aMSCs every 2 weeks collected from healthy, young, specific pathogen-free cats. Cats in pilot study 1 (six cats) received 2 × 10(6) cryopreserved aMSCs per infusion, cats in pilot study 2 (five cats) received 4 × 10(6) cryopreserved aMSCs per infusion, and cats in pilot study 3 (five cats) received 4 × 10(6) aMSCs cultured from cryopreserved adipose. Serum biochemistry, complete blood count, urinalysis, urine protein, glomerular filtration rate, and urinary cytokine concentrations were monitored during the treatment period. Changes in clinical parameters were compared statistically by means of repeated measures analysis of variance (ANOVA) followed by Bonferroni’s correction. RESULTS: Cats in pilot study 1 had few adverse effects from the aMSC infusions and there was a statistically significant decrease in serum creatinine concentrations during the study period, however the degree of decrease seems unlikely to be clinically relevant. Adverse effects of the aMSC infusion in cats in pilot study 2 included vomiting (2/5 cats) during infusion and increased respiratory rate and effort (4/5 cats). Cats in pilot study 3 did not experience any adverse side effects. Serum creatinine concentrations and glomerular filtration rates did not change significantly in cats in pilot studies 2 and 3. CONCLUSIONS: Administration of cryopreserved aMSCs was associated with significant adverse effects and no discernible clinically relevant improvement in renal functional parameters. Administration of aMSCs cultured from cryopreserved adipose was not associated with adverse effects, but was also not associated with improvement in renal functional parameters

In vivo and in vitro assessment of mirtazapine pharmacokinetics in cats with liver disease.

BackgroundLiver disease (LD) prolongs mirtazapine half-life in humans, but it is unknown if this occurs in cats with LD and healthy cats.Hypothesis/objectivesTo determine pharmacokinetics of administered orally mirtazapine in vivo and in vitro (liver microsomes) in cats with LD and healthy cats.AnimalsEleven LD and 11 age-matched control cats.MethodsCase-control study. Serum was obtained 1 and 4 hours (22 cats) and 24 hours (14 cats) after oral administration of 1.88 mg mirtazapine. Mirtazapine concentrations were measured by liquid chromatography with tandem mass spectrometry. Drug exposure and half-life were predicted using limited sampling modeling and estimated using noncompartmental methods. in vitro mirtazapine pharmacokinetics were assessed using liver microsomes from 3 LD cats and 4 cats without LD.ResultsThere was a significant difference in time to maximum serum concentration between LD cats and control cats (median [range]: 4 [1-4] hours versus 1 [1-4] hours; P = .03). The calculated half-life of LD cats was significantly prolonged compared to controls (median [range]: 13.8 [7.9-61.4] hours versus 7.4 [6.7-9.1] hours; P < .002). Mirtazapine half-life was correlated with ALT (P = .002; r = .76), ALP (P < .0001; r = .89), and total bilirubin (P = .0008; r = .81). The rate of loss of mirtazapine was significantly different between microsomes of LD cats (-0.0022 min-1 , CI: -0.0050 to 0.00054 min-1 ) and cats without LD (0.01849 min-1 , CI: -0.025 to -0.012 min-1 ; P = .002).Conclusions and clinical importanceCats with LD might require less frequent administration of mirtazapine than normal cats

Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

<p>Abstract</p> <p>Background</p> <p><it>Penicillium marneffei </it>is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in <it>P. marneffei </it>that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of <it>P. marneffei</it>.</p> <p>Results</p> <p>Whole cell proteins from the early stages of mould and yeast development in <it>P. marneffei </it>were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated <it>RanA</it>, was subsequently cloned and characterized. The <it>P. marneffei </it>RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous <it>Aspergillus </it>proteins.</p> <p>Conclusion</p> <p>This study clearly demonstrates the utility of proteomic approaches to studying dimorphism in <it>P. marneffei</it>. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in <it>P. marneffei</it>.</p

Feline chronic kidney disease: novel approaches to etiology, specific therapy and supportive care

2012 Summer.Includes bibliographical references.Chronic kidney disease is one of the leading causes of morbidity and mortality in geriatric cats, affecting conservatively 30% of the population; an estimated 24 million cats nationwide in the United States. Despite the common nature of the disease, its etiology is yet unknown, and there is no definitive cure short of renal transplantation. The goals of the research described in this dissertation were to explore possible etiologies of chronic kidney disease and to develop novel treatment strategies to help cats afflicted with this disease. The first part of this project investigated a possible etiology for CKD; renal aging as manifested by telomere shortening and cellular senescence. In these studies telomere length and cellular senescence were assessed in cats with CKD in comparison to young healthy and geriatric healthy controls. Using a TELI-FISH assay to measure telomere length in specific renal cell populations, significantly shorter telomeres were found in the renal proximal and distal tubular cell population of CKD cats compared to young normal or geriatric normal cats. There was no difference between CKD cats and normal cats when liver or skin telomere length was measured. Additionally, β-galactosidase assay revealed increased cellular senescence in the kidneys of CKD cats in comparison to young normal. CKD cats tended to have increased β-galactosidase staining in comparison to normal geriatric cats, but this did not reach statistical significance. Neither telomere length nor cellular senescence were correlated with age, but the normal geriatric population available for assessment was small. It was concluded that telomere shortening and cellular senescence are present in feline CKD; future studies will be necessary to determine cause and effect aspects of this relationship. Demonstration of an association between telomere shortening, cellular senescence and feline CKD could be the foundation of new treatment strategies. Cats with CKD frequently have poor appetites and nutritional management of these patients is important. Mirtazapine is an appetite stimulant and anti-nausea medication that has recently gained popularity in veterinary medicine and anecdotally appears to be helpful for the management of appetite. However, no pharmacokinetic or pharmacodynamic information exists on the drug in cats. The aims of the second part of these studies were a) the assessment of the pharmacokinetics and pharmacodynamics of commonly prescribed doses of mirtazapine in normal cats, elderly cats and cats with CKD, and b) a placebo-controlled blinded crossover clinical trial to assess the efficacy of mirtazapine in CKD cats. These studies demonstrated that there are differences in the metabolism of mirtazapine between young normal cats, geriatric normal cats and CKD cats. Based on the pharmacokinetic studies, young cats could receive daily mirtazapine at a low dose without significant likelihood of drug accumulation whereas CKD cats should receive the drug every other day due to delayed clearance. In a subsequent clinical trial, mirtazapine significantly increased appetite, activity and weight in CKD cats when administered at a low dose every other day for three weeks. Additionally, a significant decrease in vomiting was noted. This demonstrated that mirtazapine does have significant appetite stimulating and anti-nausea effects in CKD cats. The information gathered in this body of work will help clinicians prescribe mirtazapine more effectively with a decreased incidence of unwanted drug side effects. Most importantly, it will help improve the quality of life and potentially prognosis of cats suffering from CKD. Most treatments for CKD are palliative in nature and do not directly address the underlying pathology. CKD is characterized by tubulointerstitial inflammation, fibrosis and progressive loss of renal function. Mesenchymal stem cell (MSC) therapy is thought to be anti-inflammatory, and has the potential to improve or stabilize renal function in animals with renal failure, based on evidence from rodent model studies of induced renal disease. At present, there is little published work regarding the use of MSC for treatment of naturally occurring CKD. The last section of this body of work focuses on the evaluation of MSC therapy as a novel treatment strategy for cats with CKD. A series of pilot studies was performed; a pilot study of intrarenal injection of autologous stem cells and two pilot studies of intravenously injected allogeneic cryopreserved MSC. Urinary cytokines were measured to assess intra-renal inflammation, fibrosis and vascular health and the possible effects of MSC injection on these factors. We determined that MSC could be successfully harvested and cultured from bone marrow and adipose sources, but the latter was preferred for ease of collection, expansion and superior yield. Intrarenal injection did not induce immediate or longer-term adverse effects. Two CKD cats that received intrarenal adipose-derived MSC experienced modest improvement in GFR and a mild decrease in serum creatinine concentration. In the allogeneic cryopreserved intravenous study, six cats received 2 x 106 MSC per injection and experienced a significant decrease in serum creatinine with negligible side effects. Five cats received 4 x 106 MSC per injection and side effects included vomiting during infusion and increased respiratory rate. Variable decreases in serum creatinine, increases in GFR by iohexol clearance and changes in urinary cytokines were seen. Despite the mild improvement in creatinine seen in some of the cats, none had improvement to the extent described in rodent models. While MSC therapy potentially holds promise for palliation of CKD, additional work is necessary to determine if this therapy can be manipulated to increase its efficacy. The work described in this dissertation has increased our knowledge of the biology of renal aging and its relationship to CKD. In addition it has assessed the effect of two novel treatment strategies on cats with CKD. This information will directly improve the lives of cats with CKD as well as providing a strong foundation for further research in this area

Assessment of peritubular capillary rarefaction in kidneys of cats with chronic kidney disease

Abstract Background Hypoxia is a key driver of fibrosis and is associated with capillary rarefaction in humans. Objectives Characterize capillary rarefaction in cats with chronic kidney disease (CKD). Animals Archival kidney tissue from 58 cats with CKD, 20 unaffected cats. Methods Cross‐sectional study of paraffin‐embedded kidney tissue utilizing CD31 immunohistochemistry to highlight vascular structures. Consecutive high‐power fields from the cortex (10) and corticomedullary junction (5) were digitally photographed. An observer counted and colored the capillary area. Image analysis was used to determine the capillary number, average capillary size, and average percent capillary area in the cortex and corticomedullary junction. Histologic scoring was performed by a pathologist masked to clinical data. Results Percent capillary area (cortex) was significantly lower in CKD (median 3.2, range, 0.8‐5.6) compared to unaffected cats (4.4, 1.8‐7.0; P = <.001) and was negatively correlated with serum creatinine concentrations (r = −.36, P = .0013), glomerulosclerosis (r = −0.39, P = <.001), inflammation (r = −.30, P = .009), and fibrosis (r = −.30, P = .007). Capillary size (cortex) was significantly lower in CKD cats (2591 pixels, 1184‐7289) compared to unaffected cats (4523 pixels, 1801‐7618; P = <.001) and was negatively correlated with serum creatinine concentrations (r = −.40, P = <.001), glomerulosclerosis (r = −.44, P < .001), inflammation (r = −.42, P = <.001), and fibrosis (r = −.38, P = <.001). Conclusions and Clinical Importance Capillary rarefaction (decrease in capillary size and percent capillary area) is present in kidneys of cats with CKD and is positively correlated with renal dysfunction and histopathologic lesions

Serum concentrations of gabapentin in cats with chronic kidney disease

ObjectivesThe purpose of this study was to assess serum concentrations of gabapentin in cats with chronic kidney disease (CKD) vs clinically healthy cats.MethodsFive healthy cats were enrolled in a pharmacokinetic study. A single 20 mg/kg dose of gabapentin was administered orally and blood was obtained at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 8, 12, 24 and 36 h via a jugular catheter. Serum gabapentin concentrations were measured using liquid chromatography coupled to tandem mass spectrometry. Non-compartmental pharmacokinetic analysis was performed. The same five healthy cats plus 25 cats with stable International Renal Interest Society stage 2 (n = 14) and 3 (n = 11) CKD were enrolled in a limited sampling study. Cats in both groups received a single 10 mg/kg dose of gabapentin, and serum gabapentin concentrations and compliance scores were obtained 3 and 8 h post-administration.ResultsCats with CKD had significantly higher dose-normalized serum gabapentin concentrations than normal cats at 3 h (P = 0.0012 CKD vs normal 10 mg/kg; P = 0.008 CKD vs normal 20 mg/kg) and 8 h (P &lt;0.0001 CKD vs normal 10 mg/kg; P &lt;0.0001 CKD vs normal 20 mg/kg). Both 3 and 8 h dose-normalized serum gabapentin concentrations were significantly correlated with serum creatinine (3 h: P = 0.03, r = 0.39; 8 h: P = 0.001, r = 0.57) and symmetric dimethylarginine (3 h: P = 0.03, r = 0.41; 8 h: P = 0.007, r = 0.48). There was a significant correlation between 3 h serum gabapentin concentrations and compliance scores (P = 0.0002, r = 0.68).Conclusions and relevanceCats with CKD that received 10 mg/kg of gabapentin had significantly higher dose-normalized serum concentrations than normal cats that received 20 mg/kg, supporting the need to dose-reduce in this patient population

Drug exposure and clinical effect of transdermal mirtazapine in healthy young cats: a pilot study

Objectives The objective of this study was to measure drug exposure and clinical effects after administration of transdermal mirtazapine (TMZ) in healthy cats. Methods Phase I: seven healthy research cats received (1) 3.75 mg and 7.5 mg TMZ once aurally with 48 h serum sampling (serum samples were obtained via the jugular catheter at 0, 0.5, 1, 2, 5, 9, 12, 24, 36 and 48 h); (2) 7.5 mg TMZ and placebo daily aurally for 6 days then 48 h serum sampling; (3) 1.88 mg mirtazapine orally once with serum sampling at 1, 4 and 8 h. Phase II: 20 client-owned cats were enrolled in a randomized, double-blind, placebo-controlled, three-way crossover clinical effect study. Treatments consisted of 6 days of aural 7.5 mg TMZ or placebo gel at home, and 1.88 mg mirtazapine orally once in the clinic. Owners documented appetite, rate of food ingestion, begging activity and vocalization daily at home. On day 6, food consumed, activity and vocalization were documented in hospital, and trough and peak serum mirtazapine levels were obtained. Serum mirtazapine and gel concentrations were measured using liquid chromatography/tandem mass spectrometry. Results Phase I: administration of TMZ achieved measureable serum mirtazapine concentrations. Area under the curve0-48 of multidose 7.5 mg TMZ was significantly higher than single-dose 1.88 mg oral mirtazapine (OMZ) ( P = 0.02). Phase II: client-owned cats administered TMZ had a significant increase in appetite ( P = 0.003), rate of food ingestion ( P = 0.002), activity ( P = 0.002), begging ( P = 0.002) and vocalization ( P = 0.002) at home. In hospital there was a significant increase in food ingested with both TMZ and OMZ compared with placebo ( P &lt;0.05). Gel concentrations ranged from 87%-119% of target dose. Conclusions and relevance TMZ 7.5 mg daily achieves measureable serum concentrations and produces significant appetite stimulation despite variance in compounded gel concentrations, but side effects denote a lower dose is indicated