Diana L. Donnelly-Roberts

Longitudinal Changes in Measured Glomerular Filtration Rate, Renal Fibrosis and Biomarkers in a Rat Model of Type 2 Diabetic Nephropathy

Background: Obese ZSF-1 rats display many features of human type II diabetes including nephropathy (DN). The study aimed to further understand the relevance of this model to DN, for which glomerular filtration rate (GFR), renal fibrosis and several urinary/tissue biomarkers was followed over 24 weeks in ZSF-1 rats. Methods: Intact/sham or uninephrectomized male and female ZSF-1 rats were studied. GFR was measured by transdermal clearance of fluorescein isothiocyanate-sinistrin. Urine was collected every 2-4 weeks for biomarker analysis. Renal tissue was examined histologically for fibrosis and for levels of inflammatory and fibrotic genes. Results: Male obese ZSF-1 rats demonstrated metabolic syndrome and proteinuria. Female counterparts were hyperlipidemic with delayed proteinuria, but were not hyperglycemic. Kidney hyperfiltration was observed in male obese rats in weeks 2-4 after surgery, and subsequently declined to levels significantly lower than controls. Tubulointerstitial/glomerular fibrosis in male obese rats was significantly elevated by week 12 post surgery and continued to expand in the ensuing weeks, particularly in uninephrectomized rats. Female rats had less severe fibrosis. Except for epidermal growth factor which decreased, the levels of several key inflammatory, injury and fibrotic factors were elevated in both tissue (mRNA) and urine (protein) of male obese rats. Conclusion: Male obese ZSF-1 rats represent an important DN model, manifesting key pathophysiological features including metabolic syndrome, proteinuria, progressive tubular and glomerular fibrosis, and transient hyperfiltration followed by progressive decline in renal function. Uninephrectomy significantly accelerated disease progression. Females were less severe in disease manifestation. Several urinary and tissue biomarkers were identified in the male obese rats that tracked with disease progression.

Antibodies against an α-bungarotoxin-binding peptide of the α-subunit of the acetylcholine receptor

Abstract Polyclonal and monoclonal antibodies were raised against a peptide comprising residues 173–204 of the α-subunit of the acetylcholine receptor. The polyclonal and pooled monoclonal antibodies inhibited up to 50% of 125 I-α-bungarotoxin binding to peptide 173–204. Some of the antibodies recognized native receptor but did not significantly affect α-bungarotoxin binding. Epitope mapping revealed that the antibodies are directed against residues 183–194 indicating this region is a major determinant of toxin binding. This region is most likely conformationally constrained in the native receptor.

ABT-418: A Novel Cholinergic Channel Activator (ChCA) for the Potential Treatment of Alzheimer’s Disease

Evidence is accumulating to suggest that compounds which activate neuronal nicotinic acetylcholine receptors (nAChRs) may have potential benefit in the treatment of dementia, especially Alzheimer’s disease (AD) (for review: Arneric and Williams, 1994). The focus of this chapter is to summarize the preclinical pharmacology of ABT-418 [(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole], a novel analog of (-)-nicotine that is in clinical development for the treatment of AD. ABT-418 is a cholinergic channel activator (ChCA) with cognitive enhancement and anxiolytic-like activity possessing a substantially reduced side-effect profile compared to (-)-nicotine (Americ et al., 1994; Decker et al., 1994a). The enhanced preclinical safety profile and excellent transdermal delivery across species are consistent with the early Phase I clinical studies (Sebree et al., 1993).

Sodium dodecyl sulfate- and carbamylcholine-induced changes in circular dichroism spectra of acetylcholine receptor synthetic peptides

The effect of sodium dodecyl sulfate (SDS) on the conformation of acetylcholine receptor alpha-subunit synthetic peptides was investigated by circular dichroism. In the presence of SDS (0.01-0.02%), the affinity of a 173-204 32 residue peptide and a 172-227 56 residue peptide for the competitive antagonist alpha-bungarotoxin increases about 10-fold to the nanomolar range. Circular dichroism spectroscopy of these peptides revealed significant changes in the secondary structure of the peptides in the presence of SDS at concentrations below the critical micelle concentration. It is concluded that SDS induces a conformation of the peptides that is conductive to high affinity binding. Carbamylcholine, an acetylcholine analog, produced small but significant changes in the spectrum of the 173-204 peptide. This change could be the result of agonist-induced conformational changes in this region of the acetylcholine receptor alpha-subunit or to changes in the asymmetric environments of aromatic chromophores in the binding site. These studies demonstrate that synthetic peptides alone are capable of retaining significant functional activity and contain significant secondary structure.

ABT-089: An Orally Effective Cholinergic Channel Modulator (ChCM) with Cognitive Enhancement and Neuroprotective Action

Recent evidence suggests the existence of a diversity of neuronal nicotinic acetylcholine receptor (nAChRs) subtypes with a wide distribution in brain, that each subtype may be involved in mediating specific functions/behaviors, and that these subtypes have a defined pharmacology that may be selectively targeted (Arneric et al., 1995a). The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favorable side-effect profiles than existing agents which generally exhibit low selectivity. This broader class of agents, collectively called cholinergic channel modulators (ChCMs) selectively activate some subtypes of nAChRs (i.e. Cholinergic Channel Activators, ChCAs) or inhibit the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs).

Losartan improves renal function and pathology in obese ZSF-1 rats

Abstract Background: Losartan, a blocker of the angiotensin II type I receptor, is an important part of the standard of care for diabetic nephropathy (DN). The obese ZSF-1 rats display many aspects of the clinical features of human Type II DN. The current study was designed to examine the treatment effects of losartan on obese ZSF-1 rats and to evaluate the impact of the onset of dosing on efficacy. Methods: The rats (7–10 weeks) underwent a right uninephrectomy (Unx) or sham surgery. Losartan (3, 10, 30 mg/kg) was dosed 3 or 9 weeks post-Unx and continued for 12 weeks. Results: Treatment with losartan reduced urinary protein excretion and blood lipids (triglyceride and cholesterol) dose-dependently in both studies. The glomerular filtration rate (GFR) was significantly lower in obese ZSF-1 rats compared with those in lean rats, and losartan was efficacious against this endpoint, in particular with the earlier onset of treatment. Losartan also decreased tubulointerstitial fibrosis, and similar to GFR, earlier treatment conferred beneficial actions even at the lowest dose of 3 mg/kg. Several urinary biomarkers were elevated in the obese ZSF-1 rats, but the levels of sTNFR1, TIMP-1, L-FABP and KIM-1 were the only markers decreased by losartan. Conclusions: Losartan was renoprotective in the ZSF-1 rats with DN, improving both the pathological and functional parameters of the disease. Importantly, the data also highlight the importance of treatment at earlier stages of the disease for protecting against decline in the GFR and the development of fibrosis.

Short-term oral gavage administration of adenine induces a model of fibrotic kidney disease in rats

INTRODUCTION The adenine model of kidney disease typically involves dietary delivery of adenine over several weeks. This model can be variable in its disease progression and can result in significant mortality. In the current study, the amount of adenine delivered to rats was controlled by utilizing oral gavage administration over a short period in an attempt to induce robust renal pathology while addressing variability and viability of the animals. METHODS Adenine (150 or 200 mg/kg) was administered via oral gavage for 10 consecutive days, and assessed over a total of 20 days. RESULTS Both adenine dose groups manifested pathophysiological features of kidney disease such as proteinuria, elevated serum creatinine and BUN, and tubulointerstitial fibrosis. The animals also displayed a decline in glomerular filtration rate. Renal mRNA expression of genes associated with injury, inflammation, and fibrosis (i.e., Col1a1, Acta2, Serpine1, Timp1, Fn-Eda, Tgfb1, Ccl2, Nlrp3, Aqp1 and Ccnd1) were elevated as were urinary biomarkers that have translational utility (i.e., clusterin, KIM-1, MCP-1, OPN, NGAL, B2M, calbindin, and cystatin C). All disease endpoints were more pronounced in the 200 mg/kg group, however, while measures of tissue fibrosis were sustained, there was partial recovery by day 20 in functional readouts. No mortality was observed in either dose group. DISCUSSION Short-term delivery of adenine via precise gavage delivery induced a robust model with hallmarks of fibrotic kidney disease, had limited variance between animals, and no animal morbidity within the 20 days studied. This model represents a methodical alternative to long-term dietary dosing of adenine.