LR-90 prevents dyslipidaemia and diabetic nephropathy in the Zucker diabetic fatty rat.

AIMS/HYPOTHESIS: Previous studies have shown that LR-90, a new inhibitor of AGE formation, prevented the development of experimental type 1 diabetic nephropathy. In this study, we examined the effects of LR-90 in the Zucker diabetic fatty (ZDF) rat, a model of type 2 diabetes and metabolic syndrome, and investigated the mechanisms by which it may protect against renal injury. METHODS: Male ZDF rats were treated without or with LR-90 from age 13 to 40 weeks. Metabolic and kidney functions and renal histology were evaluated. AGE accumulation and the production of the receptor for AGE (AGER) were measured. Profibrotic growth factors, extracellular matrix proteins and intracellular signalling pathways associated with glomerular and tubular damage were also analysed. RESULTS: LR-90 dramatically reduced plasma lipids in ZDF rats, with only modest effects on hyperglycaemia. Renal AGE, AGER and lipid peroxidation were all attenuated by LR-90. LR-90 significantly retarded the increase in albuminuria and proteinuria. This was associated with reduction in glomerulosclerosis and tubulointerstitial fibrosis, concomitant with marked inhibition of renal overproduction of TGF-beta1, connective tissue growth factor, fibronectin and collagen IV. Additionally, LR-90 downregulated the activation of key mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappaB) in the renal cortex. CONCLUSIONS/INTERPRETATION: These results support our earlier studies on the renoprotective effects of LR-90 on type 1 diabetic nephropathy and provide further evidence that LR-90, an AGE inhibitor with pleiotrophic effects, may also be beneficial for the prevention of type 2 diabetic nephropathy, where multiple risk factors, such as hyperglycaemia, dyslipidaemia, obesity, insulin resistance and hypertension, contribute to renal injury.

LR-90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats.

AIMS/HYPOTHESIS: Advanced glycation and lipoxidation endproducts have been implicated in the pathogenesis of diabetic complications, including diabetic nephropathy. LR-90, a new advanced glycation endproduct inhibitor, was investigated for its effects on the development of renal disease in diabetic rats. METHODS: Diabetic animals were randomly allocated into groups receiving LR-90 or vehicle (untreated). Age- and weight-matched non-diabetic rats were studied concurrently. Body weight, plasma glucose, glycated haemoglobin, urinary albumin and creatine excretions were measured serially. Kidney histopathology, AGE accumulation in cells and tissues, protein oxidation, were also examined. In vitro assays were used to assess the possible mechanism of action of LR-90. RESULTS: LR-90 inhibited the increase in albumin and creatinine concentrations, and concentrations of circulating AGE in diabetic rats without any effect on glycaemic control. LR-90 treated-rats also showed higher body weights than untreated diabetic rats. LR-90 prevented glomerulosclerosis, tubular degeneration and collagen deposition in the kidney. AGE-induced cross-linking and fluorescence of tail collagen were reduced by LR-90 treatment. LR-90 also decreased AGE accumulation in kidney glomeruli and nitrotyrosine deposition in the renal cortex. In vitro, LR-90 was capable of reacting with reactive carbonyl compounds and was a more potent metal chelator than pyridoxamine and aminoguanidine. CONCLUSION/INTERPRETATION: LR-90 reduces in vivo AGE accumulation, AGE-protein cross-linking and protein oxidation, and could be beneficial in preventing the progression of diabetic nephropathy. The AGE inhibitory and therapeutic effects of LR-90 could be attributed, at least in part, to its ability to react with reactive carbonyl species and/or potent metal chelating activity that inhibits glycoxidative-AGE formation.

Brain-derived neurotrophic factor spares choline acetyltransferase mRNA following axotomy of motor neurons in vivo.

Choline acetyltransferase (ChAT) is a functional and specific marker gene for neurons such as primary motor neurons that synthesize and release acetylcholine as a neurotransmitter. In adult mammals, transection of the peripheral nerve results in a loss of immunoreactivity for ChAT in the injured motor neurons without affecting their cell number. Using a quantitative RNase protection assay, we have investigated dynamic changes in ChAT mRNA levels following axotomy of motor neurons in the brainstem of adult rats. One week after transection of the left hypoglossal nerve, levels of ChAT mRNA in the ipsilateral side of the hypoglossal motor nucleus decreased dramatically to around 10% when compared to the uninjured contralateral side. When cut axons were chronically exposed to brain-derived neurotrophic factor (BDNF) for 1 week, ChAT mRNA levels were maintained at 63% of control levels. Thus, BDNF can abrogate the injury-induced loss of ChAT mRNA in mature motor neurons in vivo. In contrast, neither neurotrophin 4/5 nor nerve growth factor could prevent the decrease in message. This effect of BDNF on ChAT mRNA levels following peripheral injury to motor neurons demonstrates the existence of regulatory pathways responsive to neurotrophic factors that can "rescue" or "protect" cholinergic gene expression.

Target regulation of a motor neuron-specific epitope.

In the adult rat nervous system, motor neurons are recognized specifically by a monoclonal antibody, MO-1. Because binding by MO-1 is lost following axotomy, contact with the target may regulate this motor neuron-specific epitope. To test this hypothesis, we examined the recovery of MO-1 immunoreactivity in hypoglossal neurons following unilateral damage to the hypoglossal nerve. During the first week following nerve crush, neurons in the ipsilateral hypoglossal nucleus lost all immunoreactivity for MO-1. Antibody binding returned with time, and by 4 weeks, 80% of the injured neurons had recovered the MO-1 epitope. Since motor neurons reinnervate their original targets readily following nerve crush, it appears that MO-1 binding is recovered when motor neurons return to their original target muscles in the tongue. When the hypoglossal nerve was cut and inserted into a foreign muscle nearby (the sternomastoid muscle), the MO-1 epitope was not detected in the injured neurons, even when examined 6 weeks after surgery. However, if the sternomastoid muscle was denervated prior to insertion of the hypoglossal nerve, thus allowing the hypoglossal nerve to synapse with this foreign target, increasing numbers of hypoglossal neurons reacquired MO-1 immunoreactivity with time. Our results suggest that the MO-1 epitope is only expressed in motor neurons that are in synaptic contact with skeletal muscle. Thus, a property that distinguishes mature motor neurons from other neuronal phenotypes appears to be regulated by direct synaptic interaction with the postsynaptic target.

Distinct neurotrophic responses of axotomized motor neurons to BDNF and CNTF in adult rats.

In adult mammals, transection of the hypoglossal nerve results in a dramatic loss of choline acetyltransferase (ChAT) in the hypoglossal motor neurons without affecting their cell number. This injury-induced reduction in ChAT is prevented when brain-derived neurotrophic factor (BDNF) is applied to the proximal end of the transected nerve. In contrast, application of ciliary neurotrophic factor (CNTF) has no such effect, even though both factors are known to rescue developing motor neurons from cell death. These results suggest that BDNF may regulate the phenotypic expression of ChAT in mature motor neurons, and indicate that the sensitivity and response of motor neurons to such neurotrophic agents change with development.

A motor neuron-specific epitope and the low-affinity nerve growth factor receptor display reciprocal patterns of expression during development, axotomy, and regeneration.

Somatic motor neurons begin to express the transmitter synthesizing enzyme, choline acetyltransferase (ChAT) and the low-affinity nerve growth factor receptor (NGFR) during embryonic development. However, as motor neurons mature in postnatal life, they lose immunoreactivity for NGFR and acquire a motor neuron-specific epitope that is recognized by the monoclonal antibody, MO-1. The present study was undertaken to examine the effect of nerve injury in adult rats on these three developmentally regulated markers in two populations of somatic motor neurons. Unilateral transection, ligation, or crushing of the sciatic nerve resulted in a loss of MO-1 binding and a concomitant rise in immunoreactivity for NGFR within axotomized motor neurons in lumbar levels of the spinal cord. These changes, detectable within 5 days following nerve injury, are reversed with reinnervation, but persist if reinnervation is prevented by chronic axotomy. Thus, regulation of the expression of NGFR and the MO-1 epitope appears to be critically dependent upon interactions between motor neurons and target muscles. These observations are also consistent with the idea that during regeneration, neurons may revert to a developmentally immature state; in motor neurons, this state is characterized by the presence of NGFRs and the absence of the MO-1 epitope. Transection of the hypoglossal nerve, a purely motor nerve, resulted in a similar loss of MO-1 binding and a selective rise in NGFR immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus. In addition, immunoreactivity for ChAT was also lost in axotomized hypoglossal motor neurons. In contrast, injury to the sciatic nerve, which bears both sensory and motor axons, did not result in any detectable change in ChAT immunoreactivity in spinal motor neurons.

Laminin and s-laminin are produced and released by astrocytes, Schwann cells, and schwannomas in culture.

Components of the extracellular matrix (ECM) have been implicated in the regulation of neuronal migration, axonal growth, and synaptogenesis. We have examined cultures of glial cells, Schwann cells, and schwannomas for the expression of two components of the ECM, laminin and s-laminin, using immunohistochemical and Western blot techniques. Laminin is a potent promotor of neurite outgrowth in cultures of both central and peripheral neurons, and is present in all ECMs. In contrast, s-laminin (for synaptic laminin), a recently described homolog of laminin, is highly localized at the neuromuscular synaptic cleft (Sanes and Chiu, Cold Spring Harbor Symp. Quant. Biol. 1983;48:667-678; Chiu and Sanes, Dev. Biol. 1984;103:456-467) and shows selective adhesivity for motor neurons (Hunter et al. Cell 1989;59:905-913). While the distribution of these ECM components have been well documented in situ, the sources of these extracellular molecules are unclear. We report that astrocytes cultured in serum-free medium maintain an organized ECM that only bears laminin immunoreactivity; s-laminin appears to be sequestered intracellularly. However, both molecules are found in the astrocyte conditioned medium. Thus, under these growth conditions, astrocytes produce and release laminin and s-laminin, but only incorporate the former into an ECM. In contrast, neither molecule is present in comparable cultures of oligodendrocytes. Although no established ECM is seen in cultures of Schwann cells or schwannomas, laminin and s-laminin immunoreactivity are present within cells and in the conditioned media. These results indicate that certain populations of non-neuronal support cells and cell lines can produce and release both synaptic and extrasynaptic components of the ECM. The assembly of these different molecules into an organized basal lamina may require the presence of additional factors or interaction with neurons.