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BACKGROUND: Accumulation of glomerular macrophages, proliferation of mesangial cells (MCs), and deposition of extracellular matrix proteins are pathobiological hallmarks of glomerulonephritis. We previously reported that a clinically available nonselective inhibitor of cyclic 3',5'-nucleotide phosphodiesterase, pentoxifylline (PTX), inhibits proliferation of cultured rat MCs, as well as collagen production by these cells. In this study, we investigated the in vivo effects of PTX on rat anti-Thy1 disease, a model of mesangial proliferative nephritis. METHODS: Anti-Thy1 nephritis was induced in Sprague-Dawley rats by injecting mouse anti-rat Thy1 antibodies intravenously. Nephritic rats were randomly assigned to receive PTX (0.1 g/kg/day) or vehicle (phosphate-buffered saline) and were sacrificed at various time points. Paraffin kidney sections were stained with hematoxylin and periodic acid-Schiff reagents for glomerular histology. Frozen kidney sections were stained by monoclonal antibodies against proliferating cell nuclear antigen, ED-1, and alpha-smooth muscle actin and were visualized by color development from a horseradish peroxidase reaction. Monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and various extracellular matrix mRNAs were analyzed by Northern blotting. Urine protein concentrations were determined by Lowry's method. RESULTS: Nephritic rats treated with PTX excreted less urinary protein on day 5 of nephritis than vehicle-treated nephritic rats. In periodic acid-Schiff-stained kidneys from PTX-treated nephritic rats, there was attenuation of both glomerular cellularity and glomerular sclerosis compared with vehicle-treated nephritic rats. PTX decreased the augmented glomerular mRNA levels of MCP-1 and ICAM-1 at two hours and on day 1 of nephritis. Immunoreactive staining showed that PTX reduced the number of proliferating glomerular macrophages on days 1, 2, and 3, but not at two hours of nephritis, compared with vehicle-treated nephritic rats. On day 5, PTX decreased the number of activated proliferating MCs and attenuated the glomerular mRNA levels of type I (alpha1), type III (alpha1), and type IV (alpha1) collagen and fibronectin compared with vehicle-treated nephritic rats. CONCLUSION: The administration of PTX to rats with anti-Thy1 disease reduces accumulation and proliferation of glomerular macrophages, attenuates proteinuria, suppresses activation and proliferation of MCs, and ameliorates glomerular sclerosis. These results suggest that PTX may have a suppressive effect in acute phases or relapses of mesangial proliferative glomerulonephritis.

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Aldosterone secretion in most patients with aldosterone-producing adenomas (APAs) is typically unresponsive to angiotensin II stimulation (AII-unresponsive, AII-U). In some patients, however, plasma aldosterone increases in response to AII stimulation (AII-responsive, AII-R). This differential aldosterone responsiveness could be related to the levels of type 1 AII receptors (AT1R) in the APA. To test this hypothesis, plasma aldosterone levels in response to upright posture and/or sequential high- and low-salt diets were measured by radioimmunoassay in nine patients with APAs. AT1R mRNA levels in the adenomas were quantified by competitive reverse transcription-polymerase chain reaction and correlated to the cellular composition of the adenoma. Two patients were categorised as AII-R by an increase of plasma aldosterone greater than 50% over the baseline. The remaining seven patients who had blunted plasma aldosterone responses were classified as AII-U. Histologically, the AII-R APAs consisted predominantly of zona glomerulosa (ZG)-like cells (> 90%), while the AII-U APAs contained zona fasciculata (ZF)-like cells ranging from 28 to 72%. There was an inverse relationship between the levels of AT1R mRNA in the APA and the percentage of ZF-like cells in the adenoma (n = 9, r = 0.73, P < 0.05). In situ hybridisation findings demonstrated that AT1R mRNA was more uniform and intensive in ZG-like cells than in ZF-like cells. These results suggest that heterogenous aldosterone responsiveness to angiotensin in APAs is histologically dependent and related to the differential expression of AT1R mRNA in the adenoma.

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Dorsal root ganglion (DRG) sensory neurones from adult rats are known to lose their capsaicin sensitivity in vitro if they are cultured without nerve growth factor (NGF). Here we show similar results following peripheral nerve transection in vivo, which deprives DRG sensory neurones of target-derived NGF. By measuring capsaicin-stimulated 45Ca uptake into DRG neurones which had been briefly cultured, capsaicin sensitivity was shown to decrease in neurones whose axons had been previously severed in vivo. Conversely, during experimental inflammation of the rat paw, there is an increase in the supply of NGF to neurones innervating the inflamed area. In this case, however, no significant increase in capsaicin sensitivity could be demonstrated in briefly cultured neurones which had previously innervated an inflamed limb. This suggests that expression of capsaicin sensitivity in DRG is maximal at levels of NGF found in normal animals.

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Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increases in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA.

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A retrograde labelling technique combined with a cobalt uptake assay in cultured adult rat dorsal root ganglion (DRG) neurons were applied to study the distribution of capsaicin sensitivity in relation to different peripheral targets. This study shows that there are regional differences between skin, skeletal muscle and urinary bladder; 20-30% of skin afferents, 40% of muscle afferents and 60% of bladder afferents were found to be capsaicin-senditive. This may reflect differences in the proportion of chemosensitive afferents innervating different peripheral tissues.

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The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000 - 3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.