Recent studies have identified that first-line renoprotective agents that interrupt the renin-angiotensin system not only reduce BP but also can attenuate advanced glycation end product (AGE) accumulation. This study used in vitro, preclinical, and human approaches to explore the potential effects of these agents on the modulation of the receptor for AGE (RAGE). Bovine aortic endothelial cells that were exposed to the angiotensin-converting enzyme inhibitor (ACEi) ramiprilat in the presence of high glucose demonstrated a significant increase in soluble RAGE (sRAGE) secreted into the medium. In streptozotocininduced diabetic rats, ramipril treatment (ACEi) at 3 mg/L for 24 wk reduced the accumulation of skin collagen-linked carboxymethyllysine and pentosidine, as well as circulating and renal AGE. Renal gene upregulation of total RAGE (all three splice variants) was observed in ACEi-treated animals. There was a specific increase in the gene expression of the splice variant C-truncated RAGE (sRAGE). There were also increases in sRAGE protein identified within renal cells with ACEi treatment, which showed AGE-binding ability. This was associated with decreases in renal full-length RAGE protein from ACEi-treated rats. Decreases in plasma soluble RAGE that were significantly increased by ACEi treatment were also identified in diabetic rats. Similarly, there was a significant increase in plasma sRAGE in patients who had type 1 diabetes and were treated with the ACEi perindopril. Complexes between sRAGE and carboxymethyllysine were identified in human and rodent diabetic plasma. It is postulated that ACE inhibition reduces the accumulation of AGE in diabetes partly by increasing the production and secretion of sRAGE into plasma.

Abstract. NF-B-dependent pathways play an important role in macrophage infiltration and kidney injury. NF-B is regulated by angiotensin II (AII). However, the role of this pathway in diabetic nephropathy has not been clearly delineated. First, the activation of NF-B, monocyte chemoattractant protein-1 (MCP-1), and macrophage infiltration in the diabetic kidney were explored, in a temporal manner. The active subunit of NF-B, p65, was elevated in the diabetic animals in association with increased MCP-1 gene expression and macrophage infiltration. Second, the effects of treatment for 4 wk with the AII type 1 receptor antagonist valsartan, the AII type 2 receptor antagonist PD123319, or pyrrolidine dithiocarbamate, an inhibitor of NF-B and on these parameters were assessed. These treatments were associated with a reduction in p65 activation, MCP-1 gene expression, and macrophage infiltration. These findings demonstrate a role for activation of NF-B, in particular the p65 subunit, in the pathogenesis of early renal macrophage infiltration in experimental diabetes. In the context of the known proinflammatory effects of AII, it is postulated that the renoprotection conferred by angiotensin II receptor antagonism is at least partly related to the inhibition of NF-Bdependent pathways.

Blockade of advanced glycation end product (AGE) accumulation with alagebrium with concomitant angiotensin converting enzyme inhibition was tested for effects on renal function and on other postulated mediators of diabetic renal disease including the renin-angiotensin system, AGEs, mitochondrial and cytosolic oxidative stress, and intracellular signaling molecules. Sprague Dawley rats were rendered diabetic with streptozocin and followed consecutively for 32 wk with nondiabetic controls. Groups were treated with ramipril (1 mg/kg⅐d; wk 0 -32); alagebrium (10 mg/kg⅐d; wk 16 -32); or a combination of both. Although individual treatments had significant effects on albuminuria, no further improvements were seen with combination therapy. Changes in urinary vascular endothelial growth factor excretion mirrored those seen in albuminuria. Diabetes was associated with suppression of circulating angiotensin II in the context of increased circulating and renal levels of the AGE, carboxymethyllysine. All treatments attenuated circulating but not renal carboxymethyllysine levels. The renal gene expression of AGE receptor 1 and soluble receptor for advanced glycation end products were markedly reduced by diabetes and normalized with alagebrium. Diabetes induced renal mitochondrial oxidative stress, which was reduced with alagebrium. In the cytosol, both therapies were equally effective in reducing reactive oxygen species production. Increases in membranous protein kinase C activity in diabetes were attenuated by all treatments, whereas diabetes-associated increases in nuclear factor-B p65 translocation remained unaltered by any therapy. It is evident that renin-angiotensin system blockade and AGE inhibition have specific effects. However, many of their downstream effects appear to be similar, suggesting that their renoprotective benefits may ultimately involve common pathways and key points of convergence, which could be important targets for new therapies in diabetic nephropathy. (Endocrinology 148: 886 -895, 2007)

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It has been hypothesized that the biochemical process of advanced glycation plays an important part in the pathogenesis of diabetic vascular complications [1] and particularly in diabetic nephropathy [2]. This hypothesis was based primarily on the renoprotective effects observed with aminoguanidine (AG), an inhibitor of advanced glycation end-product formation (AGE) [3]. Aminoguanidine, however, has other actions including inhibiting nitric oxide synthase, and thus, nitric oxide production [4]. Nitric oxide has direct effects on renal haemodynamics in diabetes [5,6] and therefore it has been difficult to ascertain if the benefits of aminoguanidine relate primarily to inhibiting renal AGE accumulation.We compared the effect of AG in diabetic rats with ALT-946, N-(2-Acetamidoethyl) hydrozinecarboximidamide hydrochloride, a more potent inhibitor of AGE-derived protein modification than AG. ALT-946 is also a poor inhibitor of nitric oxide synthase with fewer toxic side effects than AG (unpublished results). We have reported previously that AG Diabetologia (2001) AbstractAims/hypothesis. ALT-946, an inhibitor of advanced glycation with a minimal inhibitory effect on nitric oxide synthase, was compared with aminoguanidine in experimental diabetic nephropathy. Methods. In vitro and in vivo assays were used to assess the ability of ALT-946 to inhibit AGE-protein cross-link formation. Diabetic animals were randomly allocated into groups receiving aminoguanidine for 32 weeks, ALT-946 or vehicle (untreated). As a delayed intervention protocol, an additional diabetic group was treated with ALT-946 from week 16 to week 32 of the study. Non-diabetic rats were studied concurrently. Systolic blood pressure, body weight, plasma glucose, glycated haemoglobin and urinary albumin excretion were measured serially. Accumulation of advanced-glycation end products in the kidney was assessed by immunohistochemistry.Results. The ALT-946 inhibitor was more potent than aminoguanidine in inhibiting AGE-protein crosslinking both in vitro and in vivo. Increased albuminuria observed in diabetic rats was attenuated in all three treatment groups. We found no difference in body weight, blood pressure or glycaemic control with any of the treatments. The untreated diabetic group had a twofold increase in glomerular staining for advanced-glycation end products compared with the diabetic groups which received treatment. Conclusion/interpretation. ALT-946 is a potent inhibitor of advanced renal glycation end-product accumulation and reproduces the renoprotective effects of aminoguanidine. Therefore, ALT-946 should be considered as a treatment for preventing or retarding diabetic nephropathy. [Diabetologia (2001) 44: 108± 114]

A method is described to obtain intramolecular cleavage rates for the hammerhead ribozyme during in vitro transcription. By avoiding RNA purification and renaturation, the potential for formation of inactive conformations of the RNA is minimized. By showing that an intramolecular hammerhead and a closely related intermolecular hammerhead cleave at the same rate under a given set of conditions, we confirm that both reactions probably have the same ratelimiting step. An in vitro selection strategy was used to isolate active hammerheads from a library of molecules where six randomized nucleotides replaced stem-oop II. The sequence and number of nucleotides which replace stem-loop II have large effects on hammerhead cleavage activity. The relative activities of three sequences selected from the intramolecular library are the same when the sequences are transferred into an intermolecular hammerhead background.The hammerhead is an RNA structural motif of about 50 nucleotides that is embedded within the sequences of certain plant pathogenic RNAs and is responsible for self-cleavage activity (1-3). Intramolecular hammerhead cleavage has been studied by isolating uncleaved RNA from in vitro transcription reactions and introducing it into the desired reaction buffer (4,5). Because hammerheads often self-cleave quite efficiently during transcription, it is possible that the small fraction of uncleaved RNA might contain mutations as a result of misincorporation and thus may not have the same cleavage properties as the presumed RNA sequence. In addition, when the RNA is introduced into the reaction buffer, a rate-limiting conformational change may precede cleavage. To avoid these problems, a common approach to study hammerhead cleavage under defined conditions has been to divide the hammerhead into separate RNAs and combine them to initiate the reaction (6, 7). However, because this introduces an additional assembly step in the reaction pathway, it is critical to show that closely related intramolecular and intermolecular hammerheads cleave at the same rate. Indeed, based on the rapid cleavage of an intramolecular hammerhead (8), Forster and Symons (9) have suggested that intermolecular hammerhead cleavage rates may reflect formation of the active intermolecular complex and not the chemical step of the reaction.To compare intramolecular and intermolecular hammerhead cleavage rates under identical conditions, a method to measure intramolecular cleavage rates in transcription reactions has been developed. To be certain that the method is effective with a variety of hammerheads, a library of hammerheads was prepared where six randomized nucleotides replaced stem-loop II. It is known that the sequence of stem-loop II is not strictly conserved (2, 9, 10) and replacement of stem-loop II with shorter sequences reduces but does not abolish cleavage activity (10)(11)(12)(13). By selecting active molecules from the library, hammerheads with a range of cleavage activities were isolated. Cleavage rates were then measured in both...

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Telomerase is a reverse transcriptase that maintains chromosome ends. The N-terminal half of the catalytic protein subunit (TERT) contains three functional domains (I, II, and III) that are conserved among TERTs but not found in other reverse transcriptases. Guided by an amino acid sequence alignment of nine TERT proteins, mutations were introduced into yeast TERT (Est2p). In support of the proposed alignment, mutation of virtually all conserved residues resulted in loss-of-function or temperature sensitivity, accompanied by telomere shortening. Overexpression of telomerase component Est3p led to allele-specific suppression of the temperature-sensitive mutations in region I, suggesting that Est3p interacts with this protein domain. As predicted by the genetic results, a lethal mutation in region I resulted in loss of Est3p from the telomerase complex. We conclude that Est2p region I is required for the recruitment of Est3p to yeast telomerase. Given the phylogenetic conservation of region I of TERT, this protein domain may provide the equivalent function in all telomerases.