Both isoforms of ketohexokinase are dispensable for normal growth and development.

Dietary fructose intake has dramatically increased over recent decades and is implicated in the high rates of obesity, hypertension, and type 2 diabetes (metabolic syndrome) in Western societies. The molecular determinants of this epidemiologic correlation are incompletely defined, but high-flux fructose catabolism initiated by ketohexokinase (Khk, fructokinase) is believed to be important. The Khk gene encodes two enzyme isoforms with distinctive substrate preferences, the independent physiological roles of which are unclear. To investigate this question, and for testing the importance of Khk in metabolic syndrome, isoform-selective genetic lesions would be valuable. Two deficiency alleles of the mouse Khk gene were designed. The first, Khk(3a), uses targeted "knock-in" of a premature termination codon to induce a selective deficiency of the minor Khk-A isoform, preserving the major Khk-C isoform. The second, the Khk(Δ) allele, ablates both isoforms. Mice carrying each of these Khk-deficiency alleles were generated and validated at the DNA, RNA, and protein levels. Comparison between normal and knockout animals confirmed the specificity of the genetic lesions and allowed accurate analysis of the cellular distribution of Khk within tissues such as gut and liver. Both Khk(3a/3a) and Khk(Δ/Δ) homozygous mice were healthy and fertile and displayed minimal biochemical abnormalities under basal dietary conditions. These studies are the first demonstration that neither Khk isoform is required for normal growth and development. The new mouse models will allow direct testing of various hypotheses concerning the role of this enzyme in metabolic syndrome in humans and the value of Khk as a pharmacological target.

Influence on energy kinetics and histology of different preservation solutions seen during cold ischemia in the liver.

BACKGROUND AND PURPOSE: Cold flush preservation prolongs tissue viability during ischemia. However, there is little understanding of the effects of various preservation fluids on events during this period. A study of cold ischemia in rat livers was undertaken to compare biochemical and histological changes over time, using three preservation solutions: University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), and Leeds solution (LS) under development at our institution. Leeds solution is a phosphate-based sucrose solution that like UW contains the impermeant lactobionate and the metabolite allopurinol (1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) which acts as a competitive inhibitor of xanthine oxidase, stopping the breakdown of hypoxanthine to xanthine by oxidizing it to alloxanthine, inhibiting both the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid. MATERIALS AND METHODS: At various time points, samples were analyzed for adenosine triphospate (ATP) and metabolites by high-performance liquid chromatography as well as for histological changes. RESULTS: In all livers, ATP, ADP, and AMP degraded over 4 hours. In UW and LS groups, degradation beyond hypoxanthine was halted, and it continued in the HTK group. This blockade led to a significant reduction in the accumulation of xanthine and uric acid. Histological analysis showed protected architecture and maintenance of reticulin scaffolds in the UW and LS groups, whereas tissue breakdown was seen from earlier time points in the HTK group. Additionally, throughout ischemia, signs of pathological injury were more pronounced with UW- than with LS-preserved tissue. CONCLUSIONS: These results implied that cold ischemia in the liver is characterized by dynamic biochemical changes coincident with pathological injury which are initiated from the time of organ perfusion and influenced by the choice of the perfusion fluid. Allopurinol in UW and LS appears to be critical. We hypothesized that it may also affect the degree of subsequent reperfusion injury. The data supported the assertion that LS offerred improved preservation over UW, adding to the impetus to shorten ischemic times in clinical transplantation.

Histidine-tryptophan-ketoglutarate and delayed graft function after prolonged cold ischemia.

BACKGROUND: Several articles have compared histidine-tryptophan-ketoglutarate solution (HTK) with other preservation solutions in both liver and kidney transplantation, and the results suggest that HTK is as good or better than the criterion standard University of Wisconsin solution (UW) for short periods of cold ischemia, such as in live donation, but that it is not so efficient for longer periods of cold ischemia, causing a higher incidence of delayed graft function. OBJECTIVE: To evaluate energy levels, metabolites, and histologic findings to determine why HTK is inefficient for longer periods of cold ischemia. METHODS: Rat livers were perfused with either HTK or UW, and at various times, tissue samples were obtained for analysis of adenine triphosphate and metabolites using high-performance liquid chromatography or for histologic analysis. RESULTS: The high energy charge observed with HTK-perfused livers plateaued after 5 minutes, and by 60 minutes began to decrease, following the same trend as other samples. The plateau is due to excess available glucose; however, after 1 hour, it is beginning to be consumed. Low levels of uridine, required for glycogen synthesis, are found in HTK-perfused livers, which suggests that at reperfusion, there is none available, whereas the higher concentrations found in UW-perfused livers may be advantageous after reperfusion. This will be especially detrimental to use of HTK because glycogen is used up rapidly because of the presence of alpha-ketoglutarate in the solution, enabling continuation of the tricarboxylic acid cycle. CONCLUSIONS: Overall, HTK seems to do well for the first 2 hours, after which any advantage observed initially starts to disappear. A liver perfused in HTK and transplanted after more than 1 hour reacts like an organ from an individual who has been starved, because of the low energy charge and absence of a glycogen store or ability to synthesis glycogen because of lack of uridine. Livers perfused with UW demonstrate higher levels of uridine and do not lose their glycogen content to the same extent as HTK-perfused livers. These findings explain in part why HTK sometimes causes delayed graft function after longer periods of cold ischemia.

Alterations of glomerular basement membrane charge and structure in diabetic nephropathy.

We examined glomerular basement membrane anionic site distribution identified by cationic gold in seven patients with insulin-dependent and four patients with non-insulin-dependent diabetes mellitus, presenting a spectrum of clinical and glomerular changes. Anionic sites were investigated by pretreatment of tissue with glycosaminoglycan-degrading enzymes prior to cationic gold staining. The distribution of chondroitin sulphate proteoglycans--a previously unrecognized glomerular basement membrane component--and type IV collagen was examined by immunoelectron microscopy to identify structural changes in the basement membrane. Findings were compared with those of non-diabetic patients showing minor proteinuria and morphologically normal glomerular basement membranes. Two patients, originally diagnosed as having diabetic nephropathy were also examined at 19 weeks and 5 years after renal transplantation. Characteristic redistribution of type IV collagen and chondroitin sulphate proteoglycans was noted in thickened glomerular basement membrane segments (> 400 nm) of diabetic patients and those with renal transplants. Extension of anionic sites deep into the glomerular basement membrane at pH 2.5, together with loss of interna sites at pH 5.8 is unique to diabetic nephropathy. Reduced charge density was apparent in some patients due to thickening of the glomerular basement membrane, although the number of anionic sites per unit length of membrane was actually increased. Thus, charge aberration in diabetic nephropathy is due to displacement rather than loss of anionic sites. Removal of more than 90% of these sites by heparitinase, confirms their association with heparan sulphate proteoglycans. Similar derangement of anionic sites in all patients with diabetic nephropathy irrespective of the degree of proteinuria, suggests that a heparan sulphate proteoglycan-related charge barrier plays a minor role in controlling permeability of the diabetic glomerular basement membrane.

Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold.

We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.

Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using epipolarization microscopy.

We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.