Current kidney allocation rules and their impact on a pediatric transplant center.

In 2005, kidney allocation rules in the United States were updated to enhance access to kidneys from young adult deceased donors (DDs) for pediatric recipients. We studied how this rule change affected transplant activity at our pediatric center. We retrospectively compared kidney transplant activity at our center since the rule change (until December 31, 2007) to before the change (n = 36 each), focusing on those recipients directly affected by it, that is, younger than 18 years. There were no significant differences in recipients' age, gender or ethnicity before versus after the rule change. Percentages of preemptive transplants and retransplants were similar in both groups, as was the percentage of sensitized patients. There was a significant decrease in overall, but not DD, mean donor age. Mean wait time for DD kidneys decreased for pediatric recipients. Increases were found in percentage of DD transplants and in mean HLA mismatches after the rule change. Patient and short-term graft survival were not significantly different. These data suggest that the allocation rule change was not only followed by improvement in overall access to kidney transplantation for children, but also by decreases in living donor transplants and HLA matching. Larger studies are needed to evaluate the long-term impact of the change.

Substituted hydrophobic and hydrophilic residues at methionine-68 influence the chaperone-like function of alphaB-crystallin.

Amino acid residues 57-69 in alphaB-crystallin have been implicated as a target protein binding site. Moreover, a direct correlation between the extent of alpha-crystallin hydrophobicity and chaperone-like activity has been demonstrated. The purpose of this study was to mutate a moderately hydrophobic residue Met-68 (M-68) in the above region to strongly hydrophobic and hydrophilic residues and show whether chaperoning ability is affected with or without structural changes. Mutation of M-68 to Val, Ile or Thr did not result in significant changes in molecular mass and secondary and tertiary structures. However, the Val and Ile mutants showed significant improvement and the Thr mutant showed substantial loss in chaperone activity. Differences in chaperone function in the absence of any structural changes confirmed that the hydrophobicity or hydrophilicity of the substituted amino acid in the putative target protein binding site was the only contributing factor.

Advanced glycation end products in human senile and diabetic cataractous lenses.

The authors prepared water-soluble (WSF), urea-soluble (USF), alkali-soluble (ASF), sonicated (SF), sonicated insoluble (SIF) and membrane (MF) fractions of lens proteins from human senile and diabetic cataractous lenses and age-matched clear lenses. Levels of advanced glycation end products (AGEs) including carboxymethyl lysine (CML), a glycoxidation product, were determined by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA). Distribution of AGEs in the various protein fractions was ascertained by SDS-PAGE and Western blotting. An overall increase in the levels of AGEs in diabetic cataractous lenses as compared to senile cataractous lenses and clear lenses has been observed. ASF and SF, both of which originated from the urea-insoluble fraction, showed the highest levels of AGEs. However, no clear-cut differences in CML levels were seen among clear lenses and senile and diabetic cataractous lenses. AGEs were found to be distributed mostly in the high molecular aggregates in all the fractions. These data suggest that AGEs contribute to protein aggregation and subsequent insolubilization.

Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function.

An autosomal dominant congenital cataract in human is associated with mutation of Arg-116 to Cys (R116C) in alpha A-crystallin. To investigate the molecular basis of cataract formation, rat alpha A-crystallin cDNA was cloned into pET-23d(+), and the site-directed mutants S142C (similar to wild-type human alpha A) and R116C/S142C or R116C (similar to human R116C variant) were generated. These were expressed in E. coli and the recombinant alpha A-crystallins purified by Sephacryl size-exclusion chromatography. The chaperone-like function of mutant R116C determined at 37 degrees C with insulin and alcohol dehydrogenase as target proteins was about 40% lower than those of wild-type and mutant S142C. Based on size-exclusion chromatography data, the oligomeric size of the R116C mutant was about 2000 kDa at 25 degrees C, 1400 kDa at 37 degrees C, and 900 kDa at 45 degrees C. In comparison, alpha A-wild-type and alpha A-S142C ranged from 477 to 581 kDa. Heat stability studies corroborated the effect of temperature on the dynamic quaternary structure of the R116C mutant. Circular dichroism spectra showed secondary and tertiary structural changes, and ANS fluorescence spectra showed loss of surface hydrophobicity in the R116C mutant. These findings suggest that the molecular basis for the congenital cataract with the alpha A-R116C mutation is due to the generation of a highly oligomerized alpha A-crystallin having a modified structure and decreased chaperone-like function.

Intrapolypeptide disulfides in human alphaA-crystallin and their effect on chaperone-like function.

We report studies on the role of protein-protein disulfides (PSSP) in the age-related loss of chaperone activity of alpha-crystallins. AlphaL-crystallin fraction was isolated from human lenses of different ages and the chaperone-like activity was determined before and after treatment with glutathione reductase (GR) and NADPH. The results confirmed an age-dependent decrease in chaperone-like function and significant improvement of this function by GR treatment. Electrospray ionization mass spectrometric (ESIMS) analysis of alphaA-crystallin suggested the presence of very little protein-glutathione mixed disulfides. ESIMS analysis of Asp-N digests of alphaA-crystallin revealed that nearly all the remaining portion of Cys-131 and Cys-142 of alphaA-crystallin was present in the form of intrapolypeptide disulfide bonds. These results show for the first time that predominantly disulfide bonds formed during aging contribute to the age-dependent loss in chaperone activity of alpha-crystallin in human lenses.

Evidence of a glycemic threshold for the development of cataracts in diabetic rats.

PURPOSE: This study was aimed to establish a possible correlation between the levels of plasma glucose and degree of lens opacification. Levels of glycation- and glycoxidation-products in different lens protein fractions were also estimated with an aim to determine the involvement of these products in lens opacification. METHODS: A wide range of hyperglycemia was induced by injecting different doses of streptozotocin to 1 month old rats and lenses were examined on the 75th, 90th and 150th day post-injection. Lens opacification was measured by Scheimpflug imaging and densitometry. Levels of plasma glucose and glycated hemoglobin were measured after overnight fasting. On 90th day, levels of Amadori products in lens water soluble (WS) fraction were measured by affinity chromatography. Similarly, advanced glycation end products (AGEs) in lens WS, urea soluble (US) and alkali soluble (AS) fractions were measured immunochemically using a monoclonal antibody against the major glycoxidation product, carboxymethyl lysine (CML). RESULTS: Different dosages of streptozotocin injection resulted in a broad range of plasma glucose levels in the rats which were grouped into three groups on the basis of their plasma glucose levels: mildly diabetic (< 170 mg/dl plasma glucose), moderately diabetic (190-350 mg/dl) and severely diabetic (> 400 mg/dl). On the 75th, 90th and 150th day post-injection, only the moderately and severely diabetic rats developed cataracts whereas lenses of the mildly diabetic rats remained clear. As seen on 90th day, levels of glycated hemoglobin and Amadori products in lens WS fraction increased significantly in the moderately and severely diabetic groups whereas in the mildly diabetic rats these levels remained more or less same as in the control group. Levels of CML in WS fractions remained unchanged between control rats and different diabetic groups, while US fractions showed a decrease in CML in both the moderately and severely diabetic groups compared to the controls and the mildly diabetic group. Interestingly, AS fractions contained the highest level of CML; the moderately and severely diabetic groups showed about 2-fold higher levels than the controls and the mildly diabetic group. CONCLUSIONS: This study strongly supports the existence of plasma glycemic threshold above which incidence of diabetic cataract formation increases exponentially. This threshold level seems to be at approximately 180 mg/dl or 10 mM plasma glucose. Significant increase in the levels of glycation and glycoxidation products mainly in cataract lenses suggests that glycation and glycation-mediated oxidation play an important role in the development of diabetic cataracts.

Influence of protein-glutathione mixed disulfide on the chaperone-like function of alpha-crystallin.

In an earlier report we showed that incubation of alpha-crystallin with oxidized glutathione results in significant loss of its chaperone-like activity. In the present study, we determined the effect of protein-glutathione mixed disulfides (PSSG), formed at Cys-131 in bovine alphaA-crystallin, and Cys-131 and Cys-142 in human alphaA-crystallin, on the function of alpha-crystallin as a molecular chaperone. After incubation of calf and young human alphaL-crystallin fractions with oxidized glutathione, levels of PSSG were determined by performic acid oxidation of the mixed disulfides followed by reversed-phase high pressure liquid chromatography separation of phenylisothiocyanate-derivatized glutathione sulfonic acid. Levels of PSSG increased from 0.01 to 0.14 nmol/nmol (20 kDa) in bovine alphaL-crystallin and from 0.022 to 0.25 nmol/nmol in human alphaL-crystallin. The presence of glutathione adducts at Cys-131 and Cys-142 were confirmed by mass spectral analysis. The chaperone-like activity was determined by the heat denaturation assay using betaL-crystallin as the target protein. To examine the reversibility of the effect of mixed disulfides on chaperone activity, studies were done before and after reduction with the glutathione reductase system. Increased levels of PSSG resulted in lower chaperone activities. Treatment with the glutathione reductase system led to 80% reduction in PSSG levels with a concomitant recovery of the chaperone activity. These results suggest that cysteine(s) in the alphaA-crystallin subunit play an important role in the function of alpha-crystallin as a molecular chaperone.

The role of alpha- and epsilon-amino groups in the glycation-mediated cross-linking of gammaB-crystallin. Study of three site-directed mutants.

In the previous report we demonstrated that gammaB-crystallin is glycated predominantly at the N-terminal alpha-amino group (Casey, E. B., Zhao, H. R., and Abraham, E. C. (1995) J. Biol. Chem. 270, 20781-20786). To investigate the possible role of alpha- and epsilon-amino groups of gammaB-crystallin in glycation-mediated cross-linking, Lys-2 or Lys-163, or both, were mutated to threonine by site-directed mutagenesis in bovine gammaB-crystallin cDNA. Wild type and mutant gammaB-crystallins were expressed in Escherichia coli cells. Cross-linking studies were performed by incubating wild type and mutant gammaB-crystallins with glyceraldehyde, ribose, and galactose followed by SDS-polyacrylamide gel electrophoresis under reducing conditions. When both of the lysines of gammaB-crystallin were mutated to threonines (gammaB-K2T/K163T), the quantity of cross-linked products was greatly reduced, indicating that, despite the fact that the alpha-amino group is a major glycated site, epsilon-amino groups play a predominant role in cross-linking. Therefore, cross-linking ability depends not only upon the level of glycation but also upon which amino group is glycated. Steric hindrance may decrease the cross-linking ability of the alpha-amino group. Our results also show that Lys-2 and Lys-163 play almost equal roles in cross-linking of gammaB-crystallin. By incubating carbonic anhydrase, a protein with a blocked N terminus, and our novel "no lysine" gammaB (gammaB-K2T/K163T) with sugar, we were able to show for the first time that significant cross-linking occurs between lysines and non-lysine sites. The fact that pentosidine and imidazolysine, formed from ribose and methylglyoxal, respectively, were present in the cross-linked gammaB-crystallins revealed the existence of Lys-Arg and Lys-Lys cross-linking.

Identification of hydrogen peroxide oxidation sites of alpha A- and alpha B-crystallins.

The alpha-crystallins are the most abundant structural proteins of the lens and, because of their chaperone activity, contribute to the solubility of the other crystallins. With aging, the lens crystallins undergo a variety of modifications which correlate with a loss of solubility and the development of cataract. A recent study demonstrating that alpha-crystallins exposed in vitro to FeCl3 and H2O2 exhibit decreased chaperone activity, implicates metal catalyzed oxidations of alpha-crystallins in this loss of solubility. The present study has determined that alpha-crystallins incubated with FeCl3 and H2O2 are modified by the nearly complete oxidation of all methionine residues to methionine sulfoxide, with no other detectable reaction products. The modifications were identified from the molecular weights of peptides formed by enzymatic digestion of the alpha-crystallins and located by tandem mass spectrometric analysis of the fragmentation pattern of the mass spectra of the fragments from peptides with oxidized methionine is loss of 64 Da, which corresponds to loss of CH3SOH from the methionine sulfoxide. These fragments are useful in identifying peptides that include oxidized methionine residues.

Immunochemical evidence for increased formation of advanced glycation end products and inhibition by aminoguanidine in diabetic rat lenses.

Water-soluble and urea-soluble protein fractions from control and streptozotocin-diabetic rats were analyzed for AGEs with a CML-specific monoclonal anti-AGE antibody and a polyclonal anti-AGE antibody. AGEs, CML in particular, were significantly increased in the diabetic rats whereas aminoguandine treatment resulted in significant decrease in AGEs. The data also confirm that CML, a glycoxidation product, is a major epitope of AGE structures in lenses.

Sites of glycation of beta B2-crystallin by glucose and fructose.

We determined the sites of glycation of bovine beta B2-crystallin by glucose and fructose. After incubation with glucose or fructose, glycated tryptic peptides were purified by affinity chromatography/reverse-phase HPLC and identified by electrospray ionization mass spectrometry (ESIMS). The results gave evidence of glycation at lysine 10, 75, 100, 107, 120, 139, 167 and 171 by both glucose and fructose, while glycated lysine 119 and 47 or 67 were detected only after fructosylation. We conclude that glucose and fructose have similar glycation specificity.

Inhibition of cataracts in moderately diabetic rats by aminoguanidine.

The effect of aminoguanidine (AG), an inhibitor of advanced glycation, on the development of cataracts was studied in diabetic rats. Rats were made diabetic with streptozotocin, and based on the level of plasma glucose they were grouped as moderately (< 350 mg dl-1 plasma glucose) and severely (> 350 mg dl-1 plasma glucose) diabetic. One half of the animals in each group received AG (25 mg kg-1 body weight each day), intraperitoneally, starting from the day of streptozotocin injection. Progression of lens opacification was recorded using Fundus and Scheimpflug photography at regular time intervals. On the ninetieth day all the rats were killed and the levels of advanced glycation end products (AGE) was determined by measuring the non-tryptophan fluorescence of the lens soluble and insoluble fractions. Densitometric analysis of Scheimpflug images showed that in diabetic rats lens opacification progressed in a biphasic manner, an initial slow progression for the first 60 days, followed by a steep increase during next 30 days. Moderately and severely diabetic rats developed lens opacities more or less at the same time. AGE fluorescence in the lens soluble fractions increased three-fold and seven-fold in the moderately and severely diabetic rats, respectively; whereas in insoluble fractions there was a 30% and three-fold increase in the moderately and severely diabetic rats, respectively. Although AG treatment inhibited the AGE fluorescence of lens soluble and insoluble fractions by about 56% and 75% in moderately diabetic and by 19% and 52% severely diabetic rats, respectively, the development of cataracts was delayed only in the moderately diabetic rats. These results thus suggest that the effect of AG is indeed inhibition of the formation of AGEs. However, in the severely diabetic rats the beneficial effect of AG is overwhelmed by the excessive accumulation of AGEs.

Scheimpflug densitometric analysis of cataracts in diabetic rats: correlation with glycation.

Glycation has been implicated in cataract formation. Our earlier studies showed that crystallin glycation enhances oxidation and aggregation, whereas MIP26 glycation affects the membrane permeability. Scheimpflug densitometric analysis has been used to quantify the lens opacification. In this study, we measured the progressive changes in lens opacification and correlated them with protein glycation in streptozotocin-induced diabetic rats. The lens opacification progressed in a biphasic manner: an initial slow increase between 0 and 60 days, followed by a steep increase between 60 and 90 days of diabetes. There was a strong correlation between lens opacification and lens crystallin and MIP26 glycation. The correlation was relatively weak with plasma glucose. This study suggests that glycation of lens crystallin and MIP26 plays a significant role in the development of lens opacification in diabetic rats.

Role of glycine 1 and lysine 2 in the glycation of bovine gamma B-crystallin. Site-directed mutagenesis of lysine to threonine.

To determine the role of Gly-1 and Lys-2 of bovine gamma B-crystallin in glycation and cross-linking, Lys-2 was changed to Thr by site-directed mutagenesis. A polymerase chain reaction was used to perform site-directed mutagenesis on the third codon (AAG-->ACG) of bovine gamma B-crystallin cDNA. The wild type and mutant cDNAs were cloned into pMON5743 and expressed in JM101 Escherichia coli cells, and the identity of gamma B-crystallin was confirmed by Western blotting after purification by cation exchange high performance liquid chromatography. Glycation of gamma B-crystallin by [14C]glucose was reduced significantly due to the mutation of Lys-2, supporting the view that Lys-2 is a major glycation site. Peptide mapping showed the presence of two major labeled peptides containing N-terminal sequences, and in the mutant these peptides had longer retention times and reduced radioactivity. Amino acid analysis, after glycation with [14C]glucose, revealed N-terminal glycine as the most predominant glycation site. Lys-2 was glycated slower than Gly-1 but faster than Lys-163. Glycation with DL-glyceraldehyde showed an important role for both Gly-1 and Lys-2 in the glycation-mediated gamma B-crystallin cross-linking.

Effect of germanium-132 on galactose cataracts and glycation in rats.

Germanium compounds have been shown to be effective in preventing the formation of advanced glycation end-products and for reversible solubilization of glycated proteins. As protein glycation has been proposed to play a role in lens opacification, we initiated studies to evaluate the effects of 2-carboxyethyl germanium sesquioxide (germanium compound 132 or Ge-132) on galactose-induced cataractogenesis. For this study young Sprague-Dawley rats were fed a 50% galactose diet. One group of rats received topical saline and another group was administered Ge-132 in saline four times a day. The lenses were periodically examined with an ophthalmoscope and at desired intervals processed for light and scanning electron microscopy. Our observations, beginning at 3 days and continuing to 21 days of galactose feeding, exhibited the characteristic galactose-induced morphological alterations, which include the formation of vacuoles, cysts, membrane disruption and swelling of fibers and epithelial cells as well as disorganization of the bow in lenses of rats in both groups. However, in the majority of rats administered Ge-132 these alterations were delayed as compared to the lenses of rats administered saline. Our findings show that, although the initiation, progression and pattern of lens opacification in rats receiving saline and Ge-132 were similar, in the majority of lenses the progression and establishment of mature cataracts in the Ge-132 group of rats were delayed. Analysis of the water-soluble and water-insoluble lens-protein fractions for glycated proteins showed increased levels of the Amadori products and advanced glycation related fluorescent products in galactosemic rats treated with saline eye drops. In rats receiving the topical Ge-132 treatment the levels of these glycation products were substantially reduced to levels lower than control values. Prevention of glycation seems to be a mechanism by which cataract progression is delayed.

Diabetes affects alpha-crystallin chaperone function.

In vitro glycation was previously shown to influence alpha-crystallin chaperone function. In the present study we show that this function is compromised in diabetes. The alpha H, alpha L and the total alpha fractions were isolated by gel permeation chromatography from the water-soluble protein of streptozotocin-diabetic as well as age-matched normal rats. Based on the beta L-crystallin thermal denaturation assay the chaperone function was significantly decreased in the diabetic rats.

Decreased molecular chaperone property of alpha-crystallins due to posttranslational modifications.

We studied the effect of oxidation, mixed disulfide formation and glycation of alpha-crystallins on their molecular chaperone property. The ability of alpha-crystallins to protect heat-induced denaturation and aggregation of beta L-crystallin was significantly diminished by these modifications. alpha-Crystallin from senile human lenses also showed significant loss of chaperone-like property. Age-dependent increase in posttranslationally modified alpha-crystallins is the likely cause for this change.

Site selectivity in the glycation of alpha A- and alpha B-crystallins by glucose.

We determined the site selectivity of glycation by glucose (glucosylation) in alpha A- and alpha B-crystallins using two independent approaches. HPLC purified 14C-glucose labeled chymotryptic peptides and affinity chromatography/HPLC purified fully glycated peptides were identified by FAB-MS. Lys 11 and 78 of alpha A-crystallin and Lys 90 and/or 92 of alpha B-crystallin were the fast reacting sites of glucosylation.

Glycation mediated lens crystallin aggregation and cross-linking by various sugars and sugar phosphates in vitro.

Glycation of lens crystallins results in protein conformational changes, oxidation, browning and aggregation. Though glucose is the major sugar, other sugars and sugar phosphates generated as intermediates of metabolic pathways are present in the lens, albeit at low concentrations. In this study we incubated bovine lens soluble fraction with various sugars and sugar phosphates (5mM for 10 days). The reactivity was in the order trioses > tetroses > pentoses > hexoses. High molecular weight (HMW) aggregates were also formed at a comparable rate. Increased levels of fluorescence were associated with the HMW aggregates with fast reacting sugars. The phosphorylated derivatives were only slightly more reactive than their respective sugars. Interestingly, fructose-1,6-diphosphate was more reactive and cross-linked more readily than fructose-6-phosphate. Gel electrophoresis under reducing and nonreducing conditions showed formation of disulfide linked protein aggregates with slow reacting sugars such as glucose and non-disulfide covalent linked protein aggregates with fast reacting sugars such as erythrose. In contrast, if 0.1 m DTT was present in erythrose incubations (a fast reacting sugar), the HMW aggregate formation was significantly reduced. In order to show the reactivity among the slow reacting hexoses, we incubated lens proteins with 1 M hexoses for 30 days and the results showed that galactose was more reactive and showed higher cross-linking than fructose and glucose. These results thus indicate that relatively low levels of some sugars and sugar phosphates in the lens could be compensated by enhanced lens protein cross-linking and the combined effect could be rather significant with respect to cataractogenesis.