Tomas Welbourne

Advanced glycation end products: a Nephrologist's perspective.

Advanced glycation end products (AGEs) are a heterogeneous group of molecules that accumulate in plasma and tissues with advancing age, diabetes, and renal failure. There is emerging evidence that AGEs are potential uremic toxins and may have a role in the pathogenesis of vascular and renal complications associated with diabetes and aging. AGEs are formed when a carbonyl of a reducing sugar condenses with a reactive amino group in target protein. These toxic molecules interact with specific receptors and elicit pleiotropic responses. AGEs accelerate atherosclerosis through cross-linking of proteins, modification of matrix components, platelet aggregation, defective vascular relaxation, and abnormal lipoprotein metabolism. In vivo and in vitro studies indicate that AGEs have a vital role in the pathogenesis of diabetic nephropathy and the progression of renal failure. The complications of normal aging, such as loss of renal function, Alzheimers disease, skin changes, and cataracts, may also be mediated by progressive glycation of long-lived proteins. AGEs accumulate in renal failure as a result of decreased excretion and increased generation resulting from oxidative and carbonyl stress of uremia. AGE-modified beta(2)-microglobulin is the principal pathogenic component of dialysis-related amyloidosis in patients undergoing dialysis. Available dialytic modalities are not capable of normalizing AGE levels in patients with end-stage renal disease. A number of reports indicated that restoration of euglycemia with islet-cell transplantation normalized and prevented further glycosylation of proteins. Aminoguanidine (AGN), a nucleophilic compound, not only decreases the formation of AGEs but also inhibits their action. A number of studies have shown that treatment with AGN improves neuropathy and delays the onset of retinopathy and nephropathy. N-Phenacylthiazolium bromide is a prototype AGE cross-link breaker that reacts with and can cleave covalent AGE-derived protein cross-links. Thus, there is an exciting possibility that the complications of diabetes, uremia, and aging may be prevented with these novel agents.

Glutamate transport and renal function

Brush border γ-glutamyltransferase-glutaminase activity and the high-affinity glutamate transporter EAAC1 function as a unit in generating and transporting extracellular glutamate into proximal tubules as a signal that modulates intracellular glutamine/glutamate metabolism, paracellular permeability, and urinary acidification. The reported presence of a second glutamate transporter, GLT1, on the antiluminal tubule surface points to specific functional roles for each subtype in physiological and pathophysiological processes.Brush border gamma-glutamyltransferase-glutaminase activity and the high-affinity glutamate transporter EAAC1 function as a unit in generating and transporting extracellular glutamate into proximal tubules as a signal that modulates intracellular glutamine/glutamate metabolism, paracellular permeability, and urinary acidification. The reported presence of a second glutamate transporter, GLT1, on the antiluminal tubule surface points to specific functional roles for each subtype in physiological and pathophysiological processes.

Role of hippurate in acidosis induced adaptation in renal γ-glutamyltransferase

Abstract Metabolic acidosis results in an adaptation in renal γ-glutamyltransferase (γ-GT) and a doubling of hippurate excretion. The greater rate of γ-glutamohydroxamate, γ-GHA, formation from L-glutamine, but not from glutathione, by acidotic kidney homogenates suggest an increased γ-glutamyl-enzyme complex formation and a preference for glutamine as the γ-glutamyl donor in acidosis. Hippurate added in vitro to cortical homogenates or microsomes mimics the affect of acidosis upon γ-GHA formation from glutamine. Acid extracts of urine stimulated ammonia formation from glutamine using cortical microsomes in agreement with the measured hippurate levels. Administering an exogenous hippurate load to fasting nonacidotic rats doubled ammonia excretion and the rate of γ-GHA formation by cortical homogenates. These results are consistent with the acidosis induced adaptation in renal γ-GT governed by hippurate.

Role of glucocorticoids in regulating interorgan glutamine flow during chronic metabolic acidosis

The role of glucocorticoids in external glutamine mobilization and renal utilization was evaluated in three groups of chronically acidotic rats: sham-treated controls, adrenalectomized, and adrenalectomized supplemented with triamcinolone. Chronic acidosis was induced by administering NH4Cl in their drinking solution over a three-day period. Adrenalectomized rats were supplemented by triamcinolone at a dose of 40 micrograms/100 g/d administered by pellet implantation. Interorgan glutamine flow was evaluated in the postabsorptive state by monitoring net balances across the hindquarters, gut, liver, and kidneys. In the adrenal-intact group, acidosis increased the flow of glutamine from the hindquarters to the kidneys; splanchnic bed uptake, the major glutamine sink in nonacidosis, was eliminated by virtue of hepatic reversal from net uptake to release. Adrenalectomy, in the absence of an exogenous acid load, reversed the flow of glutamine with the kidneys releasing and the hindquarters removing glutamine. Acid loading restored hindquarter glutamine release to levels seen in the intact chronically acidotic animals; however, renal extraction is much less than that exhibited by the intact animals. As a consequence, arterial glutamine concentration rose with the overflow removed by the splanchnic bed, the major glutamine sink in adrenalectomized acidotic rats. Supplementing adrenalectomized acidotic rats with triamcinolone restored glutamine extraction to values seen in intact acidotic rats. Despite the renal extraction, the large hindquarter glutamine release led to hepatic uptake and a high rate of ureagenesis. Glucocorticoids, the release of which is enhanced in metabolic acidosis, appear essential for renal glutamine extraction while playing a lesser role in modulating hindquarter glutamine release.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the acidosis induced adaptation in renal γ-glutamyltransferase

Abstract Acidosis induces an adaptation in renal γ-glutamyltransferase activity. The mechanism responsible for this adaptation was studied in isolated kidneys from control and chronically acidotic rats perfused with either γ-glutamyl-p-nitroanilide or D-glutamine. The results clearly establish that acidosis increased the utilization of both γ-glutamyl donors and that the adaptation occurs on both the luminal (urine) and antiluminal (blood) border of tubule cells. Acidotic rat kidneys exhibited an apparent Vmax for γ-glutamyl-p-nitroanilide similar to that of the control while the apparent K m was significantly reduced consistent with an increased affinity of the enzyme for the substrate in acidosis.

Glutamate transport asymmetry and metabolism in the functioning kidney

Renal glutamate extraction in vivo shows a preference for the uptake ofd-glutamate on the antiluminal and l-glutamate on the luminal tubule surface. To characterize this functional asymmetry, we isolated rat kidneys and perfused them with an artificial plasma solution containing either d- orl-glutamate alone or in combination with the system [Formula: see text]specific transport inhibitor,d-aspartate. To confirm that removal of glutamate represented transport into tubule cells, we monitored products formed as the result of intracellular metabolism and related these to the uptake process. Perfusion withd-glutamate alone resulted in a removal rate that equaled or exceeded thel-glutamate removal rate, with uptake predominantly across the antiluminal surface;l-glutamate uptake occurred nearly equally across both luminal and antiluminal surfaces. Thus the preferential uptake ofd-glutamate at the antiluminal and l-glutamate at the luminal surface confirms the transport asymmetry observed in vivo. Equimolard-aspartate concentration blocked most of the antiluminald-glutamate uptake and a significant portion of the luminall-glutamate uptake, consistent with system [Formula: see text] activity at both sites. d-Glutamate uptake was associated with 5-oxo-d-proline production, whereas l-glutamate uptake supported both glutamine and 5-oxo-l-proline formation;d-aspartate reduced production of both 5-oxoproline and glutamine. The presence of system[Formula: see text] activity on both the luminal and antiluminal tubule surfaces, exhibiting different reactivity towardl- andd-glutamate suggests that functional asymmetry may reflect two different[Formula: see text] transporter subtypes.Renal glutamate extraction in vivo shows a preference for the uptake of D-glutamate on the antiluminal and L-glutamate on the luminal tubule surface. To characterize this functional asymmetry, we isolated rat kidneys and perfused them with an artificial plasma solution containing either D- or L-glutamate alone or in combination with the system X-AG specific transport inhibitor, D-aspartate. To confirm that removal of glutamate represented transport into tubule cells, we monitored products formed as the result of intracellular metabolism and related these to the uptake process. Perfusion with D-glutamate alone resulted in a removal rate that equaled or exceeded the L-glutamate removal rate, with uptake predominantly across the antiluminal surface; L-glutamate uptake occurred nearly equally across both luminal and antiluminal surfaces. Thus the preferential uptake of D-glutamate at the antiluminal and L-glutamate at the luminal surface confirms the transport asymmetry observed in vivo. Equimolar D-aspartate concentration blocked most of the antiluminal D-glutamate uptake and a significant portion of the luminal L-glutamate uptake, consistent with system X-AG activity at both sites. D-Glutamate uptake was associated with 5-oxo-D-proline production, whereas L-glutamate uptake supported both glutamine and 5-oxo-L-proline formation; D-aspartate reduced production of both 5-oxoproline and glutamine. The presence of system X-AG activity on both the luminal and antiluminal tubule surfaces, exhibiting different reactivity toward L- and D-glutamate suggests that functional asymmetry may reflect two different X-AG transporter subtypes.

Glutamate transport asymmetry in renal glutamine metabolism

d-Glutamate (Glu) was previously shown to block l-Glu uptake and accelerate glutaminase flux in cultured kidney cells [Welbourne, T. C., and D. Chevalier. Am. J. Physiol. 272 ( Endocrinol. Metab. 35): E367-E370, 1997]. To test whether d-Glu would be taken up by the intact functioning kidney and effect the same response in vivo, male Sprague-Dawley rats were infused withd-Glu (2.6 μmol/min), and renal uptake of d- andl-Glu was determined from chemical and radiolabeled arteriovenous Glu concentration differences times renal plasma flow. The amount removed was then compared with that amount filtered to obtain the antiluminal contribution. In the controls, l-Glu uptake measured as net removal was 33% of the arteriall-Glu load and not different from that filtered, 27%; however, the unidirectional uptake was actually 58% of the arterial load, indicating that antiluminal uptake contributes at least half to the overall Glu consumption. Surprisingly, the kidneys showed a more avid removal ofd-Glu, removing 73% of the arterial load, indicating uptake predominantly across the antiluminal cell surface. Furthermore, uptake ofd-Glu was associated with a 55% reduction in l-Glu uptake, with the residual amount taken up equivalent to that filtered;d-Glu did not increase the excretion of the l-isomer. However, elevating plasma l-Glu concentration reduced uptake of thed-isomer, suggesting a shared antiluminal transporter. Thus there is an apparent asymmetrical distribution of the d-Glu transporter. Under these conditions, kidney cortexl-Glu content decreased 44%, whereas net glutamine (Gln) uptake increased sevenfold (170 ± 89 to 1,311 ± 219 nmol/min, P < 0.01) and unidirectional uptake nearly threefold (393 ± 121 to 1,168 ± 161 nmol/min, P < 0.05); this large Gln consumption was paralleled by an increase in ammonium production so that the ratio of production to consumption approaches 2, consistent with accelerated Gln deamidation and subsequent Glu deamination. These results point to a functional asymmetry (antiluminal vs. luminal) for Glu transporter activity, which potentially plays an important role in modulating Gln metabolism and renal function.

Glutamate transport and cellular glutamine metabolism : regulation in LLC-PK1 vs. LLC-PK1-F+ cell lines

The glutamate (Glu) transporter may modulate cellular glutamine (Gln) metabolism by regulating both the rates of hydrolysis and subsequent conversion of Glu to α-ketoglutarate and[Formula: see text]. By delivering Glu, a competitive inhibitor of Gln for the phosphate-dependent glutaminase (PDG) as well as an acid-load activator of glutamate dehydrogenase (GDH) flux, the transporter may effectively substitute extracellularly generated Glu from the γ-glutamyltransferase for that derived intracellularly from Gln. We tested this hypothesis in two closely related porcine kidney cell lines, LLC-PK1 and LLC-PK1-F+, the latter selected to grow in the absence of glucose, relying on Gln as their sole energy source. Both cell lines exhibited PDG suppression as the result of Glu uptake while disrupting the extracellularl-Glu uptake, withd-aspartate-accelerated intracellular Glu formation coupled primarily to the ammoniagenic pathway (GDH). Conversely, enhancing the extracellular Glu formation with p-aminohippurate and Glu uptake suppressed intracellular Gln hydrolysis while[Formula: see text] formation from Glu increased. Thus these results are consistent with the transporters dual role in modulating both PDG and GDH flux. Interestingly, PDG flux was actually higher in the Gln-adapted LLC-PK1-F+cell line because of a two- to threefold enhancement in Gln uptake despite greater Glu uptake than in the parental LLC-PK1 cells, revealing the importance of both Glu and Gln transport in the modulation of PDG flux. Nevertheless, when studied at physiological Gln concentration, PDG flux falls under tight Glu transporter control as Gln uptake decreases, suggesting that cellular Gln metabolism may indeed be under Glu transporter control in vivo.

Gamma glutamyltransferase ammonia production from glutamine: effect of physiological glutathione concentration

The formation of ammonia from physiological glutamine concentration catalyzed by gamma glutamyltransferase was studied in the presence of physiological glutathione concentration. The apparent Km for ammonia formation from glutamine was 1.6 mM some 2 fold greater than the actual plasma concentration. In the presence of 30 microM glutathione neither the apparent Km or Vmax were changed. At supraphysiological glutathione concentration, 1mM, the apparent Km was increased while the Vmax decreased to one third. Hippurate the physiological modulator of the enzymes glutaminase activity reduced the Km to 0.9 mM, the physiological range, and elevated the Vmax 2.7 fold.

Adaptation of γ-glutamyltransferase to acidosis

Abstract This study established the following points: (1) the rat kidney contains a mitochondrial (PDG) andm extramitochondrial glutamine utilizing enzymes; (2) this extramitochondrial activity exhibits a molecular weight of 70,000 identical with γ-glutamyltranspeptidase (γ-GTP) and is capable of hydrolyzing both isomers of glutamine; (3) this pathway adapts to metabolic acidosis consistent with a significant contribution to in vivo renal ammoniagenesis.