Toru Shinzato

Determination of Kt/V and Protein Catabolic Rate Using Pre- and Postdialysis Blood Urea Nitrogen Concentrations

We developed a new urea kinetic method for simultaneous determination of the Kt/V and protein catabolic rate (PCR) only from blood urea nitrogen (BUN) concentrations before and after a single dialysis session. Using this method, the parameters were calculated within 1.5 s even when a hand-held computer with a low central processing capacity is used. The total amount of urea eliminated during three dialysis sessions in 1 week is assumed to be equal to urea volume (Gw) generated over a 1-week period (Tw): [formula: see text]. Here, G is the generation rate, K is the dialyzer urea clearance, T is the dialysis time and C1, C2 and C3 are BUN during the respective dialysis session. If this equation and the equation expressing the urea kinetics during a single dialysis session are solved together, we have a solution for Kt/V and G. The thus-obtained Kt/V and G are corrected using the change in body weight. The corrected Kt/V showed a good correspondence with the parameter calculated with the classical method, and the midweek PCR derived from G determined by the present method being equivalent to the PCR averaged for a 1-week period determined by the classical methods.

Imidazolium crosslinks derived from reaction of lysine with glyoxal and methylglyoxal are increased in serum proteins of uremic patients: evidence for increased oxidative stress in uremia

Glyoxal (GO) and methylglyoxal (MGO) are reactive dicarbonyl compounds formed during autoxidation of both carbohydrates and lipids. They may react with lysine and arginine residues of proteins in Maillard or browning reactions, yielding advanced glycation or lipoxidation end products. Among these are the imidazolium crosslinks, N,N(‐di(N ϵ‐lysino))imidazolium (glyoxal‐lysine dimer, GOLD) and N,N(‐di(N ϵ‐lysino))‐4‐methyl‐imidazolium (methylglyoxal‐lysine dimer, MOLD). We have detected and measured GOLD and MOLD in human serum by electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS), using 15N4‐GOLD and 15N4‐MOLD as internal standards. In this report we show that levels of GOLD and MOLD are significantly elevated (3–4‐fold, P<0.01) in sera of non‐diabetic uremic patients, compared to age‐matched controls, and represent a major class of non‐enzymatic, Maillard reaction crosslinks in plasma proteins. These results provide strong evidence for increased non‐enzymatic crosslinking of tissue proteins by GO and MGO in uremia, implicating oxidative stress and resultant advanced glycation and lipoxidation reactions in tissue damage in uremia.

Purification and characterization of perlecan fragment in urine of end-stage renal failure patients

We found a new spot on the two-dimensional electrophoresis pattern of the urine protein from hemodialysis patients. In order to identify the protein forming this new spot, the protein was purified by five steps of chromatography. It was shown that the amino acid sequence of this new protein from the N-terminal to the 20th amino acid was identical with the sequence from the 4197th to 4216th amino acid of perlecan, which is the core protein of the proteoglycan localizing in the systemic capillary basement membranes. It was also found that the molecular weight (25,000 daltons) of this new protein was comparable to the calculated molecular weight of the molecular region of the perlecan from the 4197th amino acid to the C-terminal. Lastly, it was shown that the antibodies against this new protein reacted with the perlecan produced by human fibroblasts. All these findings indicated that the new protein is a perlecan fragment.

Thy-1 antigen mediates apoptosis of rat glomerular cells in vitro and in vivo.

Injection of anti-Thy-1 antibody into a rat induces immediate glomerular cell death and subsequent development of glomerulonephritis. Whether the immediate cell death in this model is apoptotic has yet to be determined. Recent in vivo studies on thymocyte death have elucidated that the Thy-1 molecule can activate intracellular signaling for apoptosis. This observation prompted us to re-examine whether stimulation with anti-Thy-1 antibody can provoke apoptosis in the rat glomerulus. We found that anti-Thy-1 antibody could induce laddered DNA fragmentation of isolated glomeruli and mesangial cells in culture, definite biochemical evidence for random double-stranded breaks through apoptosis. Such DNA laddering was also demonstrated in the isolated glomeruli of rats that had been infused with anti-Thy-1 antibody several hours before. Furthermore, the terminal deoxynucleotidyl-transferase-mediated oligonucleotide nick end labeling technique stained a cell in the mesangium. Although apoptosis may be considered a candidate mechanism mediating resolution of hypercellularity in the anti-Thy-1 model, we propose that it is also involved in the immediate cell death in this model.

Mass spectrometric study on the protein chemical modification of uremic patients in advanced Maillard reaction

The Maillard reaction, initiated by the nonenzymatic reaction of reducing sugar with protein, is proposed to play a significant role in protein aging and the complications of aging and diabetes. In this study, we detected and quantified some advanced glycation endproducts (AGEs) in human serum proteins of control and uremic patients by a highly selective and specific assay, electrospray ionization liquid chromatography-mass spectrometry-mass spectrometry (ESI-LC-MS-MS). From our results, levels of each AGEs in serum of uremic patients were significantly elevated, compared to age-matched controls. These results provide the evidence for increased modifications of proteins by Maillard reaction in uremia.

Mechanism of dialysis-induced hypotension

Dialysis-induced hypotension, the sharp decrease in blood pressure occurring during hemodialysis, remains one of the most difficult problems associated with hemodialysis even today. However, there is yet no established theory to explain the mechanism triggering dialysis-induced hypotension. This review attempts to offer a consistent and cohesive source of information on the hemodynamics during dialysis-induced hypotension, and then analyzes etiologic factors in such hypotension reported by various investigators. Finally, three hypotheses concerning the mechanism of dialysis-induced hypotension including our own are introduced.

Push/Pull Hemodiafiltration

Push/pull hemodiafiltration is characterized by alternate filtration and backfiltration, while sterile pyrogen-free dialysate is flowing through a hemodiafilter. During the filtration phase, uremic substances are eliminated not only by diffusive, but also by convective transport. During the backfiltration phase, dialysate is quickly pushed to the blood side (i.e. backfiltration) so as to make up for the excessive reduction in body fluid that has developed during the immediately preceding filtration phase. In the most recently improved version of push/pull hemodiafiltration, the body fluid replacement volume is over 120 liters during a 4- hour treatment. This replacement of a large amount of body fluid may be due to the increased filtration rate in the hemodiafilter resulting from failure of the complete formation of a protein gel layer on the blood side surface. The filtration time in push/pull hemodiafiltration is so short that the also short backfiltration to follow may take over before the protein gel layer is completely formed on the membrane surface. Since the filtration and backfiltration times are much shorter in push/pull hemodiafiltration than the time for blood to pass through the hemodiafilter, it is concentrated and diluted many times (approx. 25 times) before it leaves the hemodiafilter. Therefore, push/pull hemodiafiltration is functionally similar to a predilution hemodiafiltration. The reduction rate of beta-microglobulin was greater by push/pull hemodiafiltration than by hemodialysis, when a high-flux polysulfone hemodiafilter was employed. However, the difference in the reduction rate was rather small between them, because of the improved hemodiafilters, which remove so much beta2-microglobulin only by dialysis. Nevertheless, restless legs syndrome, irritability, insomnia and pruritus were alleviated after switching the treatment modality from hemodialysis to push/pull hemodiafiltration. This may indicate that these symptoms are caused by the accumulation of uremic substances larger than beta2-microglobulin.

Health-related quality of life among dialysis patients in Seattle and Aichi

We used the 36-item Short-Form Health Survey to compare health-related quality of life (HRQOL) between 104 dialysis patients in Seattle, WA, and 2,178 patients in Aichi, JAPAN: Compared with Aichi patients, Seattle patients had lower scores on three scales related to physical HRQOL: Physical Functioning (PF; P = 0.03), Role-Physical (RP; P = 0.004), and Vitality (VT; P < 0.001). However, scores related to mental HRQOL were higher for Seattle patients compared with those of Aichi patients, which included scores for Role-Emotional (RE; P = 0.005) and Mental Health (MH; P < 0.001). Scores for Bodily Pain, General Health Perception, and Social Functioning did not differ significantly between the two groups. These differences persisted even after potential confounding factors were controlled for. However, after taking into account national norm data for the United States and Japan, differences in PF and VT disappeared, whereas differences in RP, RE, and MH persisted. These results suggest that the higher scores for PF and VT in Aichi patients were partly explained by the higher physical HRQOL of the Japanese general population. Although these data may not be representative of the total dialysis populations in the United States and Japan, they suggest potential differences in HRQOL between patients in the two countries. Additional research is needed to confirm these results and understand the factors associated with these differences. The findings suggest the need for further attention to the physical limitations of US dialysis patients and the mental health of Japanese dialysis patients.

Push/Pull Hemodiafiltration: Technical Aspects and Clinical Effectiveness

Push/pull hemodiafiltration (HDF) is characterized by alternate repetition of filtration and backfiltration during hemodialysis with high-flux membrane. In the pressure-controlled push/pull (PC P/P) HDF system, which is the newest push/pull HDF system, there are about 25 repetitions of dilution and concentration of the blood while it passes through the hemodiafilter. Hence, the PC P/P is functionally close to the predilution mode of on-line HDF. In the PC P/P, body fluid is replaced usually by more than 120 L of dialysate during the 4 h treatment. In selecting a hemodiafilter for PC P/P, one must be certain that the blood flow channels in the hemodiafilter do not collapse by the positive pressure on the dialysate side in the backfiltration phase. Thus, the polyacrylonitrile hollow-fiber hemodiafilter and polysulfon hollow-fiber hemodiafilter are suitable for PC P/P. In the short term, PC P/P has been reported to be effective against joint pain, itchiness, insomnia, irritability, and restless leg syndrome experienced by hemodialysis patients. Midterm clinical effectiveness of PC P/P includes the requisite lowering of the erythropoietin dose and improvement in skin pigmentation. The albumin loss per treatment with the PC P/P was significantly lower than that with the conventional HDF approach when a protein-permeable membrane is used. In terms of the removal rate of prolactin, no significant difference was found between PC P/P and conventional HDF. On the other hand, the removal rates of myoglobin and beta2M, where molecular size was smaller than prolactin, was significantly greater with the PC P/P than with conventional HDF.

Platelet GPIIb/IIIa Is Activated and Platelet-Leukocyte Coaggregates Formed in vivo during Hemodialysis

Background/Aim: During hemodialysis, platelets and leukocytes are activated and form platelet-leukocyte coaggregates in which GPIIb/IIIa (CD41/CD61) and CD62P (P-selectin) are involved. However, it is still controversial whether platelet activation and platelet-leukocyte coaggregate formation are dependent on the dialyzer membrane material. Method: We examined the appearance of activation-dependent antibody on platelets as an index of platelet activation, and the appearance of platelet-specific antigen on leukocytes as an index of platelet-leukocyte coaggregation, during hemodialysis in 7 patients treated using regenerated cellulose (RC) membrane and next using polysulfone (PS) membrane. In order to reduce the influence of factors other than dialyzer membrane material, this study was conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate. Moreover, flow cytometric techniques with whole blood were employed, which reduce artificial cell activation during the cell or plasma separation procedure. The platelet-specific monoclonal antibodies used in this study were anti-CD61, PAC-1 (which recognizes only the conformationally activated GPIIb/IIIa) and anti-CD62P. Results: Changes in the percentage of PAC-1-positive platelets were significantly greater during hemodialysis with RC than with PS. However, changes in the percentage of CD62P-positive platelets were not significantly different between hemodialysis with RC and PS. Changes in the percentage of CD61- or CD62P-positive leukocytes were significantly greater during hemodialysis with RC than with PS. Although changes in percentage of PAC-1-positive platelets did not parallel those of CD62P-positive platelets during hemodialysis, there was a significant positive correlation between the percentage of CD61-positive leukocytes and the percentage of CD62P-positive leukocytes. Conclusion: This study, conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate in order to reduce the influence of factors other than the dialyzer membrane material, demonstrated that both the degrees of GPIIb/IIIa activation and platelet-leukocyte coaggregation were greater during hemodialysis with RC than PS.