Low health-related quality of life is associated with all-cause mortality in patients with diabetes on haemodialysis: the Japan Dialysis Outcomes and Practice Pattern Study.

AIMS: Whether health-related quality of life (HRQoL) can be accurately predicted in patients with extremely low HRQoL as a result of diabetic complications is unclear. We investigated the impact of HRQoL on mortality risk in patients with diabetes on haemodialysis. METHODS: Data from the Dialysis Outcomes Practice Pattern Study (DOPPS) were analysed for randomly selected patients receiving haemodialysis in Japan. Information regarding the diagnosis of diabetes and clinical events during follow-up was abstracted from the medical records at baseline and HRQoL was assessed by a self-reported short form (SF)-36 questionnaire. The association between physical component score and mental component score in the SF-36 and mortality risk was analysed using a Cox proportional hazard model. RESULTS: Data from 527 patients with diabetes on haemodialysis were analysed. The mortality age-adjusted hazard ratio of having a physical component score greater than or equal to the median was 0.27 [95% confidence interval (CI) 0.08-0.96] and the multivariable-adjusted mortality hazard ratio of having an mental component score greater than or equal to the median was 1.21 (95% CI 0.44-3.35). CONCLUSIONS: The physical component score derived from the SF-36 is an independent risk factor for mortality in patients with diabetes on haemodialysis who generally had very low HRQoL scores. Baseline mental component score was not predictive of mortality. Patient self-reporting regarding the physical component of health status may aid in risk stratification and clinical decision making for patients with diabetes on haemodialysis.

Depressive symptoms predict the future risk of severe pruritus in haemodialysis patients: Japan Dialysis Outcomes and Practice Patterns Study.

BACKGROUND: Recent reports suggest a cross-sectional association between psychiatric distress and pruritus in patients on haemodialysis (HD). However, no study has examined the likelihood of developing severe pruritus in patients on HD with depressive symptoms. OBJECTIVES: To evaluate the relationship between baseline depressive symptoms and subsequent risk of developing severe pruritus. METHODS: A longitudinal study with a 0.5-2.5-year follow-up period was performed using 1799 patients on HD who had no/mild pruritus at baseline, based on the Japan Dialysis Outcomes and Practice Patterns Study (1996-2004), a cohort study composed of a representative sample of patients on HD. We assessed pruritus after the follow-up period using a self-reported questionnaire and depressive symptoms using scores from the five-item version of the Mental Health Inventory (MHI-5). RESULTS: The 1799 patients had a mean age of 56.9 years, 59.5% were men, and 23.6% presented depressive symptoms. Multivariable analysis revealed that patients with depressive symptoms had significantly higher odds of developing severe pruritus during the 0.5-2.5-year follow-up period [adjusted odds ratio (AOR) 1.57, 95% confidence interval 1.22-2.01, P < 0.001]. In addition, a significant linear trend was observed between baseline MHI-5 scores and risk of developing severe pruritus, with AORs for third, second and first MHI-5 score quartiles of 1.08, 1.51 and 1.95, respectively (P for trend < 0.0001). CONCLUSIONS: Our results suggest that depressive symptoms measured by MHI-5 may predict the future risk of developing severe pruritus in patients on HD.

Direct injection of calcitriol or its analog into hyperplastic parathyroid glands induces apoptosis of parathyroid cells.

Hyperplasia of the parathyroid gland (PTG) is associated not only with excessive secretion of parathyroid hormone (PTH) but also with changes in the parathyroid cell (PTC) characteristics (i.e. hyperproliferative activity, and low contents of vitamin D and calcium-sensing receptors). Control of PTG hyperplasia is most important in the management of secondary hyperparathyroidism, but the advanced stage of hyperplasia is considered irreversible. In the present study, dialysis patients with PTG hyperplasia underwent direct injection of calcitriol or maxacalcitol (OCT) into the PTG. Ultrasonography showed that this treatment had significantly reduced PTG volume and tissue analysis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and DNA electrophoresis indicated that cellular apoptosis had been induced. The mechanism of apoptosis was evaluated in detail in uremic rats fed a high-phosphate diet. OCT or its vehicle was directly injected into the rats' PTGs. In the PTGs treated by OCT, there was a significantly increased number of TUNEL-positive PTCs and DNA electrophoresis revealed the characteristic ladder pattern of DNA fragmentation, both findings indicative of apoptosis. There was also a significant upregulation of both vitamin D and Ca-sensing receptors in the PTCs and a clear shift of the Ca-PTH response curve to the left and downward. None of these findings was observed in the PTGs treated by vehicle. This novel treatment is successful in causing regression of PTG hyperplasia. Thus, it is expected to significantly reduce the PTH level and ameliorate the abnormal bone turnover and mineral metabolism.

Processing of a precursor of 72-kilodalton type IV collagenase/gelatinase A by a recombinant membrane-type 1 matrix metalloproteinase.

Membrane-type 1 matrix metalloproteinase that is associated with the proteolytic activation of progelatinase A was expressed as a recombinant fusion protein in Escherichia coli. The recombinant enzyme cleaved the propeptide sequence of gelatinase A in a sequence-specific manner. A mutant progelatinase A that has a substitution of Asn(66)-Leu to Ile-Val was not processed at all. The processing was blocked by tissue inhibitor of metalloproteinases-2 or BB-94 but not by tissue inhibitor of metalloproteinases-1. Thus, membrane-type 1 matrix metalloproteinase is a direct activator of progelatinase A without requiring additional proteases.

Bcl-3, a member of the I kappa B proteins, has distinct specificity towards the Rel family of proteins.

Expression of the bcl-3 gene is demonstrated to be elevated in some B-cell chronic lymphocytic leukemias with a chromosomal translocation, t(14;19)(q32;q13.1). Bcl-3 protein has seven tandem ankyrin repeats that are also found in I kappa B proteins, inhibitors of Rel/NF kappa B transcription factors. In this paper, we demonstrate that Bcl-3 is a member of I kappa B family of proteins with a novel specificity. Bcl-3 preferentially associates with the p50 of NF kappa B, and the nuclear localization signal of p50 is required for this association. Bcl-3 inhibits the DNA-binding activity of p50 homodimers but not that of p50-p65 heterodimers. Transient transfection experiments revealed that appropriate expression of Bcl-3 results in inhibition of the function of p50 homodimers but not that of p50-p65 heterodimers, whereas pp40 and I kappa B gamma inhibit the function of both p50 homodimers and p50-p65 heterodimers. These studies suggest that Bcl-3 could modulate the transcription in a way different from pp40 and I kappa B gamma.

HTLV-1 Rex protein accumulates unspliced RNA in the nucleus as well as in cytoplasm.

The Rex protein of human T-cell leukemia viruses (HTLV) is a trans-acting regulator inducing the expression of gag and env mRNA containing the introns. The rex gene can also induce expression of unspliced RNA of human immunodeficiency viruses (HIV). We have analyzed the level of spliced and unspliced RNAs in nucleus and cytoplasm to understand the mechanism by which the Rex protein modulates RNA processing. With the gag gene of HTLV-1, the unspliced RNA was accumulated by Rex protein in both nucleus and cytoplasm. However, the apparent effects on nuclear unspliced RNA depended on the reporter genes: with the env gene of HTLV-1 as well as that of HIV-1, Rex did not accumulate the unspliced RNA in nucleus, but did so only in cytoplasm. These results clearly indicate that Rex protein not only activates the nuclear export of unspliced RNA, but also modulates some steps of RNA processing before the splicing, probably through stabilization of the precursor RNA.

HTLV-1 rex and HIV-1 rev act through similar mechanisms to relieve suppression of unspliced RNA expression.

Human retroviruses, human T cell leukemia viruses (HTLV) and human immunodeficiency viruses (HIV), express two classes of mRNAs; fully spliced mRNA in the early phase and intron-containing mRNA in a later phase. The expressions of HTLV-1 rex and HIV rev by early mRNAs are essential for the later phase of expression of intron-containing gag and env mRNAs. Each two cis-acting sequences seem to be involved in these regulations: HTLV-1 rex depends on a splice donor (SD) and a responsible element (RXE) at the 3' end, whereas HIV rev depends on a specific repressive sequence (CRS) and a responsible element (RRE) in the intron, but does not require an SD. For analyses of these cis-acting sequences, we inserted an HIV element RRE into an HTLV-1 construct and tested the responses to HTLV-1 rex and HIV rev regulations. The results indicated that both rex and rev could regulate RNA expression of these chimeric constructs responding to an HIV RRE. A repressive element (CRS) was dispensable, and the intronic or exonic location of RRE was not important. These observations suggest that rex and rev could be functionally equivalent to induce cytoplasmic expression of unspliced RNA which expression is suppressed either by an SD or CRS depending on the construction.

Isolation of acein-2, a novel angiotensin-I-converting enzyme inhibitory peptide derived from a tryptic hydrolysate of human plasma.

We previously described a novel angiotensin-I-converting enzyme (ACE) inhibitory peptide, designated Acein-1, that was isolated from a tryptic hydrolysate of human plasma. We now report a second such inhibitory peptide, Acein-2 obtained from the same hydrolysate. The peptide was purified by gel filtration and cation exchange chromatography followed by reversed-phase gradient and isocratic high performance liquid chromatography. Acein-2 was found to be a tripeptide, Leu-Ile-Tyr, which is thought to correspond to f(518-520) of human alpha2-macroglobulin. The synthetic tripeptide showed a potent dose-dependent inhibition of ACE, with an IC(50) value of 0.82 micromol/l. Lineweaver-Burk plots suggested that Acein-2 as well as the previously described Acein-1 are non-competitive inhibitors.

Clinical effects of maxacalcitol on secondary hyperparathyroidism of uremic patients.

Maxacalcitol (22-oxacalcitriol [OCT]) is a newly developed vitamin D analogue in Japan. OCT has shown less calcemic action and a strong suppressive effect on parathyroid hormone (PTH) in uremic rats and dogs. In uremic patients with secondary hyperparathyroidism, OCT dose-dependently suppressed PTH secretion and increased serum calcium levels. However, more than 60% of patients achieved a greater than 30% decrease in intact PTH level from baseline with long-term OCT treatment up to 1 year without an unphysiological increase in mean serum calcium levels. Long-term treatment also brought about a reduction in bone metabolic markers, including bone alkaline phosphatase, tartrate-resistant acid phosphatase, and bone gra-protein. These results suggest that although careful attention should be paid to the onset of hypercalcemia and oversuppression of PTH, OCT is one of the effective tools for the treatment of secondary hyperparathyroidism.

Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization.

Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors.

Role of uremic and endothelial factors in the development of beta 2-microglobulin amyloidosis.

To clarify the importance of uremic and endothelial factors in the development of beta 2-microglobulin (beta 2m) amyloidosis, in vitro evaluations were carried out using cultured synoviocytes and synovial tissues. Synoviocytes of patients with chronic renal failure were cultured with media containing uremic or normal serum. In cultures with uremic serum, greater proliferation of synoviocytes and stronger expression of CD68, beta 2m, interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) on synoviocytes were observed, compared with those containing normal serum. Uremic serum stimulated greater production of IL-1 beta and TNF alpha than normal serum (P < 0.05 for IL-1 beta, P < 0.01 for TNF alpha). Addition of supernatant of endothelial cell culture to medium containing uremic serum significantly accelerated production. After a three month culture of uremic synovial tissue, embedded in 3% type 1 collagen gel with uremic serum, beta 2m-positive 10 nm fibrils were recognized around the synoviocytes. In culture with uremic serum plus supernatant of endothelial cell culture, positive Congo red staining was noted. These findings indicate that uremic serum plays a significant role in the development of synovial beta 2m amyloidosis and that endothelial cell-derived factors contribute to the formation of beta 2m amyloid.

Incidence and clinical characteristics of hypoparathyroidism in dialysis patients.

The incidence and clinical characteristics of hypoparathyroidism (Hypo) were evaluated in 8188 hemodialysis (HD) and 1207 CAPD patients treated in 65 hospitals or clinics in Japan. Hypo was defined by an intact parathyroid hormone (PTH) level below 160 pg/ml, which corresponded to the low normal limit of intact PTH to maintain a normal osteoblastic surface in 40 bone biopsy specimens of Japanese dialysis patients, and patients were classified into two groups: absolute Hypo (A-Hypo), intact PTH < 60 pg/ml, and relative Hypo (R-Hypo), 60 pg/ml < or = intact PTH < 160 pg/ml. A total of 2537 (31.0%) and 2736 (33.4%) HD patients were classified into A- and R- Hypo, and 401 (31.3%) and 379 (31.4%) CAPD patients occupied A- and R-Hypo, respectively. A high incidence of Hypo was observed in the HD patients with diabetes mellitus (DM) and old age (> or = 70 years old) compared with that of a nationwide epidemiological report for dialysis patients by Japanese Society for Dialysis Therapy. Hypo patients who were treated by CAPD had a background of being younger, having a shorter duration on dialysis, and were less frequently diagnosed with DM than in those Hypo patients on HD. Bone pain and metastatic calcification were observed in approximately 20% and 25% of Hypo patients, respectively. No difference was observed in the background factors and the prevalence of signs and symptoms between the A- and R-Hypo groups, regardless of the mode of treatment (HD or CAPD). These results suggest that a very high incidence and specific backgrounds (DM and aging) of Hypo exist in Japanese dialysis patients.

Determination of bufalin-like immunoreactivity in serum of humans and rats by time-resolved fluoroimmunoassay for using a monoclonal antibody.

The existence of a mammalian natriuretic substance or endogenous digitalis-like factor, which inhibits Na+,K+-ATPase and thereby regulates body fluid volume, has been speculated for a long time but has yet to be defined. We established in the present study a simple and highly sensitive procedure to measure bufalin, a constituent of toad venom preparation and a specific inhibitor of Na+,K+-ATPase by time-resolved fluoroimmunoassay (TR-FIA) and using a monoclonal antibody. The antibody was specific to bufalin and resembled bufadienolides but showed no cross-reactivity with digitoxin and ouabain. A bufalin-like immunoreactivity was detectable in serum of humans and rats by the proposed TR-FIA. The levels of bufalin-like immunoreactivity in serum of healthy volunteers were significantly correlated with their systolic blood pressure. Moreover, bufalin-like immunoreactivity in serum of Dahl-S rats increased in parallel with a period of high-salt diet. These results suggest that increased bufalin-like immunoreactivity may be associated with certain types of hypertension.

Development of guanine analyzer to measure activity of guanylate cyclase.

A previous analyzer of adenine compounds by high-performance liquid chromatography was converted for the determination of guanine, its nucleoside and nucleotides by a post-column fluorescence derivatization with phenylglyoxal (PGO) in place of bromoacetoaldehyde. The gel filtration column (Asahipak GS-320H) was used for separation by a mobile phase consisting of 25 mM sodium citrate buffered (pH 4.0)-150 mM NaCl solution and CH3CN (85:15, v/v) containing 15 mM PGO. The separated analytes reacted with flow through PGO in a reaction coil at 90 degrees C into fluorescent derivatives. Those derivatives were detected fluorimetrically, highly selective and quantitatively. The activity of soluble guanylate cyclase (sGC) in the neuroblastoma N1E-115 cell was measured by tracing the peak height of cGMP synthesized from substrate GTP using this guanine analyzer. The sensitivity of the present method was lower than the radioisotope method. However, our modified method was simpler, safer and quicker than the radioisotope method. Furthermore, this method could trace other guanine compounds simultaneously, allowing measurement of guanine metabolizing enzymatic activity. Therefore, it will be useful for screening of effectors on sGC.

Plasma bradykinin levels during hemodialysis with PAN DX and polysulfone membranes with and without concurrent ACE inhibitor.

The effects of dialyzer membrane material and concurrent angiotensin converting enzyme (ACE) inhibitor on plasma bradykinin levels during hemodialysis were investigated by administration of 3 successive hemodialyses using a PAN DX membrane dialyzer and another 3 using a polysulfone membrane dialyzer with the order of the 2 sequences randomized, for 6 patients receiving concurrent treatment with ACE inhibitor and 6 others receiving no ACE inhibitor. With the PAN DX membrane dialyzer the plasma bradykinin concentration obtained from the dialyzer outlet was significantly higher than that from the inlet at 10 min, but not at 5 min after initiation of dialysis, whereas no significant difference between inlet and outlet bradykinin concentrations was observed at either time with the polysulfone membrane dialyzer. No significant difference was observed between the changes in plasma bradykinin concentration in cases involving concurrent ACE inhibitor and that in cases receiving no ACE inhibitor. The results suggest that the PAN DX membrane dialyzer stimulates bradykinin production, but also that its release of bradykinin is delayed, possibly because of adsorption and modified release of bradykinin by the PAN DX membrane, and that ACE inhibitor may have no significant effect on the change in plasma bradykinin levels.

Effect of dialysis membranes and ACE inhibitor on bradykinin levels during hemodialysis.

The effects of dialyzer membrane material and concurrent angiotensin converting enzyme (ACE) inhibitor on plasma bradykinin levels during hemodialysis were investigated. Six patients treated with an ACE inhibitor and 6 other patients not receiving an ACE inhibitor underwent three consecutive hemodialysis sessions with an AN-69 dialyzer and three sessions with a polysulfone dialyzer. The sequence of dialyzers (AN-69 followed by polysulfone or the reverse) was determined randomly. With the AN-69 membrane dialyzer, the plasma bradykinin level at the dialyzer outlet was significantly greater than that at the dialyzer inlet at 5 min but not at 10 min after initiation of dialysis, whereas no significant difference between inlet and outlet bradykinin concentrations was observed at either time with the polysulfone membrane dialyzer. The changes in plasma bradykinin level in patients with concurrent ACE inhibitor did not differ from those found in patients without ACE inhibitor. These results indicate that the AN-69 membrane stimulates bradykinin production at the initial stage of hemodialysis in patients with as well as without concurrent ACE inhibitor. Further study is necessary to clarify the exact role of ACE inhibitor in elevation of bradykinin levels during hemodialysis.

Modulation of parathyroid cell function by calcium ion in health and uremia.

Extracellular calcium ion concentration is the major determinant of parathyroid hormone (PTH) secretion from parathyroid cells. In dialysis patients with secondary hyperparathyroidism, higher calcium concentration is needed to suppress PTH secretion as demonstrated by the PTH-calcium curve. Such abnormal sensitivity to extracellular calcium ion has been recently explained by the decrease in number of calcium-sensing receptors, especially on cells in nodular hyperplasia, which is the advanced type of parathyroid hyperplasia in uremia. Modulation of the sensitivity of parathyroid cells to calcium has become partly possible through the use of newly developed calcimimetics.

Chiral analysis of 3-methoxy-4-hydroxyphenylglycol in human urine.

Since 3-methoxy-4-hydroxyphenylglycol (MHPG) is a neutral metabolite from norepinephrine, it will be a diagnostic marker for mental diseases such as depression. For the development of an immunoassay, the natural enantiomer of MHPG would be required to prepare its antigen and to examine the specificity of the antibody. A natural enantiomer synthesized, however, has not been obtained so far. In this paper, we attempted to enantioseparate synthetic DL-MHPG and to assign D-enantiomer from the optical rotation of MHPG purified from human urine, because endogenous norepinephrine occurs as D-enantiomer which should metabolically generate D-MHPG. Enantioseparation conditions were tested using a Ceramospher Chiral RU-1 column (4.6 x 250 mm) at a flow rate of 0.5 ml/min. The resolution was adequate for the analysis and purification of synthetic DL- and the urinary MHPGs using methanol as a mobile phase and the column temperature at 0 degrees C, where DL-MHPG was detected as two peaks. The earlier peak (peak 1) showed (-) optical rotation, while the latter gave (+) optical rotation. After being treated with beta-glucuronidase, the normal human urine was extracted with ethyl acetate and then evaporated to dryness. The residue was suspended in water and the supernatant was analyzed and purified by a reversed phase column with a multi channel detector. A peak corresponding to MHPG was collected and concentrated to dryness. The pooled residues were dissolved in methanol and enantioseparated on the chiral HPLC. The urinary MHPG appeared as a single peak which was corresponded to the earlier peak of DL-MHPG and showing (-) optical rotation. Thus, the urinary MHPG was found to be D-(-)-MHPG. Then the absolute configuration of enantioseparated MHPGs were assigned to each optical rotation, judging from the chemical data and the metabolic pathway of the urinary D-MHPG. These enantiomers will be useful for studying on biochemistry and immunoassay.

Development of enzyme immunoassay of 2'-deoxycytidine.

In order to study 2'-deoxycytidine (2'-dCyd) as a possible prognostic marker in cancer chemotherapy, an enzyme immunoassay (EIA) was developed. 2'-dCyd as a hapten was succinylated and two omicron-monosuccinyl-2'-dCyd's were purified by high performance liquid chromatography and identified by mass spectrometry and 1H-NMR. Two antigens were prepared by conjugating two omicron-succinyl derivatives with keyhole limpet hemocyanin (KLH) as a carrier. Both antigen produced specific antibodies to 2'dCyd in BALD/c mice. The spleen cells of one mouse immunized with 5'-omicron-succinyl-2'-dCyd-KLH were hybridized with myeloma cells. One monoclone selected produced a specific antibody. A convenient EIA was attained by using the monoclonal antibody.

Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7).

A simple and convenient method for measuring the activity of a recombinant human matrix metalloproteinase 7 (MMP-7, matrilysin) was developed by flow injection analysis (FIA). For this method, purified recombinant MMP-7 zymogen expressed in E. coli and the substrate peptide (MOCAc-Pro-Leu-Gly-Leu-A2pr(DNP)-Ala-Arg-NH2) were used. Following the incubation of substrate peptide with activated r-proMMP-7, the resulting fluorescent product peptide (MOCAc-Pro-Leu-GLY) was monitored with a fluorescence detector (lambda ex 328 mm, lambda em 393 mm) without chromatographic separation. In this FIA system, the analysis time is 2 min and the standard curve is linear from 5 to 100 pmol of the product peptide injected. In order to use this FIA system as a method for screening inhibitors against MMP-7, the effects of CaCl2, EDTA and of the tissue inhibitor of metalloproteinase-1, and -2, were tested. A synthetic PRCGXPD-containing peptide (BS-10) was also observed to inhibit MMP-7 activity, with an IC50 value of 104 microM. Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by the proposed system. Furthermore, to confirm the utility of this FIA system as a screening method, the inhibitory activity of the MMP-related substance in Joro spider (Nephilia clavata) venom was measured by this method. This inhibitory activity was observed in an extract of a venom diluted 1000-fold. Thus, the FIA method is not only simple and quick, but also sensitive enough to screen and analyze the inhibitory properties of a large number of test compounds.