Taro Sugimoto

The Critical Role of Src Homology Domain 2–Containing Tyrosine Phosphatase-1 in Recombinant Human Erythropoietin Hyporesponsive Anemia in Chronic Hemodialysis Patients

The molecular mechanism of anemia that is hyporesponsive to recombinant human erythropoietin (rHuEPO) in hemodialysis patients without underlying causative factors has not been investigated fully in hematopoietic stem cell system. Circulating CD34+ cells (1 x 10(4)) were isolated from rHuEPO hyporesponsive hemodialysis patients (EPO-H; n = 9), patients who were responsive to rHuEPO (EPO-R; n = 9), and healthy control subjects (n = 9). The patients with known causes of EPO hyporesponsiveness were eliminated from the current study. The cells were cultured in STEM PRO 34 liquid medium, supplemented with rHuEPO, IL-3, stem cell factor, and granulocyte-macrophage colony stimulating factor for 7 d and then transferred to a semisolid methylcellulose culture medium for performing burst forming unit-erythroid (BFU-E) colony assay. Expression of src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was assessed with Western blot analysis. In EPO-H patients, SHP-1 antisense or scrambled S-oligos were included in the culture medium, and its effects were evaluated. The number of circulating CD34+ cells was not statistically different among the three groups, and their proliferation rates were similar for 7 d in culture. However, BFU-E colonies were significantly decreased in EPO-H patients compared with EPO-R and control groups. The mRNA and protein expression of SHP-1 and p-SHP-1 was significantly increased, whereas that of p-STAT5 was reduced in EPO-H patients. The inclusion of SHP-1 antisense S-oligo in culture suppressed SHP-1 protein expression associated with p-STAT5 upregulation, increase in p-STAT5-regulated genes, and partial recovery of BFU-E colonies. In EPO-H hemodialysis patients, the EPO signaling pathway is attenuated as a result of dephosphorylation of STAT5 via upregulation of SHP-1 phosphatase activity, and SHP-1 may be a novel target molecule to sensitize EPO action in these patients.

Plasma Orexin Concentrations in Patients on Hemodialysis

Background: Orexins A and B are neuropeptides that regulate feeding behavior and are localized exclusively in neurons within and around the lateral hypothalamic area. Intracerebroventricular injection of orexin A stimulates food consumption in rats. Plasma concentrations of orexins may reflect nutritional states and may have clinical significance in patients on hemodialysis. In this study, we investigated the relationship between plasma orexin concentrations and nutritional states in patients on hemodialysis. Method: We measured plasma orexin concentrations in patients on hemodialysis (HD group, n = 67), patients with IgA nephropathy (n = 10), patients with diabetes mellitus (n = 11) and healthy controls (n = 10). We examined the relationships between plasma orexin concentrations and nutritional indices. Results: Plasma orexin A concentrations were significantly higher in the HD group than in the control group and showed a significant correlation with serum creatinine. In all subjects, there was a positive correlation between the plasma orexin A concentration and the serum creatinine concentration, but there were no correlations between these concentrations in each group. In the HD group, plasma orexin A concentrations had a significant positive correlation with the serum albumin concentration and percent creatinine generation rate (%CGR). Multiple regression analysis demonstrated that %CGR was the only independent factor associated with plasma orexin A concentrations. Conclusion: Plasma orexin A concentrations are increased in patients on hemodialysis. It is possible that the kidney plays a major role in the clearance of orexins. The plasma orexin A concentration is significantly correlated with %CGR, and it may be able to be used as a clinical marker of the nutritional state in patients on hemodialysis.

Minimal Change Nephrotic Syndrome Developing during Postoperative Interferon-Beta Therapy for Malignant Melanoma

A 64-year-old man presented with proteinuria during postoperative interferon (IFN)-β therapy against malignant melanoma. Renal pathologic findings were consistent with minimal change nephrotic syndrome (MCNS) showing extensive foot process effacement of visceral glomerular epithelial cells (podocyte). Nephrotic range proteinuria gradually regressed after stoppage of local injection of IFN-β without glucocorticoid treatment. To our knowledge this is the first report that demonstrates histological abnormalities of the glomerulus associated with postoperative IFN-β therapy for the malignant melanoma.

Plasma Adrenomedullin Levels in Patients on Hemodialysis

Adrenomedullin (AM) is a hypotensive peptide that has recently been isolated from human pheochromocytoma. In this study, we measured plasma AM concentrations in 54 patients on hemodialysis (HD) and examined the clinical significance. We also evaluated the effects of high-flux and low-flux dialysis membranes on plasma AM levels. The average value of plasma AM at pre-HD (4.44 ± 0.16 fmol/ml) was significantly elevated compared with that in 44 healthy volunteers (1.31 ± 1.41 fmol/ml) (p < 0.0001). The plasma AM concentrations at pre-HD showed a negative correlation with age and mean blood pressure (MBP) at pre-HD. The plasma AM concentrations at post-HD showed a negative correlation with MBP at post-HD and a negative correlation with the reduction rate of AM. Multiple regression analysis showed that age and MBP were independent factors associated with plasma AM at pre-HD and that MBP and reduction rate of AM were independent factors associated with plasma AM at post-HD. We investigated the differences between high-flux dialyzers (PS-UW, PS-N and FB-F) and a low-flux dialyzer (AM-BC-F), and we found that high-flux dialyzers removed plasma AM more efficiently than a low-flux dialyzer did. In addition, in 3 patients on HD, plasma AM levels decreased significantly during isovolumic dialysis using a high-flux dialyzer, despite the fact that there were no significant changes in MBP and ANP. In conclusion, elevation in plasma AM level causes a fall in MBP in patients on HD, therefore, removal of AM by HD treatment using a high-flux dialyzer contributes to the stability of blood pressure during HD.

Relationships of Serum Levels of Insulinlike Growth Factors with Indices of Bone Metabolism and Nutritional Conditions in Hemodialysis Patients

Insulinlike growth factor (IGF) I and IGF-II are synthesized in osteoblasts and stimulate proliferation, differentiation, and matrix synthesis in these cells. There is some evidence that IGFs act on bone cells not only by paracrine but also by endocrine pathways, suggesting that circulating IGFs may be of importance for the regulation of bone metabolism. On the other hand, the serum IGF-I level is also thought to be a good indicator of the nutritional conditions in hemodialysis patients. The present study was performed to analyze the correlations of circulating levels of IGF-I, IGF-II, IGF-binding protein (IGFBP) 1 and IGFBP-3 with biochemical markers of bone metabolism and parameters of the urea kinetic model which reflect nutritional conditions in hemodialysis patients. We also examined the differences between these relationships in male and female patients on hemodialysis. Sixty-two hemodialysis patients, 36 men (male group) and 26 women (female group), were included in this study. We measured the serum levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3. The bone mineral content (BMC) of the radius was measured by dual-energy X-ray absorptiometry. We calculated Kt/V, protein catabolic rate, and percent creatinine generation rate (%CGR). We also examined the relationships between serum levels of IGFs and BMC and the parameters of the urea kinetic model. It was found that the serum levels of IGF-I in the hemodialysis patients were almost the same as those in the control group. However, the serum levels of IGF-II, IGFBP-1, and IGFBP-3 in the hemodialysis patients were significantly higher than those in the control group. In the male group, the serum IGF-I levels showed a significant correlation with both serum intact parathyroid hormone levels and BMC, but no significant correlations between these indices were found in the female group. The serum levels of both IGF-I and IGF-II showed significant correlations with %CGR in the male group, but not in the female group. Stepwise multiple regression analysis was performed to clarify the relationship between serum levels of IGFs and BMC or %CGR. It was found that age, hemodialysis duration, serum intact parathyroid hormone levels, and sex were independent factors associated with BMC. The %CGR was associated independently with serum levels of IGF-I, and IGF-II and with the presence of diabetes mellitus. In conclusion, it is thought that serum levels of IGF-I and IGF-II can be used as indices of nutritional conditions in hemodialysis patients. However, the serum IGF-I level cannot be used as a marker of bone metabolism in hemodialysis patients.

Plasma Neuropeptide Y Concentrations in Patients on Hemodialysis

Background: Neuropeptide Y (NPY) is a 36-amino-acid peptide that was originally isolated from the porcine brain. NPY, in contrast to leptin, is one of the most potent appetite stimulants. In some previous studies, NPY was found to be correlated with mean blood pressure (MBP) and fluid volume in patients on hemodialysis (HD), contributing to volume-induced hypertension. However, it is still unclear which NPY-sensitive neuronal pathways are responsible for the various changes seen in response to central NPY administration. In this study we analyzed the correlation of circulating levels of NPY with parameters of nutritional conditions, and we investigated the relationships between NPY concentrations and clinical markers of fluid volume in patients on HD. We also evaluated the effects of high-flux dialysis membranes on plasma NPY levels as compared with those of low-flux membrane. Methods: Plasma NPY concentrations in patients on regular HD were measured using commercially available radioimmunoassay (RIA) kits. We examined the relationship between plasma NPY concentration and other clinical indices in patients on HD. Results: Plasma NPY concentrations were inversely correlated with the serum urea nitrogen levels (r = –0.32) as well as protein catabolic rate (PCR) (r = –0.28). Plasma NPY was also correlated with the increase in body weight between HD sessions (r = 0.29). On the other hand, plasma NPY concentrations were not correlated with MBP, atrial natriuretic peptide (ANP), or adrenomedullin (AM). The reduction rate of plasma NPY with a high-flux dialysis membrane was significantly higher than that with a low-flux dialysis membrane. Conclusions: The secretion of NPY may be enhanced in a poor state of nourishment and stress induced by fluid volume overload in patients on HD, and plasma NPY is removed by a high-flux dialyzer.

A case of congenital generalized lipodystrophy with lipoatrophic diabetes developing anti-insulin antibodies

Human amylin or islet amyloid polypeptide (IAPP) is a 37 amino acid polypeptide which is produced in the pancreatic β -cells and may be diabetogenic both through inhibition of insulin secretion and through formation of the major component of the islet amyloid associated with Type 2 diabetes [1–3]. However, the peptide may also be anti-diabetogenic, since it inhibits gastric emptying [4]. To evaluate the possible antidiabetogenic action of amylin, the human amylin analogue, pramlintide, has been developed. In pramlintide, proline was introduced in positions 25, 28 and 29 in the molecule to provide a therapeutic molecule substantially better than human amylin. Pramlintide reduces post-prandial glycaemia as shown in both short-term studies and in studies up to 4 weeks in subjects with Type 1 diabetes [5–10]. The actions of pramlintide (delayed gastric emptying accompanied by reduced circulating glucagon) resemble the effects of glucagon-like peptide (GLP1), the gut incretin hormone which exerts an anti-diabetogenic effect and which currently is explored for treatment of patients with Type 2 diabetes [11–15]. To examine whether the antidiabetogenic effect of pramlintide depends on a release of GLP-1, we explored the effect of pramlintide over a 4-week period in patients with Type 1 diabetes. Basal and meal-induced GLP-1 concentrations were studied. Nine patients with Type 1 diabetes (three males, six females; age 35.4 ± 2.3 years (mean ± SD ); diabetes duration 13.7 ± 2.5 years; HbA 1c 7.2 ± 0.3%; daily insulin dose 53.3 ± 3.2 U; no clinically diagnosed secondary complications) were studied over 4 weeks. The study protocol was approved by the ethics committee of the Karolinska Institute, Stockholm. Written informed consent was obtained from all participants. The subjects were treated for 4 weeks with four daily subcutaneous injections of 30 μ g pramlintide (Amylin Corp., San Diego, CA, USA). During the entire study period, including during the test day, the patients remained on their usual insulin treatment. A mixed meal (500 kcal, 28% protein, 22% fat and 50% carbohydrates) was served to the overnight fasted subjects before the start of pramlintide treatment and after 4 weeks. Before and at regular time points after meal ingestion, blood samples were taken for analysis of glucose (glucose oxidase), GLP-1 (radioimmunoassay analysing amidated C-terminus of GLP-1, i.e. GLP-1 of intestinal origin; [16]), glucagon (radioimmunoassay; Linco Res., St Charles, MO, USA) and pramlintide (double antibody enzymatic immunoassay; [17]). Comparisons of differences between the groups were performed using two-way analysis of variance for multiple comparisons. Baseline glucose, glucagon or GLP-1 did not change during the treatment. However, pramlintide reduced the post-prandial increase in glucose and glucagon levels (Fig. 1). The 240-min post-meal AUC glucose (suprabasal area under the curve, calculated by the trapezoid rule) was reduced from 364.2 ± 276.6 mmol/ l × 240 min to –486.5 ± 235.9 mmol/ l × 240 min by pramlintide ( P = 0.013) and the AUC glucagon over the first 90-min postmeal study period was reduced from 825 ± 201 ng/ l × 90 min to –94 ± 146 ng/ l × 90 min by pramlintide ( P < 0.001). In contrast, the post-prandial GLP-1 was not altered by pramlintide (Fig. 1c); the peak values were 20.7 ± 2.0 pmol/ l before treatment and 19.8 ± 2.5 pmol/ l after treatment and the suprabasal AUC GLP-1 was 1.57 ± 0.03 before treatment vs. 1.67 ± 0.03 nmol/ l × 240 min after 4 weeks of treatment. However, the time point at which the peak was seen was delayed after treatment with pramlintide. Thus, before treatment, GLP-1 levels peaked at 93 ± 12 min, whereas the GLP-1 peak was observed at 206 ± 19 min ( P < 0.001) after 4 weeks of treatment with pramlintide. Figure 1d shows that following subcutaneous administration of pramlintide, its peak level was observed at 15 min. Thereafter, the elimination of pramlintide followed a first order kinetic, as evidenced by a linear relation between the logarithmic values of pramlintide concentrations vs. time after peak ( r = –0.79, P < 0.001). The calculated circulating half-life of pramlintide was 69.5 ± 9.5 min. The study thus confirms previous reports that subcutaneous administration of the amylin analogue, pramlintide, reduces post-prandial glucose excursions in subjects with Type 1 diabetes [5–10]. This pronounced and rapid action is probably executed by inhibiting gastric emptying, which is a well-known action of amylin [3–5], and through prevention of the mixed meal-induced increase in glucagon levels. In contrast, we found no evidence that the anti-diabetogenic action of pramlintide is mediated by release of GLP-1. We measured total GLP-1 immunoreactivity, i.e. intact hormone and its primary metabolite. This analytical approach is required for the study of L-cell secretion because of the intravascular degradation of GLP-1 [16]. There is still a possibility that pramlintide could alter the degradation of GLP-1 and its effect mediated by increased levels of active GLP-1. However, it is unlikely that pramlintide inhibits the activity of proteases. The anti-diabetogenic action of pramlintide is more likely due to its decelerating effect on gastric emptying, which would cause glucose absorption to be effected over a longer time period and which would inhibit glucagon secretion by delaying the absorption of glucagonotropic amino acids.