Errata: Corrigendum Volume 18, Issue 4, 370, Article first published online: 13 August 2014
Dr Yoshiharu Tsubakihara, Department of Comprehensive Kidney Disease Research, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan. Email: firstname.lastname@example.org
We investigated the long-term effects of maintaining high hemoglobin (Hb) on renal function in patients with chronic kidney disease not on dialysis. Subjects (Hb < 10 g/dL and serum creatinine (Cr) 2–6 mg/dL) were randomized to either a high Hb group (N = 161, 11.0 ≤ Hb < 13.0 g/dL) receiving darbepoetin alfa or to a low Hb group (N = 160, 9.0 ≤ Hb < 11.0 g/dL) with epoetin alfa, stratified according to baseline Hb and serum Cr levels, comorbidity of diabetes, and study centers. Primary endpoints were composites of the following events: doubling of serum Cr, initiation of dialysis, renal transplantation, or death. Three-year cumulative renal survival rates (95% CI) were 39.9% (30.7–49.1%) and 32.4% (24.0–40.8%) in the high and low Hb groups, respectively (log-rank test; P = 0.111). A Cox proportional-hazards model adjusted by age, sex and the randomization factors showed a significantly lower event rate in the high Hb group (P = 0.035). The estimated hazard ratio (95% CI) for the high versus the low Hb group was 0.71 (0.52–0.98), the risk reduction was 29% in the high Hb group. Incidences of serious adverse cardiovascular events did not differ significantly between the high and low Hb groups (3.1% and 4.4%, respectively). No safety issues were noted in either group. Maintaining higher Hb levels with darbepoetin alfa better preserved renal function in patients with chronic kidney disease not on dialysis.
Treatment with erythropoiesis-stimulating agents (ESA) is clinically beneficial in patients with chronic kidney disease (CKD) not on dialysis. These patients are often anemic and ESA can eliminate the need for blood transfusion and thereby improve physical function and quality of life (QOL). Several clinical reports have also suggested that ESA therapy can even prevent the progression of renal failure. Preservation of renal function is reportedly achieved at Hb levels (11–13 g/dL) higher than the conventional target of approximately 10 g/dL for treatment with recombinant human erythropoietin (rHuEPO) in Japan (1). In contrast, recently reported large-scale overseas controlled studies (2–4). described no renal protective effects of ESA therapy being observed in a high Hb group (>13 g/dL) as compared with a low Hb group (10.5–11.5 g/dL). Furthermore, high target Hb levels may tend to increase the risk of cardiovascular events. Therefore, target Hb levels in CKD patients, receiving treatment for anemia, are still controversial. In Japan, to date, no large-scale randomized controlled trial (RCT) has been performed to investigate renal protective effects at target Hb levels in patients with CKD not on dialysis.
We conducted a 3-year RCT in Japanese CKD patients not on dialysis to test the hypothesis that long-term maintenance of target Hb levels (≥11.0 g/dL and <13.0 g/dL) with ESA may delay the progression of renal failure as compared with conventional anemia treatment targets (≥9.0 g/dL and <11.0 g/dL). The data collected during the first year of this study confirmed that there was no difference in safety between the low and high Hb groups and that the latter demonstrated a decrease in left ventricular mass index (LVMI) and an improvement in QOL scores, suggesting beneficial effects of higher Hb target levels aimed at improving cardiac function and QOL (5).
PATIENTS AND METHODS
We conducted this randomized, multicenter, open-label, parallel-group study according to the principles of the Declaration of Helsinki. Protocols were approved by the Institutional Review Board at each of the 79 participating medical institutions. All patients gave written informed consent. The study was conducted from November 2005 to February 2009.
Patients in the present study were enrolled for 4 months, from December 2005 to March 2006.
We enrolled 321 CKD patients not receiving dialysis. The patients had to be at least 20 years old and have a Hb level <10.0 g/dL at the start of the study. CKD was defined by a serum creatinine (Cr) level of 2–6 mg/dL. Key exclusion criteria included uncontrolled hypertension, congestive heart failure (above class III according to the New York Heart Association classification), malignancy, blood disease or active bleeding, and critical allergy.
Eligible patients were assigned to either of two groups by a computer program according to a minimization method: a high Hb group (11.0–13.0 g/dL) to receive darbepoetin alfa (DA; Kyowa Hakko Kirin, Tokyo, Japan) or a low Hb group (9.0–11.0 g/dL) to receive epoetin alfa (EA; Kyowa Hakko Kirin). Modulators for randomization were Hb level (<9.0 or ≥9.0 g/dL), Cr level (<4.0 mg/dL or ≥4.0 mg/dL), diabetes or non-diabetes and medical institution. The study duration was 144 weeks for both groups.
Iron supplements were administered to maintain transferrin saturation rates above 20% or serum ferritin levels above 100 ng/mL. Management targets for blood pressure were systolic <130 mm Hg and diastolic <80 mm Hg.
Evaluation of outcomes
The primary endpoints were composites of the time from baseline to the first occurrence of any of the following events: doubling of serum Cr levels, initiation of dialysis (maintenance dialysis), renal transplantation, or death.
The secondary endpoints were the time to the first occurrence of any of the above events defined as primary endpoints.
To evaluate safety and efficacy based on the maintenance of high target Hb levels, we also assessed adverse cardiovascular events.
On the basis of the results of previous studies, including those by Kuriyama et al. (1) and Gouva et al. (6), with a significance level of α = 0.05 and a statistical power of 80%, the number of subjects necessary to detect a statistically significant difference in the cumulative renal survival rate between the two groups during a 3-year follow-up was 150 for each group and 300 in total. Two interim safety and efficacy analyses were planned at week 48 and week 96. An O'Brien-Fleming alpha-spending function with truncated symmetric monitoring boundaries was used to allocate significance levels of 0.0060 and 0.0151 at the interim points, and a final significance level of 0.0472 for testing of the primary endpoint.
For the primary endpoint, the two groups were compared using the log-rank test and cumulative renal survival rates were estimated by the Kaplan–Meier method. Also, the hazard ratio between the two groups was estimated using the Cox regression model, which included covariates such as age, sex and certain allocation factors, that is, baseline serum Cr concentration, baseline Hb concentration, and diabetes. Similar analyses were planned for the secondary endpoints.
All subjects whose eligibility was confirmed, and who received at least one dose of the study drugs, were included in the analysis of efficacy and safety.
All analyses were performed using SAS V8.2 (SAS Institute, Cary, NC, USA).
Patients and baseline characteristics
In total, 322 patients meeting the eligibility criteria for this study were randomized into either the high Hb group (161 patients) or the low Hb group (161). Of these, one patient discontinued the study prior to starting the study treatment. Thus, 321 patients (161 in the high Hb group and 160 in the low Hb group) received one of the study medications.
There were no differences in baseline demographics or other baseline characteristics between the two groups (Table 1).
Urinary protein/creatinine ratio (g/g·Cr): No. patients (%)
ACEi and ARB
ACEi or ARB
Calcium channel blocker
Lipid-lowering agent (others)
Types and dosages of concomitant medications that patients were taking at baseline and that may have affected renal function remained unchanged during this study. There was no marked difference between the two groups in the frequencies of any concomitantly used drugs with possible effects on renal function at baseline, except for antiplatelet agents (Table 1).
Treatment of anemia
Figure 1 shows the time-course of mean Hb level changes in the high and low Hb groups during the 144-week duration of study drug treatment. In the high Hb group, the mean Hb levels (11.4 ± 1.4 g/dL) reached the target range within 10 weeks after the start of the study and then remained within this range through week 144. The mean Hb in the low Hb group was 9.18 ± 0.86 g/dL at baseline and maintained within the target range throughout the study. The mean weekly doses in the high and low Hb groups were DA 25–35 µg/week and EA 3500–4500 IU/week, respectively. Mean duration of the treatment (mean ± SD) was 87.5 ± 48.3 weeks and 80.4 ± 50.4 weeks for High- and Low Hb groups, respectively.
Blood pressure control
Mean systolic blood pressure (SBP) and diastolic blood pressure (DBP) over the 144-week course of the study did not differ between the high and low Hb groups; SBP remained at approximately 130 mm Hg and DBP at 70 mm Hg (Fig. 2).
Primary and secondary outcomes
The cumulative renal survival rates (95% CI) at week 144 calculated by the Kaplan–Meier method were 39.9% (30.7–49.1%) and 32.4% (24.0–40.8%) in the high and low Hb groups, respectively (log-rank test, P = 0.111; Fig. 3).
In the high and low Hb groups, 75 and 87 patients, respectively, experienced one or more of the above events defined as an endpoint by the protocol: doubling of serum Cr levels was observed in 52 and 57, dialysis was initiated in 20 and 29, and death occurred in three and one, respectively. No patient received renal transplantation during the study. The median time to the occurrence of these events was 123 and 96 weeks in the high and low Hb groups, respectively. The cumulative renal survival rates of the high Hb group exceeded those of the low Hb group at all time points throughout the study.
There were no significant differences between the groups in cumulative renal survival rates relative to the time from baseline to the first occurrence of doubling of serum Cr levels, initiation of dialysis, or death.
An intergroup comparison of renal survival rates using the Cox regression model incorporating covariates such as age, sex, and the randomization factors of baseline serum Cr levels, baseline Hb levels, the presence of diabetes as a comorbidity, showed the hazard ratio (95% CI) for the high Hb group versus the low Hb group to be 0.71 (0.52–0.98). The risk for developing the primary endpoint events was significantly lower (the risk reduction was 29%) in the high Hb than in the low Hb group (P = 0.035) (Fig. 4).
Exploratory data analysis
There were substantial numbers of patients (44 out of 161) who failed to reach the targeted Hb levels at more than half of the measurement time points during the study. Therefore, we next evaluated whether achieving the targeted Hb levels affected the outcomes. In the comparison of patients whose Hb levels were within the target range, renal survival rates were significantly better in the high Hb than in the low Hb group (P = 0.020) (Fig. 5A). No significant differences in baseline values among the subgroups were detected.
Similarly, a subgroup analysis was conducted on data from patients with a mean blood pressure (SBP/DBP) less than 130/80 mm Hg and those with 130/80 mm Hg or higher during the study. In the comparison of patients whose blood pressure was controlled below 130/80 mm Hg, renal survival rates also tended to be better in the high Hb group than in the low Hb group (P = 0.135) (Fig. 5B).
Furthermore, the exploratory analysis, including such factors as maintenance of target Hb levels, mean SBP/DBP during the study and the baseline urinary protein/creatinine ratio (<1.0 or ≥1.0 g/gCr) as covariates, in addition to the analysis using the Cox regression model, demonstrated the risk of developing the composite renal events to be significantly lower in the high Hb group than in the low Hb group (HR 0.66; 95% CI 0.47–0.93; P = 0.016) (Fig. 6).
Rates of changes in estimated glomerular filtration rate (eGFR) (95% CI) from the baseline to the final measurements at study end (or at discontinuation) were −4.71 ± 4.83 mL/min per 1.73 m2 (−5.46 to −3.96 g/m2) and −4.91 ± 4.42 mL/min per 1.73 m2 (−5.60 to −4.22 g/m2) in high- and low Hb group, respectively. Difference in the changes of mean eGFR between the two groups was 0.20 mL/min per 1.73 m2 (95% CI: −0.82 to 1.22 mL/min per 1.73 m2)
Adverse events were observed in 159 (98.8%) of the 161 patients in the high Hb group and 157 (98.1%) of the 160 patients in the low Hb group, with no statistically significant difference in incidence between the two groups.
We particularly examined the occurrence of cardiovascular events (defined in Table 2). Cardiovascular events occurred in 63 high Hb patients (39.1%) and 60 low Hb patients (37.5%) (difference not significant). The major event was hypertension or increased blood pressure.
Table 2. Cardiovascular events considered as adverse
Preferred term (MedDRA/J Version 8.1)
High Hb group (n = 161)
Number of patients (%)
Low Hb group (n = 160)
Blood pressure increased
Blood pressure decreased
Retinal vein occlusion
Arteriovenous fistula occlusion
Cardiac failure congestive
Cardiac failure chronic
Heart rate increased
Mitral valve incompetence
Also, incidences of serious adverse cardiovascular events did not differ markedly between the high and low Hb groups, i.e. five (3.1%) patients in the high Hb group and seven (4.4%) in the low Hb group. When these serious adverse cardiovascular events were grouped based on Hb levels measured during the study and according to the mean drug doses administered during the study, no apparent relationships were detected.
This study is the largest RCT focused on anemia correction with ESA conducted in Japan, to date. This 3-year follow-up study of CKD patients with anemia not on dialysis evaluated influences on renal function in patients maintained at target Hb levels between 11 g/dL and 13 g/dL versus the conventional target Hb levels in Japan of approximately 10 g/dL. The data collected during the first year of the study confirmed the safety of the study drug, as well as showing improved LVMI and QOL scores in the high Hb group (5). These promising results warrant further evaluation of the long-term effects of target Hb maintenance on renal function.
The Kaplan–Meier analysis revealed no difference between the high Hb group treated with DA and the low Hb group treated with EA in the primary composite endpoints of doubling of Cr levels, initiation of dialysis and death (Fig. 3). However, the intergroup comparison of renal survival rates using the Cox regression model with covariates of age, sex and the randomization factors of baseline serum Cr levels, Hb levels and the presence of diabetes as a comorbidity demonstrated that the risk of developing the endpoint events was significantly lower in the high Hb group than in the low Hb group (P = 0.035) (Fig. 4). Furthermore, the exploratory comparison of patients whose Hb levels were maintained within the target range at more than half of the measurement points during the study showed renal survival rates to be significantly better in the high Hb group (Fig. 5A).
Several epidemiological and observational studies have demonstrated anemia to be an independent risk factor for the progression of renal failure and/or mortality. A sub-analysis of the RENAAL study (7), an RCT evaluating the renal protective effect of angiotensin receptor antagonists in patients with renal failure caused by type II diabetes, demonstrated mild anemia with a Hb level of less than 13.8 g/dL to be an independent risk factor for the progression of renal failure. A retrospective analysis of population-based screening data in Okinawa Prefecture in Japan (8) also found anemia to be an independent risk factor for developing ESRD.
Kuriyama et al. (1) conducted a randomized controlled trial for 36 weeks comparing CKD patients with severe anemia (hematocrit (Ht) level of <30%) with and without rHuEPO treatment. The mean Cr level in these subjects was 2.9 mg/dL and the time to doubling of the Cr level was used as a primary endpoint. During the study, the mean Ht level in the non-treatment group decreased from 27.9% to 25.3%, while increasing from 27.0% to 32.1% in the treatment group, demonstrating that controlling anemia with ESA provided significant renal protection so long as patient blood pressure was also well controlled. Subsequently, Gouva et al. (6) evaluated renal survival rates by comparing patients who began rHuEPO treatment after Hb levels had decreased to less than 9.0 g/dL and those who immediately began rHuEPO treatment to increase Hb levels to 13 g/dL. In their study, renal protective effects were significantly better in the patients who immediately began treatment during a 2-year observation period.
Several mechanisms by which anemia exacerbates renal dysfunction in CKD patients have been suggested. Lieberthal et al. (9) implicated chronic ischemia associated with anemia in mediating renal tubular cell apoptosis and subsequent renal damage. More recently, Nangaku (10) also demonstrated evidence of chronic ischemia being a final common pathway of renal tubulointerstitial lesions using various animal experimental models, and suggested that anemia correction with ESA could contribute to renal protection.
On the other hand, three large RCTs investigated the renal protective effect of ESA. In the CREATE study (2), the secondary endpoint analyses showed no significant difference in the progression of renal failure between the group with Hb levels of 13–15 g/dL and those with Hb levels of 10.5–11.5 g/dL. The rate of initiating dialysis was actually significantly higher in the high Hb group. As an adverse reaction, SBP rose above 160 mm Hg in significantly more patients in the high Hb group (20% of patients in the low Hb group and 30% in the high Hb group). In our study, in contrast, the renal survival rate tended to be the highest in the subgroup of patients in the high Hb group in which blood pressure was controlled below 130/80 mm Hg (Fig. 5B). In the CREATE study, dialysis was started with the estimated glomerular filtration rate (eGFR) at approximately 12 mL/min per 1.73 m2, whereas, in our study, the mean eGFR at patients withdrawn in order to start dialysis was lower (5.16 mL/min per 1.73 m2 and mean Cr level of 8.83 mg/dL).
In the CHOIR study (3), comparison of the primary endpoint of a composite of death, myocardial infarction, hospitalization for congestive heart failure and stroke in groups with Hb levels of 13.5 g/dL and 11.3 g/dL showed the incidence to be higher in the high Hb group. Though renal survival rates were not assessed in the CHOIR study, rates for initiation of dialysis did not differ significantly between the two groups.
The Trial to Reduce Cardiovascular Events with Aranesp Therapy (TREAT) study reported in 2009 (4) was a placebo-controlled RCT involving 4038 CKD patients with diabetes not on dialysis, and anemia with a target Hb level of 13 g/dL using DA. The achieved mean Hb levels were 12.5 g/dL and 10.6 g/dL in the treatment and placebo groups, respectively. Incidences of the primary endpoints of ESRD and death did not differ between the groups. In the TREAT study, renal function at baseline was relatively preserved (serum Cr levels were 1.8–2.5 mg/dL, [median 1.8 mg/dL] and eGFR values were 27–42 mL/min/1.73 m2[median 34 mL/min/1.73 m2]). Approximately 65% of the study subjects had serious cardiovascular disorders, in marked contrast to the patients in our study. The TREAT study defined renal events as either death or ESRD. Renal events were observed in 338 and 314 patients in the treated and placebo groups, respectively. Though not specified, the reason for introduction for dialysis was most likely cardiac failure or over-hydration, given that baseline renal function was relatively preserved.
Data from these large RCTs performed in Europe and the USA may not be applicable to CKD patients in Japan due to differences in patient characteristics and reasons for initiating dialysis. The Japanese Society for Dialysis Therapy reported that maintenance dialysis was started at a mean eGFR of 5.4 mL/min per 1.73 m2 (Cr level of 8.3 mg/dL) (11). Nearly the same eGFR value (5.16 mL/min per 1.73 m2) was observed at dialysis introduction in this study. By contrast in the USA, the mean eGFR at the start of dialysis was 11.1 mL/min per 1.73 m2 according to the report by the United States Renal Data System. Therefore, in general, the primary reason for starting maintenance dialysis in Europe and the USA might be severe cardiac failure or over-hydration, rather than ESRD as in Japan. Because of these demographic differences, factors such as the rate of eGFR decline that would be less affected by subjective factors might be more appropriate as primary endpoints for assessing the protective effects of a therapeutic approach, compared to events such as initiation of dialysis or death that would be impacted by multiple, possibly subjective, factors.
The present study was randomized but not doubleblind. Therefore, subjective bias might have affected the results. Also, different ESAs were administered to patients in the two study groups, as maintaining Hb levels at around 12 g/dL with EA was not covered by public health insurance in Japan whereas the processes of DA development and approval were still underway, enabling us to use DA in the high Hb group. Since DA and EA share similar pharmacological profiles, the exception being the difference in halflife, their renal protective effects would be attributable solely to the Hb levels achieved.
The results of this 3-year follow-up study of Hb level maintenance in CKD patients with anemia not on dialysis suggest that ESA administration to achieve higher Hb levels, in combination with adequate blood pressure control, is beneficial for preservation of renal function.
This Randomized Controlled Study was sponsored by Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Registration of Clinical Trials: The Cochrane Renal Group registry: autoid 121, crg_id CRG030600049. The authors participated in this study as advisers and received consultation fees from Kyowa Hakko Kirin Co., Ltd.
[Correction added on 2 July 2012, after first online publication: Acknowledgement was added.]
We would like to express our deepest appreciation to the following members and investigators for their participation in this study.
Medical Advisor—Yusuke Tsukamoto (Shuwa General Hospital).
Clinical Pharmacology Advisor—Eiji Uchida (Showa University School of Medicine).
Investigators—Atsushi Wada (Asahikawa Red Cross Hospital), Izumi Yamaji (Teine Keijinkai Hospital), Noritomo Itami (Nikko Memorial Hospital), Jun Soma (Iwate Prefectural Central Hospital), Yoshio Taguma (Sendai Shakaihoken Hospital), Masahiko Ogihara (Ogihara Clinic), Hiroshi Hayakawa, and Masato Ikeda (Jikei University School of Medicine Aoto Hospital), Noriaki Shimada (Koto Hospital), Matsuhiko Suenaga (Tokyo Metropolitan Bokutoh Hospital), Satoshi Hijiya (Miyanomae Clinic), Sei Sasaki (Tokyo Medical and Dental University Hospital Faculty of Medicine), Yasuhiko Tomino (Juntendo University Hospital), Yasuhiko Iino (Nippon Medical School Hospital), Tatsuo Hosoya (Jikei University School of Medicine), Kouzou Kitazawa and Takanori Shibata (Showa University Hospital), Tomoko Gomi and Yuko Shibuya (Kanto Medical Center NTT EC), Kosaku Nitta (Tokyo Women's Medical University), Toshiyuki Nakao (Tokyo Medical University), Ken-ichi Oguchi (Ikegami General Hospital, Medical Corporation of Showakai), Ryoichi Ando (Japan Red Cross Musashino Hospital), Takahiro Mochizuki (Kameda Clinic), Yoshiyuki Toya (Yokohama City University Graduate School of Medicine), Eriko Kinugasa (Showa University Northern Yokohama Hospital), Akira Saito (Tokai University), Koju Kamata (Kitasato Institute Hospital), Yuichiro Hukudome (Yokosuka Kyosai Hospital), Kunihiro Yamagata (Tsukuba University Hospital), Yoshitaka Maeda (Toride Kyodo General Hospital), Hironobu Kawai (Saiseikai Maebashi Hospital), Yoshihisa Nojima (Gunma University Hospital), Shinichi Nishi (Niigata University Medical and Dental Hospital), Masashi Suzuki (Shinrakuen Hospital), Shin-ichi Tokunaga, Tadashi Ideura and Toru Ichikawa (Nagano Red Cross Hospital), Yukio Yuzawa (Nagoya University Graduate School of Medicine), Kunio Morozumi (Nagoya Daini Red Cross Hospital), Chikao Yamazaki and Akira Ito (Masuko Memorial Hospital), Hiroshi Ogawa (Shinseikai Dai-ichi Hospital), Tomohiko Naruse (Kasugai Municipal Hospital), Hirokazu Imai (Aichi Medical University School of Medicine), Satoshi Sugiyama (Fujita Health University Hospital), Daijo Inaguma (Tosei General Hospital), Satoshi Suzuki (Kainan Hospital Aichi Prefectural Welfare Federation of Agricultural Cooperatives), Hiroshige Ohashi (Gifu Prefectural General Medical Center), Shinsuke Nomura (Mie University Hospital), Atsushi Fukatsu (Kyoto University Hospital), Akira Wada (National Hospital Organization Osaka National Hospital), Tatsuya Shoji (Osaka General Medical Center), Tamotsu Minakata and Masahiko Miyamoto (Osaka Red Cross Hospital), Atsushi Yamauchi (Osaka Rosai Hospital), Masayuki Tsuchihashi (Hoshigaoka Koseinenkin Hospital), Terumasa Hayashi (Izumisano Municipal Hospital, Rinku General Medical Center), Hiroyuki Iida (Toyama Prefectural Central Hospital), Hisao Mutoh (Kanazawa Social Insurance Hospital), Shigeo Yamamoto (Kansai Rosai Hospital), Takeshi Nakanishi (The Hospital of Hyogo College of Medicine), Akihiro Yoshimoto (Institute of Biomedical Research and Innovation Hospital), Yoshihiko Saito (Nara Medical University Hospital), Haruhisa Otani (Wakayama Kidney Disease Clinic), Hirofumi Makino (Okayama University Hospital), Masafumi Taki (Shigei Medical Research Hospital), Noriaki Yorioka (Hiroshima University Hospital), Shigeaki Hayashida (Tokuyama Central Hospital), Yasuhiko Oyabu (Yamaguchi-ken Saiseikai Shimonoseki General Hospital), Jun Minakuchi (Kawashima Hospital), Akira Numata (Takamatsu Red Cross Hospital), Atsumi Harada (Matsuyama Red Cross Hospital), Kenji Yuasa (Kochi Takasu Hospital), Akiyo Shiro-zu (Kokura Daiichi Hospital), Kazuhiko Tsuruya (Kyushu University Hospital), Hideki Hirakata (Fukuoka Red Cross Hospital), Kei Hori (Munakata Medical Association Hospital), Seiya Okuda (Kurume University School of Medicine), Harumichi Higashi (St. Mary's Hospital), Akira Furusu (Nagasaki University Hospital), Kimio Tomita (Kumamoto University Hospital), Shuichi Hisanaga (Koga General Hospital), Toru Ikeda (Ikeda Hospital), and Yoshiki Shiohira (Yuaikai Tomishiro Central Hospital).
[Correction added on 2 July 2012, after first online publication: Appendix was added.]