This was a multi-site randomised controlled trial for prevention of diabetic nephropathy progression among 112 type 2 diabetic patients, who were aged 30 to 70 years and had overt nephropathy. The trial was conducted from 1 December 1997 to 30 April 2006. After a baseline run-in period (3 months), the patients were monitored for 5 years. The protocol was approved by the institutional review boards of each centre. All participating patients provided written, informed consent. Before the present study, 41 diabetic patients with overt nephropathy had been randomly assigned to normal protein intake (n
21) and low protein intake (n
20) groups. This 1 year feasibility trial was completed by 34 patients. Daily protein intake in the feasibility study was 1.22
0.25 g kg−1
(normal) and 0.92
0.43 g kg−1
(low protein) and the difference was statistically significant (p
0.05). Based on these data, sample size for the present study was calculated. To achieve 90% power with a 5% significance level, we found that least 31 participants per group would be necessary. To account for drop-out due to trial duration, a 100 participants (50 per group) were planned for analysis.
The participants were Japanese men and women, aged 30 to 70 years. All had type 2 diabetes (defined according to World Health Organization criteria) of at least 5 years duration and were being treated by diet or by diet plus oral hypoglycaemic agents or insulin injection. Other inclusion criteria were: (1) urinary protein excretion more than 1 g/day but less than 10 g/day; (2) urinary albumin excretion rate of more than 200 µg/min at least twice in a 1 year period; (3) serum creatinine below 176
µmol/l; (4) at least simple diabetic retinopathy; and (5) on normal-protein diet (1.2 g kg−1
). Potential participants were excluded if they had: type 1 diabetes; other renal diseases; body weight less than 80% of ideal body weight; clinically significant illness such as congestive heart failure, hepatic disease, recent myocardial infarction and stroke, and urinary tract infection; or if they were being treated with a low-protein diet (0.8 g kg−1
) and/or ACE inhibitors (ACE-I) or angiotensin II receptor blockers (ARBs). Hypertension was defined as blood pressure≥140/90 mmHg or use of anti-hypertensive drugs.
Randomisation and intervention
During the 3 month screening period, the participants continued to take a normal-protein diet (1.2 g kg−1
) and their usual medications. They were then randomly assigned at a central location to follow either a low-protein diet (0.8 g kg−1
) or a normal-protein diet (1.2 g kg−1
) with the appropriate energy intake for each participant without masking.The methods of minimisation for allocation were applied according to age, sex, serum creatinine, estimated GFR (eGFR), and urinary albumin and protein levels during the screening period. Both groups were instructed to meet the registered dietitian for 30 min every 3 months to assess and counsel dietary issues. After randomisation we followed the participants for approximately 3.5 years (1–5 years). Every 3 months, all participants completed a 3 day food record to assess daily protein, energy and sodium intake. For this purpose, we used the fourth revised and enlarged edition of Standard tables of food composition in Japan
]. The dietary protein intake was also assessed by urinary urea nitrogen excretion during 24 h urine collection every 3 months, using the formula of Maroni et al. [15
]. To achieve dietary protein goals, dietary regimens were modified every 3 months or more as needed. The estimated protein intake during the study represents the mean of all measurements after randomisation.
Blood and urine samples were brought to the central laboratory (SRL, Tokyo, Japan) and each clinical parameter was measured using the Hitachi 7170 analyzer (Hitachi High-Technologies, Tokyo, Japan) unless otherwise specified. GFR was estimated using the following modified MDRD formula for Japanese participants 
: eGFR (ml min−1
)=175×[serum creatinine (μmol/l)/88.4]−1.154
0.741×(0.742 if female), where serum creatinine estimated by an enzymatic method was calibrated. Creatinine clearance from a 24 h timed urine collection was calculated and corrected to a body surface area of 1.73 m2
. Urinary excretion of protein and albumin was measured every 3 months in 24 h timed urine samples using an immunoturbidity assay and a pyrogallol red–molybdate complex (LX60000; Eiken Chemical Co., Tokyo, Japan), respectively. Urinary nitrogen was measured by an enzymatic ultraviolet method every 3 months. Blood samples were obtained every 3 months to measure: renal function (blood urea nitrogen, creatinine, Na, K, Cl, uric acid) by an autoanalyser; lipids (total cholesterol, triacylglycerol, HDL-cholesterol) by an enzymatic colorimetric method and a direct inhibition method, respectively; transferrin by an immunoturbidity assay (BN-II; Dade Boehring, Marbug, Germany); serum glucose by a glucose oxidase method; and HbA1c
by ion exchange HPLC (ADAMS A1c HA-8160; Aarkray, Kyoto, Japan).
The primary outcomes were: (1) the annual change in eGFR and creatinine clearance; (2) the incidence of doubling of serum creatinine; and (3) the time to doubling of baseline serum creatinine. The secondary outcomes included the proportion of patients with ESRD requiring haemodialysis and the annual changes in urinary protein and albumin excretion. Quality of life was assessed annually using the SF-36 [17
].The secondary analysis, which was not based on a direct comparison of randomised groups, was performed to assess the biological dose–response relationship between actual protein intake and progression of type 2 diabetic nephropathy.
An independent data and safety monitoring board monitored the study. The Lan–DeMets alpha spending-function method was used to adjust for interim analyses once a year. Four formal interim analyses were performed during the study period. The p
value for one interim analyses was set at p
0.01. Data handling and trial management were coordinated centrally by EPS (Tokyo, Japan).The mean dietary protein intake between the low- and normal-protein diet groups was analysed using Wilcoxon’s rank sum test. Dietary protein intake in the low- and normal-protein diet groups during the study was analysed by repeated measures ANOVA.Analyses of the primary and secondary outcomes were performed according to the intention-to-treat principle; we included data from all randomised patients with the exception of the 24 participants lost or excluded between randomisation and study termination (Fig. ). For continuous variables, the mean and standard deviation were calculated. Because of the skewed deviation, values for albuminuria and proteinuria are given as medians and interquartile ranges. In calculating the slopes of the rates of change of eGFR and creatinine clearance, linear regression analysis was used and included the data of patients who reached an endpoint. A minimum of 1 year follow-up with at least two measurements of serum and urinary creatinine during the study period were aggregated in the slope analysis. Primary outcome values between groups were assessed by an analysis of covariance model, with low-protein diet as a factor and baseline urinary protein, serum creatinine, HbA1c
, systolic blood pressure and daily protein intake, in addition to age and sex, as covariates. The incidence of doubling of serum creatinine was compared with the χ2
test. The times to doubling of baseline serum creatinine and its components were compared by Kaplan–Meier survival curves and the log-rank test. Baseline serum creatinine was adjusted using Cox proportional hazards models with terms for the diet assignment. Secondary outcomes were compared with the χ2
test (for non-parametric data) or repeated measures ANOVA (for continuous data).
In secondary analysis, the differences between achieved protein intake and renal functions were determined using Pearson’s correlation coefficient and Spearman’s rank/correlation coefficient. To identify the factors associated with the doubling of serum creatinine, the potential risk factors such as systolic blood pressure, protein intake, sodium intake, HbA1c
and total cholesterol were included in the Cox proportional hazards model, adjusting for sex, age, urinary albumin excretion and serum creatinine.All statistical tests were two-sided. For the final analysis of the primary endpoints and all other endpoints, a p
value of 0.05 or less was considered to indicate significance. Data were analysed using SAS 8.2 (Statistical Analysis System, Cary, NC, USA).
Fig. 1 Design of the trial. Fifteen patients were lost during follow-up because they moved away or withdrew informed consent within 1 year of follow-up. Nine patients were excluded from analysis because they had less than two measurements of urinary (more ...)