Materials and Methods
Source of Data and Study Population
We used the data of the nationwide, biopsy-based cohort study of DKD, provided by the Ministry of Health, Labour and Welfare and the Japan Agency for Medical Research and Development. The rationale and study designs are available elsewhere.[15,16] Briefly, the source population for this study included 895 patients with Type 2 diabetes aged 30–82 years who underwent clinical kidney biopsy at 18 main hospitals across Japan between 1985 and 2016. The study population had a pathological diagnosis with DKD as the only kidney disease diagnosis. The majority of study population was under the care of each hospital or its satellite clinics every 3 months and was followed up from the date of biopsy until the earliest date of: (i) CKD progression [defined as initiation of hemodialysis, peritoneal dialysis, kidney transplantation, death from uremia, a doubling of serum creatinine or a decrease of estimated glomerular filtration rate (eGFR) by ≥50%]; (ii) all-cause death; or (iii) censoring (censoring for loss to follow-up or administrative censoring occurring on the end of December 2019).
Since our primary focus was to evaluate the association of serum Hb concentration with tubulointerstitial lesions in kidney biopsy specimens and the risk for progression of CKD, in the early stages of CKD, we included patients who had an eGFR ≥60 mL/min/1.73 m2 at kidney biopsy. The nationwide, biopsy-based cohort study of DKD was approved by the institutional review boards of each hospital and conducted in accordance with the declaration of Helsinki.
Measurements and Definitions
Clinical characteristics, medication and laboratory data were gathered from the medical records at the time of kidney biopsy. The obtained data included age, gender, body mass index (BMI), known duration of diabetes, Hb A1c, eGFR (computed using the Modification of Diet in Renal Disease study equation for Japanese), urine albumin-to-creatinine ratio (UACR), systolic blood pressure (BP), diastolic BP, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, medication usage including renin–angiotensin system (RAS) blocker, glucose-lowering agent (oral hypoglycemics and/or insulin) and statin, medical history of CVD and history of smoking. Type 2 diabetes was defined as having diabetes onset after the age of 30 years. Diabetes duration was defined as the time from the date of diabetes onset to the date of kidney biopsy.
Pathological findings were evaluated according to the Renal Pathology Society Diabetic Nephropathy Classification. In brief, glomerular lesions were classified as follows: (i) Class I was glomerular basement thickening and only mild, non-specific changes on light microscopy; (ii) Class II was mild (IIa) or severe (IIb) mesangial expansion without either nodular lesions or global sclerosis in >50% of the glomeruli; (iii) Class III was nodular lesions without global sclerosis in >50% of the glomeruli; and (iv) Class IV was global sclerosis in >50% of the glomeruli. Pathological findings other than glomerulus were also evaluated as follows: interstitial lesions [interstitial fibrosis and tubular atrophy (Grades 0–3) and interstitial inflammation (Grades 0–2)] and vascular lesions [arteriolar hyalinosis (Grades 0–2) and large vessels arteriosclerosis (Grades 0–2)]. These procedures were conducted by three pathologists.
The outcome of interest was progression of DKD defined as a composite of new onset ESKD (dialysis, kidney transplantation or death from renal cause), a doubling of serum creatinine or a decrease of eGFR by ≥50%. The occurrence of DKD progression was ascertained by the medical records.
The study population was divided into four groups according to baseline (at the time of kidney biopsy) quartiles of serum Hb concentration: first quartile group, ≤12.0 g/dL; second quartile group, 12.1–13.3 g/dL; third quartile group, 13.4–14.5 g/dL; and fourth quartile group, ≥14.6 g/dL. Baseline clinical and pathological characteristics were quantified using median and interquartile range for continuous variables and percentages for categorical variables. Then, these characteristics were compared across serum Hb concentration quartiles, with P-values for trend calculated by a nonparametric test for trend across ordered groups developed by Cuzick. Spearman rank correlation was performed to examine intercorrelations between serum Hb concentration quartiles and pathological lesions. A Cox proportional hazards regression model that was stratified according to the quartiles of baseline serum Hb concentration and that was adjusted for the baseline covariates was used to estimate the hazard ratio (HR) and 95% confidence intervals (CIs) for the outcome. The fourth quartile group was designated as the reference quartile group to compare with other quartile groups. Global Chi-squared likelihood ratio, Akaike's information criterion (AIC) and Bayesian information criterion (BIC) were used to assess the incremental prognostic value of serum Hb concentration over known risk factors of DKD progression (age, gender, BMI, known duration of diabetes, systolic BP, Hb A1c, eGFR, UACR and RAS blocker use).
We conducted subgroup analyses to assess the robustness of our findings. First, we calculated the cumulative incidence rates of DKD progression by gender, across serum Hb concentration quartiles, because the gender difference in serum Hb concentration has been reported in healthy adults. Second, we calculated the cumulative incidence rates of DKD progression in patients with normo (UACR <30 mg/g Crea) and microalbuminuria (UACR 30–300 mg/g Crea), across serum Hb concentration quartiles.
For all descriptive analysis, we used the Stata version 15.1 (StataCorp, College Station, TX, USA). All statistical tests were two sided, and we considered P < 0.05 to be statistically significant.
Nephrol Dial Transplant. 2022;37(3):489-497. © 2022 Oxford University Press