Combining glomerular basement membrane and tubular basement membrane assessment improves the prediction of diabetic end-stage renal disease

2.1 Patient selection and study design

Patients with diabetes who underwent renal biopsy at the West China Hospital of Sichuan University from January 2011 to October 2018 were retrospectively analyzed in this study. The indications for renal biopsy have been detailed described previously.¹² They were type 2 diabetes mellitus (T2DM) patients with renal damage who lacked absolute contraindications. Especially T2DM patients with obvious glomerular hematuria and/or short diabetic duration, or with sudden onset overt proteinuria met the requirement of renal biopsy.¹³ We used the American Diabetes Association criteria for T2DM diagnosis.¹⁴ DN, which was defined based on the 2015 standard of An et al,¹⁵ was diagnosed by at least two renal pathologists according to the Renal Pathology Society (RPS) classification.⁶ Adult patients with T2DM and biopsy-confirmed DN who were followed up at our hospital for over 1 year were considered to be eligible for this study. Patients who met the exclusion criteria were excluded from this study: coexisting nondiabetic renal diseases, including membranous nephropathy and immunoglobulin A nephropathy; systemic disease such as antineutrophil cytoplasmic antibody associated vasculitis, anti-GBM disease, and lupus nephritis; entered into ESRD before renal biopsy; no glomeruli observed in electron micrographs; RPS glomerular class IV or global sclerosis; or other types of DM (Figure 1). In sum, a total of 110 patients with T2DM and associated DN and complete TBM/GBM data were enrolled in this study. Ten nondiabetic age-matched patients with minimal change disease (MCD) were retrospectively selected as controls for comparison. All patients provided informed consent and the institutional review boards of Sichuan University approved this study. This study was also in compliance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

2.2 Clinical and laboratory information

Clinical and pathologic data were abstracted from electronic medical records at the time of renal biopsy; data included age, sex, body mass index, systolic or diastolic blood pressure, presence of diabetic retinopathy, and use of renin-angiotensin aldosterone system (RAAS) blockade. We evaluated the eGFR using the creatinine-based Chronic Kidney Disease Epidemiology Collaboration equation.¹² We defined hematuria as more than five erythrocytes per high-power field in at least two of three consecutive urine tests without urinary infection and no urinary tract malignancy or stone.¹⁶ Treatment was defined as the use of the RAAS blockade, glucose-lowering agents, and statins for more than half of the follow-up period. Patient follow-up examinations were performed 2-4 times per year based on the patient's individual condition.

2.3 Pathological classification

Renal biopsy tissues for light microscopy, immunofluorescence, and electron microscopy were prepared by standard procedures at West China Hospital and examined by expert nephropathologists. Sections for light microscopy were stained with hematoxylin-eosin, periodic acid-Schiff, Masson's trichrome, and periodic acid-Schiff silver methenamine. Light microscopy sections were assessed by two nephropathologists (L.L., H.X.) without the knowledge of clinical outcomes. Renal biopsy evaluation was scored according to the RPS DN classification.⁶

2.4 Measurement of GBM and TBM thickness

GBM and TBM thickness was determined by unbiased morphometry on electron microscope images.⁷ When determining GBM thickness, globally sclerotic glomeruli or collapsed glomeruli were excluded. One to two glomeruli of every kidney specimen were randomly selected and GBM thickness was evaluated by the Haas' direct measurement/arithmetic mean method, which was described in detail in our previous study.⁷

The TBM of the proximal tubules was selected for study because of the relative ease of identification of this structure, thus providing consistency of comparison within and between groups.¹⁷ Proximal tubular basement membrane thickness was determined from the orthogonal intercept, which was described previously.^{17, 18} In summary, electron micrographs with print magnifications of 7000-15 000 were obtained to measure the thickness of the proximal segment of the proximal tubule. A calibration grid was photographed at the same time to determine the precise magnification. Subsequently, a grid with perpendicular line segments was superimposed over each micrograph. We used a harmonic ruler on a log reciprocal scale to determine the orthogonal thickness of the TBM when a grid line intersected the TBM and the proximal tubular epithelial cell interface. To examine the entire section for the presence of a proximal TBM, the microscope stage controls were moved systematically by an equal distance. For each patient, 60-100 tubular profiles were finally assessed.

2.5 Renal outcomes

ESRD was the end point. ESRD was defined as an eGFR rate < 15 mL/min/1.73 m², kidney transplantation, or the need for chronic renal replacement therapy.^{15, 19} All the patients were followed until April 2020.

2.6 Statistical analysis

Clinical and pathologic data and outcomes were expressed as counts and percentages for categorical variables. Continuous variables are expressed as the mean and SD when normally distributed, or as the median and interquartile range (IQR) when not. In patients with differing TBM and GBM thickness, differences in continuous variables were analyzed using one-way analysis of variance, followed by the Bonferroni or Tukey methods for multiple comparisons, or the Kruskal-Wallis H test, as appropriate. Categorical variables were analyzed using the chi-square test or Fisher's exact test.¹²

Survival curves for TBM and/or GBM thicknesses were obtained using the Kaplan-Meier method. Log-rank tests was used to determine if there was a statistically significant difference for renal survival rate among comparison groups. Data for hemoglobin A1c (HbA1c) were lacking for four patients (these four patients were included in multivariable analyses). First, the differences in clinical parameters between patients with or without missing values were examined to check whether the values were missed randomly. Then, multiple imputation methods were applied for multivariable models. The proportional hazard assumption in Cox models was tested to determine whether the dataset satisfied the basic assumptions of Cox analyses. The Cox proportional hazards model was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for ESRD. GBM or TBM thickness was first analyzed as a continuous variable with HRs stemmed from per SD increment of natural log-transformed and then analyzed as a categorical variable with the GBM^loTBM^lo group regarded as reference. In the multivariable Cox proportional hazard models, each HR was adjusted for age, sex, eGFR, proteinuria, HbA1c, and pathological parameters (including RPS glomerular classification, IFTA, interstitial inflammation, arteriosclerosis, and arteriolar hyalinosis) at the time of renal biopsy. Age and sex were chosen on the basis of biological plausibility. HbA1c was selected to represent previous glycemic control and it was suggested in the literature to be associated with a higher risk of renal outcome.²⁰ The pathological covariates evaluated by light microscopy were considered as potential confounders because they were associated with ESRD in previous studies.^{16, 21} Parameters with P < 0.05 in the multivariable Cox proportional hazard models were considered significant prognosis predictors. Correlations between clinical parameters and GBM or TBM thickness were evaluated using linear regression analyses.

All statistical analyses were performed using Stata version 14.0 (StataCorp LLC, College Station, TX, USA). Statistical tests were considered significant at P < 0.05.

3.1 Baseline demographics and clinical characteristics

A summary of the demographics and baseline characteristics of the 110 patients with DN enrolled in the study is presented in Table 1. The mean age of the participants was 51 years (SD, 11 years), and the cohort comprised 30 women (27.3%) and 80 men (72.7%). The median duration of T2DM was 60 months (IQR, 24-120 months), the median baseline eGFR was 91.9 mL/min/1.73 m², and the median 24-hour proteinuria was 1.80 g/d.

The median GBM and TBM thickness in the patients with DN of 681 nm (Figure 2) and 1200 nm, respectively, were significantly increased compared with that in the nondiabetic controls with MCD of 380 and 574 nm, respectively. According to the median membrane thickness, 54 patients had a GBM thickness < 681 nm, and 56 patients had a GBM thickness ≥ 681 nm. In total, 53 patients had a TBM thickness < 1200 nm, and 57 patients had a TBM thickness ≥ 1200 nm. Consequently, the patients were assigned to three groups: GBM^loTBM^lo group (GBM <681 nm and TBM <1200 nm, n = 38), GBM^hiTBM^lo/GBM^loTBM^hi group (GBM ≥681 nm and TBM <1200 nm, or GBM <681 nm and TBM ≥1200 nm, n = 40), and GBM^hiTBM^hi group (GBM ≥681 nm and TBM ≥1200 nm, n = 32). Compared with the patients in the GBM^loTBM^lo group, those in the GBM^hiTBM^lo/GBM^loTBM^hi and GBM^hiTBM^hi groups had more severe proteinuria, albumin/creatinine ratio (ACR), and greater serum creatinine but lower hemoglobin, serum albumin, and eGFR (P < 0.05). Patients in the GBM^hiTBM^hi group had the lowest hemoglobin and serum albumin concentrations but the greatest proteinuria among the three groups. There were no significant differences in blood pressure or the prevalence of diabetic retinopathy among the three groups. The usage of hypoglycemic agents, RAAS inhibitors, or statins did not significantly differ among the three groups.

3.2 Baseline pathological characteristics

Regarding pathological parameters, the severity of the RPS glomerular class, IFTA, and interstitial inflammation were increased in patients in the GBM^hiTBM^hi group compared with those in the GBM^loTBM^lo and GBM^hiTBM^lo/GBM^loTBM^hi groups (P < 0.01 and 0.04, respectively). Conversely, there were no significant differences in the degree of arteriolar hyalinosis or arteriosclerosis among the three groups (Table 2).

3.3 Correlations between GBM/TBM thickness and clinicopathological findings

Liner regression analysis revealed that TBM thickness was negatively associated with the eGFR (R² = 0.15, standard β = −0.38, P < 0.001), serum albumin concentration (R² = 0.10, standard β = −0.30, P < 0.01), and hemoglobin concentration (R² = 0.13, standard β = −0.34, P < 0.001) but was positively associated with proteinuria (R² = 0.10, standard β = 0.30, P < 0.01, Figure 3A–D), and ACR (R² = 0.12, standard β = 0.34, P < 0.01). Similarly, GBM thickness was negatively associated with the eGFR (R² = 0.07, standard β = −0.26, P < 0.01) but positively associated with proteinuria (R² = 0.07, standard β = 0.24, P = 0.02). However, the duration of diabetes was not significantly associated with TBM or GBM thickness.

TBM thickness was significantly positively correlated with GBM thickness (R² = 0.20, standard β = 0.45, P < 0.001, Figure 3E). TBM thickness also displayed a positive correlation with glomerular classification (R² = 0.13, standard β = 0.36, P < 0.01) and the IFTA score (R² = 0.06, standard β = 0.23, P = 0.01). Furthermore, GBM thickness was positively associated with the RPS glomerular classification, IFTA, and interstitial inflammation. However, arteriosclerosis and arteriolar hyalinosis were not significantly associated with TBM or GBM thickness.

3.4 Effect of combining TBM thickness with GBM thickness assessment for the prognosis of renal outcomes

During a median follow-up of 27 months (range 6-94 months), 23 (21.9%) patients progressed to ESRD. Five patients were lost to follow-up and only had baseline clinical and pathologic data. When stratified by GBM thickness, the Kaplan-Meier survival analysis showed that patients with greater GBM thickness (≥681 nm) had poorer renal survival rates than those with lesser GBM thickness (<681 nm; log-rank test P = 0.01, Figure 4A). Additionally, the Kaplan-Meier survival analysis showed that patients with greater TBM thickness (≥1200 nm) displayed poorer renal survival rates than those with lesser TGM thickness (<1200 nm; log-rank test P = 0.03, Figure 4B).

The percentages of patients in the GBM^loTBM^lo, GBM^hiTBM^lo/GBM^loTBM^hi, and GBM^hiTBM^hi groups who progressed to ESRD were 4.3%, 34.8%, and 60.9%, respectively (P = 0.01). The survival curves of the three groups, indicating the time to ESRD, are presented in Figure 4C. The Kaplan-Meier survival analysis showed that patients in the GBM^hiTBM^hi group had poorer renal survival rates than those in the GBM^loTBM^lo and GBM^hiTBM^lo/GBM^loTBM^hi groups (log-rank test P < 0.01).

Univariate and multivariable adjusted HRs for ESRD are presented in Table 3 according to the baseline GBM and TBM thickness, expressed as a continuous variable. Univariate Cox proportional hazard analysis showed that greater GBM thickness was associated with progression to ESRD (HR, per 1 SD of natural log-transformed GBM thickness, 1.08; 95% CI, 1.02-1.84), whereas greater TBM thickness was significantly associated with progression to ESRD (HR, per 1 SD of natural log-transformed TBM thickness, 1.89; 95% CI, 1.18-3.04). Additionally, patients in the GBM^hiTBM^lo/GBM^loTBM^hi and GBM^hiTBM^hi groups had higher HRs of 1.26 (95% CI, 1.03-8.55) and 1.98 (95% CI, 1.52-11.65) compared with the GBM^loTBM^lo group as the reference, respectively. Following adjustment for age, sex, baseline eGFR, proteinuria, HbA1c, and pathological parameters, including RPS glomerular classification, IFTA, interstitial inflammation, arteriosclerosis, and arteriolar hyalinosis, in multivariable Cox models, greater GBM (HR, per 1 SD of natural log-transformed GBM thickness, 1.21; 95% CI, 1.17-2.38) and TBM thickness (HR, per 1 SD of natural log-transformed TBM thickness, 1.90, 95% CI, 1.18-2.87) remained significantly associated with ESRD. When GBM and TBM thickness were combined, patients in the GBM^hiTBM^lo/GBM^loTBM^hi and GBM^hiTBM^hi groups had adjusted HRs of 1.49 (95% CI, 1.21-9.75) and 3.07 (95% CI, 2.87-12.78) compared with the GBM^loTBM^lo group, respectively. Their HRs for progressing to ESRD increased when combining TBM thickness with GBM thickness. Moreover, a traditional clinical parameter like eGFR independently predicted progression to ESRD in the multivariable Cox hazard models (HR, 0.60; 95% CI, 0.45-0.81).