Supplementary Table 1. Criteria for selection of normal control individuals among healthy volunteers Medical parameters Cut-off values BMI (kg/m 2 ) 25.0 Waist (cm) (Men and Women) (Men) 85, (Women) 90 Systolic blood pressure (mmhg) < 130 Diastolic blood pressure (mmhg) < 85 Fasting plasma glucose (mg/dl) < 110 Total cholesterol (mg/dl) < 220 Triglycerides (mg/dl) < 150 LDL cholesterol (mg/dl) < 140 HDL cholesterol (mg/dl) 40 AST (U/L) 40 ALT (U/L) 40 γ-gt (U/L) (Men) 70, (Women) 30 Uric acid (mg/dl) 7.0 Plasma creatinine (mg/dl) (Men) < 1.05, (Women) < 0.8 egfr (ml/min/1.73m 2 ) 60-104 ACR (mg/g Cr) < 10 ACR, urinary albumin creatinine ratio; AST, aspartate aminotransferase; ALT, alanine aminotransferase; Cr, urinary creatinine; egfr, estimated glomerular filtration rate; γ-gt, γ-glutamyl transferase. Supplementary Table 2. Urinary markers in the normal control individuals C-megalin (fmol/g Cr) 145 (87-233) A-megalin (pmol/g Cr) 73 (35-106) Albumin (mg/g Cr) 5.2 (3.7-6.4) NAG (IU/g Cr) 2.1 (1.4-3.1) α 1 -microglobulin (mg/g Cr) 2.2 (1.4-3.5) β 2 -microglobulin (μg/g Cr) 79 (61-102) Data are medians (interquartile range). Cr, urinary creatinine; NAG, N-acethyl-β-D-gulcosaminidase. Supplementary Table 3. Urinary A- and C-megalin levels in the normal control individuals and 68 patients with type 2 diabetes Normal control Type 2 diabetes individuals Normoalbuminuria Microalbuminuria Macroalbuminuria Number 160 39 16 13 Urinary markers C-megalin (fmol/g Cr) 145 (187-233) 330 (218-523) 486 (345-663) 1315 (765-2,575) A-megalin (pmol/g Cr) 73 (35-106) 115 (89-172) 122 (84-202) 63 (45-174) Albumin (mg/g Cr) 5.2 (3.7-6.4) 7.4 (5.6-15.0) 59.8 (46.1-112.7) 4970.3 (569.2-8,966.0) Data are median (interquartile range). Cr, urinary creatinine; Macroalbuminuria, >300 (mg/g Cr); microalbuminuria, 30-299 (mg/g Cr); normoalbuminuria, <30 (mg/g Cr).
Supplementary Table 4. Characteristics of 52 patients with type 2 diabetes Number 52 RBC count ( 10 4 /mm 3 ) 446.5 ± 49.3 Sex (% men) 61.5 Hemoglobin (g/dl) 13.8 ± 1.5 Age (years) 66.5 ± 10.9 Hematocrit (%) 41.5 ± 4.2 BMI (kg/m 2 ) 24.9 ± 5.5 MCV (fl) 93.1 ± 4.8 MCH (pg) 30.9 ± 1.6 MCHC (%) 33.2 ± 0.9 Clinical parameters WBC count (cell/μl) 6,115 ± 1,549 Systolic blood pressure Platelet count ( 10 4 /μl) 19.5 ± 4.1 129 ± 15.0 (mmhg) Diastolic blood pressure Medications, % (n/n) 78.2 ± 9.8 (mmhg) Antihypertensive A1C (%) 6.9 ± 0.8 ACE inhibitors 29 (15/52) Total cholesterol (mg/dl) 187.2 ± 30.1 ARBs 60 (31/52) Triglycerides (mg/dl) 119.7 ± 54.1 Diuretics 13 (7/52) LDL cholesterol (mg/dl) 106.8 ± 30.0 Others 50 (26/52) HDL cholesterol (mg/dl) 60.0 ± 26.7 Antihyperglycemic ALT (U/L) 27.5 ± 16.1 Insulin 10 (5/52) AST (U/L) 26.0 ± 7.3 Sulfonylurea 44 (23/52) γ-gt (U/L) 43.1 ± 65.2 Glinide 4 (2/52) Total protein (g/dl) 7.7 ± 0.4 Biguanide 27 (14/52) Creatinine kinase (U/L) 123.3 ± 87.8 Thiazolidine 19 (10/52) α-gulucosidase inhibitor Lactate dehydrogenase (U/L) 191.9 ± 40.7 56 (29/52) Alkaline phosphatase (U/L) 220.2 ± 75.5 Antidyslipidemic Cholinesterase (U/L) 318.5 ± 76.1 Statins 31 (16/52) Blood urea nitrogen (mg/dl) 14.8 ± 4.6 Fibrates 4 (2/52) Plasma creatinine (mg/dl) 0.80 ± 0.21 egfr (ml/min/1.73m 2 ) 70.6 ± 14.6 Urinary markers Uric acid (mg/dl) 5.5 ± 1.3 C-megalin (fmol/g Cr) 502 (367-683) Na (meq/l) 139.9 ± 2.4 A-megalin (pmol/g Cr) 130 (87-178) K (meq/l) 4.3 ± 0.3 Albumin (mg/g Cr) 18.1 (8.6-59.0) Cl (meq/l) 102.8 ± 3.1 NAG (IU/g Cr) 7.6 (5.5-13.3) Ca (mg/dl) 9.3 ± 0.4 α 1 -microglobulin (mg/g Cr) 6.2 (3.3-13.4) Pi (mg/dl) 3.3 ± 0.5 β 2 -microglobulin (μg/g Cr) 113 (66-256) Data are mean ± SD or median (interquartile range). ALT, alanine aminotransferase; ARB, angiotensin II receptor blocker; AST, aspartate aminotransferase; Cr, urinary creatinine; egfr, estimated glomerular filtration rate; γ-gt, γ-glutamyl transferase; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; NAG, N-acethyl-β-Dgulcosaminidase; Pi, inorganic phosphate; RBC, red blood cell; WBC, white blood cell.
Supplementary Figure 1. Comparison of urinary megalin levels between first- and second-void urine specimens A and B: Urinary A- (A) and C-megalin (B) levels were measured in type 2 diabetic patients with normoalbuminuria (closed circles, n=10, urinary albumin<30 mg/g creatinine), microalbuminuria (open triangles, n=5, 30 mg/g urinary albumin<300 mg/g creatinine), and macroalbuminuria (closed triangles, n=4, 300 mg/g creatinine urinary albumin). C and D: Urinary A- (C) and C-megalin (D) levels were measured in first- (open bars) and second-void urine specimens (closed bars) of 3 patients with type 2 diabetes on the same day. Each specimen was analyzed three times. Key: Cr, urinary creatinine
Supplementary Figure 2. Stability of urinary megalin at 37 ºC for 12 hrs. Measurement of urinary A- (A) and C-megalin (B) concentrations were not changed in urine specimens obtained from 3 patients with type 2 diabetes before (open bars) and after incubation at 37 ºC for 12 hrs (closed bars). Each specimen was used for the measurements three times. Supplementary Figure 3. Measurement of urinary A- and C-megalin levels of urine specimens before and after storage at -80 ºC for 2-4 yrs. Urinary A- (A) and C-megalin concentration (B) in urine specimens obtained from patients with chronic kidney disease was consistent before (Pre) and after (Post) storage at -80 ºC for 2-4 yrs (n = 19).
Supplementary Figure 4. Immunoblot assay of normal human kidney lysates with monoclonal antibodies against human megalin. Normal human kidney lysates (15 µg/lane) were subjected to immunoblot assay with previously characterized polyclonal antibody against human megalin (1) (Reference 20) and monoclonal antibodies, A5 (2), A12 (3), C25 (4) and C37 (5) (100 ng/ml each) in the absence (-) or presence (+) (100-fold molar excess of each monoclonal antibody) of glutathione S- transferase (GST), GST-ligand-binding domain 1 (LBD1) or GST-cytoplasmic tail (CT) proteins, showing the specificity of the monoclonal antibodies against megalin. Supplementary Figure 5. Megalin ELISA calibration standard curves (A): A-megalin ELISA; (B): C-megalin ELISA; (C): F-megalin ELISA Key: S/N ratio, signal/noise ratio (of the chemiluminescent relative light units)
Supplementary Figure 6. The ectodomain form of megalin is present in the soluble urine fraction but the full-length form is in both soluble and insoluble fractions. Original urine specimens (Ori) were separated into supernatants 1 (S1) and precipitants 1 (P1) by low-speed centrifugation, and the S1 were further separated into supernatants 2 (S2) and precipitants 2 (P2) by ultracentrifugation. A and B: Urine specimens from controls (n=10, open circles) and 11 patients with type 2 diabetes and normoalbuminuria (closed circles), microalbuminuria (open triangles), or macroalbuminuria (closed triangles) were subjected to A- (A) and C-megalin (B) assays. C and D: A- and C-megalin assays correlated stoichiometrically in P1 (C) and P2 (D) of urine samples from the controls (n=10) and the patients with type 2 diabetes (n=11). Key: Cr, urine creatinine
Supplementary Figure 7. Association between urinary C-megalin levels and egfr (<60 ml/min/1.73 m2) in patients with type 2 diabetes (n=21) Key: Cr, urinary creatinine; egfr, estimated glomerular filtration rate; rs, Spearman s rank-correlation coefficient (vs. 1/eGFR)
Supplementary Figure 8. The effect of albumin to A- and C-megalin ELISA in urine A and B: The recombinant megalin standard was added to a normoalbuminuric urine specimen and measured by A- (A) and C-megalin (B) ELISA with incrementally increased addition of human serum albumin (HSA). The final concentrations of the added standard were 100 (open bars), 200 (shaded bars) and 400 pmol/l (closed bars) for A-megalin ELISA (A) and 1 (open bars), 3 (shaded bars) and 11 pmol/l (closed bars) for C-megalin ELISA (B). Both A- and C-megalin ELISA measurements remained at ~85% of the control level (2), even at the maximum albumin concentration (7). 1, the original urine specimen with no addition of the recombinant standard. The albumin concentration was 5, 5, 71, 335, 947, 1,889 and 2,831 mg/g Cr for 1, 2, 3, 4, 5, 6 and 7, respectively. C and D: The recombinant megalin standard was added to 5 macroalbuminuric urine specimens and A- (C) and C-megalin (D) ELISA were carried out. The values were obtained by subtracting the endogenous A- (C) and C-megalin values (D) from the raw measurements for each urine specimen. Neither A- or C-megalin measurements were affected by different albumin concentrations in urine specimens. The final concentrations of the added standard were 34 (open bars), 140 (shaded bars) and 560 pmol/l (closed bars) for A-megalin ELISA (C) and 1.1 (open bars), 6.2 (shaded bars) and 63 pmol/l (closed bars) for C-megalin ELISA (D). Data are presented as mean ± SD (n = 3). Key: Cr, urinary creatinine.