Utility of glomerular and tubular markers in establishing early renal involvement in type II diabetes mellitus

Clinical Biochemistry, Vol. 29, No. 4, pp. 385-388, 1996 Copyright © 1996 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/96 $15.00 + .00 ELSEVIER

S0009-9120(96)00004-5

Utility of Glomerular and Tubular Markers in Establishing Early Renal Involvement in Type II Diabetes Mellitus ABDULKERIM BEDIR, 1 I. (~ETIN OZENER, 2 BAHATTIN ADAM, ~ and KAYA EMERK 3 2

1Department of Biochemistry, Medical Faculty of Ondokuzmayis University, Samsun, Terkiye; Department of Nephrology and 3 Department of Biochemistry, Medical Faculty of Marmara University, Istanbul, Turkey

Introduction

Diabetic nephropathy, one of the major long-term complication of diabetes, develops in 30 to 40% of type 1 diabetic patients, and terminal renal failure occurs within 7 years afLer onset of the renal disease (1). The very first clinical marker of unfavorable renal involvement seems to be the so-called "microalbuminuria," which is commonly considered hemodynamic in origin (2) and/or due to a biochemical der a n g e m e n t in glomerular b a s e m e n t m e m b r a n e (GBM) composition, leading to an abnormal charge permselectivity (3). Experimental diabetes impairs heparan sulfate (HS) synthesis (4) and decreases the degree of heparan sulfate sulphation (5). Both reduced concentration and reduced sulphation of heparan sulfate may lead to increased glomerular permeability due to loss of the glomerular basement membrane fixed charge, (6). Thus, diabetes-induced alterations of cellular and basement membrane heparan sulfate may represent an important pathogenic mechanism in the development of diabetic nephropathy. Serum alpha-amylase (1,4-a-D-glucanglucanohydrolase, EC 3.2.1.1) is ]produced mainly in the salivary gland and the pancreas. Both isoenzymes have identical molecular size (29 A) but different net charges in plasma, the salivary fraction being more anionic (pl 5.9-6.4) than the pancreatic fraction (pl

Correspondence: Abdulkerim Bedir, MD, Department of Biochemistry, Medical Faculty of Ondokuzmayis University, Samsun 55139, Tiirkiye. Manuscript received October 3, 1995; revised and accepted January 8, 1996. Abbreviations: GBM, glomerular basement membrane; Cr, creatinine; NIDD, non-insulin dependent diabetes; LAP, leucine aminopeptidase; DN, diabetic nephropathy; HbA1, glycated hemoglobin; GAG, glycosaminoglycan;HS, heparan sulfate; DMB, 1,9-dimethylmethylene blue; S/P ratio, the ratio of salivary to pancreatic amylase; Alb, albumin; P-amylase, pancreatic amylase; S-amylase, salivary amylase. C L I N I C A L B I O C H E M I S T R Y , V O L U M E 29, A U G U S T 1996

7.0). It has been shown that their excretion rates into urine are altered in several nephropathies (7) and in diabetes (8), probably due to loss of the negative charges normally present in the GBM. Leucine aminopeptidase (LAP) is an enzyme from the brush border of the proximal tubular cells and the estimation of its u r i n a r y activity has been reported to be useful in diagnosis of certain renal diseases (9). The present study was carried out to see whether or not urinary LAP excretion, the altered renal handling of those two amylase isoenzymes, and urinary excretion of GAG molecules could be used as a simple procedure to disclose tubular involvement and the loss of charge selectivity of GBM during early diabetic glomerular disease in type II diabetics. Materials a n d m e t h o d s REAGENTS AND PROCEDURES

DMB (1,9-dimethylmethylene blue) and heparan sulfate were from Aldrich (Milwaukee, WI) and Sigma (St. Louis, MO), respectively. All other chemicals used in the assay were purchased from Merck. DMB- Tris assay

The color reagent was made by mixing 10 parts of the color used in the original procedure (10) with 1 part of 2 mol/L Tris (base) solution. Heparan sulfate (50 mg/L solution) was used as standard in this assay. Absorbance was measured at 500 nm 75 s after mixing 30 ~L of standard solution or urine and 325 ~L of color reagent, and corrected for sample blank and reagent blank. Measurements were performed at 37 °C. SUBJECTS

Fifty-nine patients (18 male and 41 female aged 42 to 85 years) diagnosed with NIDD were studied. 385

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ET AL.

The healthy nondiabetic subjects were 7 males and 18 females aged 28-94 years. Fasting blood and second morning urine samples were obtained in the morning t h a t each patient attended the Endocrinology and Nephrology clinics for periodic revision of their diabetes. Urine (after centrifugation at 1000 x g for 10 min) and serum samples were stored at -70 °C until analysis. METHODS

All measurements, except HbA1, were carried out by Technicon RA-XT analyzer (Technicon Inc.). Albumin (Alb) and LAP activity in urine were measured by i m m u n o t u r b i d i m e t r y (Mikroalb, Ames, Bayer) and kinetically (LAP, Merck), respectively. Total and pancreatic amylase were measured zeroorder kinetically using a-amylase EPS and Pancreatic a-amylase EPS kits (Boehringer M a n n h e i m GmbH). Urinary creatinine and serum glucose were determined by the Jaff@ method and glucose oxidase method, respectively. We measured HbAD by the microcolumn method, depending on cation exchange using the Glycated Hemoglobin (HbA1) kit (Sigma). Amylase isoenzymes and LAP activities, and GAG and albumin concentrations in urine were expressed as units per mmol of creatinine and milligrams per mmol of creatinine, respectively (11). Comparison and correlation between control and NIDD data were done by the Mann-Whitney U test and the Spearman r a n k test, respectively. The maxim u m distance between the distributions was calculated by the Kolmogorov-Smirnov test. For evaluating frequencies, chi-square test was used. A p value <0.05 was considered significant.

Results Table 1 shows data on the control group and the NIDD groups. The age and sex distributions of all

groups were similar. The clinical status of the patients can be seen from the data regarding glycemia and glycated hemoglobin. We took the 95th percentiles as upper limits of LAP/Cr (1.27 U/mmol Cr) and S/P ratio (1.15) in healthy population. In the normoalbuminuric stage (Group A), urinary LAP/Cr was abnormal in 46% of the patients (×2: 5.22, p < 0.05 with Yates' correction), whereas S/P ratio was abnormal in 35% (×2: 4.02, p < 0.05 with Yates' correction). Five normoalbuminuric patients showed an abnormality of both LAP/Cr and S/P ratio; 7 were positive for LAP/Cr alone, but only 4 for S/P ratio. Furthermore, the prevalence of abnormal urinary LAP/Cr was 61% in the first 10-year period of NIDD, and t h a t of microalbuminuria was 35.5% (×2: 4.13, p < 0.05). During the same period, the prevalence of abnormal S/P ratio was found to be 31%, no different from the frequency of microalbuminuria statistically with the chi-square test. We also found LAP/Cr abnormality of 87.5% and S/P abnormality of 37.5% in the microalbuminuric patients. For patients with abnormal LAP/Cr, the frequency of S/P abnormality was found to be 43%. On univariate analysis, the strongest correlation of S/P ratio was with LAP/Cr followed by S-amylase/ Cr in type II diabetics (Table 2). No correlation was found between P-amylase/Cr, u r i n a r y GAG/Cr, albuminuria, and fasting blood glucose. Moreover, we found t h a t LAP/Cr has a significant correlation with both albuminuria (rs: 0.41, p < 0.005) and fasting blood glucose levels (rs: 0.52, p < 0.005). The cumulative distributions of the S/P in urine of controls and NIDD patients were compared and the m a x i m u m distance between the distributions was found to be statistically significant (p < 0.05) for both group A and group B compared to controls, and not significant for group C compared to group B. Comparisons of cumulative frequencies of LAP/Cr in

TABLE 1 Population P a r a m e t e r s a n d Analytical Values*

Parameter

Controls

N u m b e r of subjects

25

Age (years) Sex (M/F) Fasting blood glucose (mmol/L) Hb A1 Albuminuria (mg/mmol Cr) LAP/Cr (U/mmol Cr) Urinary S/P ratio Urinary GAG/Cr (mg/mmol Cr)

Group A

Group B

Group C

(Normoalbuminuric)

(Microalbuminuric)

(Macroalbuminuric)

26

23

10 56 ± 14t 3/7

52 ± 23 7/18 4.9 ± 0.3

61 ± 10t 7/19 8.16 ± 3.75

61 ± l l t 8/15 11.3 ± 3.5§

0.061 ± 0.021 0.85 ± 0.5

0.092 ± 0.027 II 0.92 ± 0.98t

0.114 ± 0.029 ~ 8.5 ± 4.9§

0.139 ± 0.037§

0.79 ± 0.28

1.21 ± 0.45#

1.92 ± 0.91§

1.88 ± 1.2#

0.55 ± 0.39 2.73 -+ 1.29

0.94 ± 0.565 2.55 ± 2.04t

1.65 ± 2.39 2.12 ± 1.97t

1.02 ± 1.15t 2.35 ± 1.55t

9.1 -+2.3§ 94.6 ± 76.3§

* P a r a m e t e r values are given as the m e a n ± SD. ~, are non-significant, $,p < 0.005, §,p < 0.0001; ll, p < 0.01 compared to controls. 9, p < 0.05; # , p < 0.001 compared to controls.

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CLINICAL BIOCHEMISTRY, VOLUME 29, AUGUST 1996

GLOMERULAR AND TUBULAR MARKERS IN DIABETES TABLE 2

Spearman Rank Correlations of Urinary S/P Ratio, LAP/Cr Excretion, and Fasting Blood Glucose in Type II Diabetics First Variable Second Variable

S/P Ratio rs

S-amyla~,e/Cr

P-amylase/Cr Albuminuria Fasting blood glucose Urinary GAG/Cr LAP/Cr

P

LAP/Cr rs

P

0.36

< 0.02

0.39

-0.18 0.09 0.2 -0.09 0.39

ns ns ns ns < 0.01

O.18 0.41 0.52 -0.03 -

different groups were done; p < 0.005 for controls v s group A, p < 0.001 for group A v s group B, and no difference for group B vs group C were found. Discussion

The use of the renal handling of two endogenous proteins of smaller sizes t h a n albumin (45000 v s 68000 daltons), different net electric charges, and catalytic activity t h a t allows easy m e a s u r e m e n t , makes it possible to explore the status of negative charges in the GBM. With a decrease in concentration of negative components of GBM, as can be seen in the diabetic kidney, the salivary isoenzyme would be less rejected and, consequently, excreted in higher quantities into urine. Our results showed t h a t NIDD produced an increase of S/P ratio in both group A (normoalbuminuric stage) and B (microalbuminuric stage), but not in group C (macroalbuminuric stage). A significant correlation of S/P ratio with only S-amylase/Cr excretion (rs: 0.36, p < 0.02) suggests t h a t it m a y be due to an increase in the excretion of salivary isoenzyme. We distinguished at least 3 different conditions in the patients with normoalbuminuria; abnormality of LAP/Cr plus S/P (19%), abnormality of only LAP/Cr (27%), and abnormality of only S/P (16%). We have been continuing to study the importance and clinical reflections of those a b n o r m a l i t i e s in the larger NIDD population. There exist no correlation between albuminuria and S/P ratio, but a significant correlation between LAP/Cr and S/P ratio raises the possibility t h a t factors in addition to barrier charge defects contribute to the development, magnitude, and composition of proteinuria early in the course of diabetic glomerular disease. Such factors may include: 1. a progressive decrement in fractional reabsorption as the filtered protein load increases due to glomerular hyperfiltration (2), 2. proximal tubular dysfunction as observed in our study by a significant correlation of albuminuria with LAP/Cr (rs: 0.41, p < 0.005), and 3. a concomitant loss of barrier sizeselectivity (3). GAG excretions in edl groups showed no differences from controls. According to these results, the CLINICAL BIOCHEMISTRY, VOLUME 29, AUGUST 1996

< 0.01

ns < 0.005 < 0.005 ns -

Fasting Blood Glucose rs

0.35

0.22 0.3 -0.12 -

P

< 0.02 ns

< 0.05 ns

m e a s u r e m e n t of urine GAG excretion, thus, may not be useful for evaluating GBM lesions in type II diabetics. In cell culture studies, it was found t h a t LAP expression is susceptible to mitogens, in particular, and shows a close relationship with the stimulation of protein kinase C. However, it was also shown in other research (12) t h a t protein kinase C m a y be induced by increased levels of glucose v i a the diacylglycerol pathway. In the light of these data, our finding t h a t fasting blood glucose shows an import a n t correlation with LAP/Cr excretion m a y gain some meaning and stress the importance of glycemic control in diabetics. The altered LAP/Cr and S/P ratio shows a distribution along the duration of the disease t h a t is statistically different from t h a t of microalbuminuria. Their higher prevalances appeared in the earlier phase of the disease (normoalbuminuric term). Although we found t h a t there were some differences between the prevalances of LAP/Cr or S/P ratio and m i c r o a l b u m i n u r i a in the first 10-year period of NIDD, only the difference between LAP/Cr and mic r o a l b u m i n u r i a was significant statistically. We suggest t h a t both LAP/Cr and S/P ratio, together, may be helpful in assessing the tubular and glomerular changes in the early stage of diabetes, particularly in the normoalbuminuric phase. References

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6. Kanwar YS, Linker A, Farquhar MG. Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion. J Cell Biol 1980; 86: 688-693. 7. Piccoli A, Zaninotto M, Mussap M, et al. Pancreatic to salivary amylase clearance in assessing glomerular charge selectivity. Progr Med Lab 1990; 4: 452. 8. Recio F, Villamil F, Recio C, Ferret C. Utility of filtration markers to monitor the quality of glomerular function. Clin Nephrol 1992; 38(Suppl 1): $8-S13. 9. Jung K, Scholz D. An optimized assay of alanine ami-

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nopeptidase activity in urine. Clin Chern 1980; 26: 1251-4. 10. De Jong JGN, Wevers RA, Laarakkers C, Poorthuis BJHM. Dimethylmethylene blue-based spectrophotometry of glycosaminoglycans in untreated urine: a rapid screening procedure for mucopolysaccharidoses. Clin Chem 1989; 35: 1472-1477. 11. Hofmann W, Guder WG. A diagnostic program for quantitative analysis of proteinuria. J Clin Chem Clin Biochem 1989; 27: 589-600. 12. Guzman NJ, Crews FT. Regulation of inositol transport by glucose and protein kinase C in mesangial cells. Kidney Int 1992; 42: 33-40.

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