Study on the relation between renal tubular disorders and. gomerular dysfunction in the early phase of insulin-dependent diabetes

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1 57 Study on the relation between renal tubular disorders and gomerular dysfunction in the early phase of insulin-dependent diabetes mellitus in children TADASHI ASAMI, TOKUSHI NAKANO, KAORU SAKAI Department of Pediatrics, School of Medicine, Niigata University Key words: insulin dependent diabetes mellitus, diabetic nephropathy, microalbuminuria, NAG, ƒà 2-microglobulin Abstract To study the relations between renal tubular disorder and glomerular dysfunction in the early phase of insulin-dependent diabetes mellitus (IDDM), we performed concomitant measurements of urinary ƒà- D-N-acetyl glucosaminidase (NAG), ƒà2-microglobulin (BMG), and microalbumin in 29 of pediatric patients with IDDM, 15 normal controls, and 83 patients with non-diabetic ketoacidosis. Urinary NAG levels were significantly elevated in the IDDM patients compared with controls. Urinary BMG levels were also elevated in IDDM, however, they were not as prominent as NAG levels. Although urinary microalbumin levels were elevated in the IDDM patients, statistical analysis did not show any significant difference between the IDDM patients and controls. Urinary NAG and BMG concentrations were also increased in patients with non-diabetic ketoacidosis, suggesting a toxic effect of ketone bodies to renal tubular cells. Statistically significant correlations were noted both between urinary NAG and microalbumin and between urinary BMG and microalbumin. These results suggest that, in early phase of IDDM, microalbuminuria is preceded by elevations in urinary NAG and BMG levels, and that keton bodies have deleterious effects on renal tubular cells. Introduction Glomerular lesion has been a major concern of diabetic nephropathy since the description of nodular glomerulosclerosis by Kimmelstiel and Wilson [1], which has been supported by recent reports on the presence of microalbuminuria in the earlier phase of diabetes mellitus [2, 3,4,5]. While these reports stress the importance of early glomerular lesion development, a distinctive glycogenic vacuolization of renal tubular cells in diabetic patients has long been recognized as a lesion pathognomonic of diabetes [6]. There have also been several studies on renal tubular involve ment in insulin-dependent diabetes mellitus (IDDM) [7,8,9,10]. Milteny et al. [9] reported that the urinary excretion of ƒà2-microglobulin Received October 2, 1991 (BMG) and that of certain enzymes including ƒà-d-n-acetylglucosaminidase (NAG) were signi ficantly increased during ketoacidosis in patients with IDDM and that tubular reabsorption of electrolytes was reduced in poorly controlled diabetics. In addition, a recent report that some disorders might develop in both renal tubule and glomerulus after a short duration of IDDM [10] raises the possibility of a more important role for tubular disorder, than hitherto considered, in the development of diabetic nephropathy. However, since there have been few studies in which concomitant measurements of renal tubular proteins and albumin were carried out in the urine samples from patients both with IDDM and with non-diabetic ketoacidosis, two points remain uncertain: 1) Does ketoacidosis itself affect renal tubular function; and 2) Which is an earlier sign of renal involvement, tubular proteins or albumin, Japanese Journal of Nephrology Vol. 34, No. 1,

58 Tadashi Asami, et al. especially in younger IDDM patients? We studied both tubular and glomerular proteins in children with IDDM without overt nephropathy.

2 58 Tadashi Asami, et al. especially in younger IDDM patients? We studied both tubular and glomerular proteins in children with IDDM without overt nephropathy. Subjects and Methods Twenty nine patients with IDDM (12 males and 17 females), with a mean age of 11 years and 5 months (range, 2 to 24), were included in this study. The duration of the disease was between 1 and 11 years (3.8 }3.1 years, mean }SD). At the time of examination, all of the IDDM patients had glucosuria (1,840 }2,950mg/dl in the first-voided morning urine samples) and a mean increased HbAlc level (9.8 }2.7%). Serum creatinine levels were normal (0.43 }0.10mg/dl, mean }1SD n= 29) ranging from 0.30 to 0.60mg/dl, and blood urea nitrogen were also normal (15.2 }4.5mg/dl) ranging from 7 to 23mg/dl. Other urinary findings including protein, red blood cells, and ketone bodies were negative in all of the subjects upon examination. To examine the effect of postural change on the excretion of urinary NAG, BMG, and albumin, two urine samples were obtained on the same day: FVM-urine; the urine voided first in the morning, and OPC-urine; the urine voided at the out-patient clinic of our hospital. To evaluate the effect of ketone bodies on renal tubules, urine samples from 83 patients with non-diabetic ketoacidosis (auto intoxication or cyclic vomiting) also examined for NAG and BMG. Ketoacidosis was diagnosed when urinary ketone body levels were higher than 0.4 nmol/l as described below. Fifteen healthy school children were used as controls with informed consent obtained from one or both parents. Urine samples were kept frozen at ph 4.8 for NAG and at ph 7.5 for BMG until assay, respectively. NAG levels were measured in simul taneously taken urine and serum samples from 14 randomly selected IDDM patients to examine whether urinary NAG levels reflect circulating levels of NAG. NAG values were measured by our method [11]. In brief, 0.lml of urine sample, kept frozen in 1.0ml of 0.1M-phosphate buffer solution at 4.8, was added 1.0ml of substrate solution (4-methylumbelliferyl-N-acetyl-ƒÀ-Dglucosaminide), and incubated at 37 Ž for 60min. The reaction was stopped by addition of 3.0ml of 0.17M-glycine carbonate buffer. The fluores, cence of the reaction mixtures was read at 450nm (excitation wave at 365nm) against a standard solution. BMG was measured by enzymeimmuno assay (Phadezyme EIA Kit, Shionogi) and albumin by radioimmunoassay (Albumin RIA Kit, Phar macia). Urinary creatinine concentrations were determined by an autoanalyzer system of our hospital. The concentrations of urinary ketone bodies were measured using a commercially available kit (Keton-Test, Sanwa) and levels above 0.4nmol/l of urine were defined as ketone-uria [12]. Statistical analysis was performed by Student's t-test combined with the Cochran-Cox correction method. Results Urinary NAG levels (Figure 1, 2) The levels of urinary NAG in the patients were significantly elevated both in FVM- and OPC-urine as compared with those in the controls (Figure 1). The values for FVM-urine and those for OPCurine were not statistically different. Elevations of urinary NAG levels above the normal range (60u/ml) were noted in 7 of 29 (24.1%) IDDM patients, while this was not observed in any of the controls. Urinary NAG levels were lower than 60 u/ml (normal upper limit), ranging between 8.4 and 54.6u/ml, in 18 of 18 (100%) IDDM patients who had no glucosuria at the time of determina tion. In 26 of 29 (89.7%) IDDM patients, the NAG/creatinine (u/mgcr.) ratios in FVM-urine increased to levels + 2SD above the values of the controls, and 15 of 29 (51.7%) for OPC-urine. There was no significant correlation between the NAG values in urine and those in serum (r=0.03), and between the duration of the disease and the urinary NAG level. In 22 of 83 (26.5%) patients with non-diabetic ketoacidosis, the urinary NAG levels also increased above the normal range (Figure 2). Urinary BMG levels (Figure 2, 3) Urinary BMG levels were elevated in the IDDM patients as compared with those in the controls, however, statistical analysis revealed a significant p value only for BMG/gCr. in FVM-urine (Figure 3). High urinary BMG levels above the normal range (200ƒÊg/ml) were found in 6 of 29 (20.7%) IDDM patients. No significant differences were 58 Japanese Journal of Nephrology Vol. 34, No. 1, 1992

3 Renal tubular and glomerular dysfunction in IDDM 59 Fig. 1. Urinary NAG levels in patients with IDDM. The values are expressed in units/ml of urine and units/mg of urinary creatinine. Shaded column represents the mean value in patients with IDDM (insulin-dependent diabetes mellitus), and open column that of normal controls. Urinary NAC levels in IDDM were significantly elevated both in the urine firstly voided in the morning (FVM-urine) and in the urine voided at our outpatient clinic after walking (OPC-urine) compared with those in controls. No significant differences were found between the values in the FVM-urine and in the OPC-urine. noted between the values for FVM-and OPCurines. In non-diabetic ketoacidosis, 26 of 83 (31.3%) patients also showed elevated urinary BMG levels above the normal range (Figure 2). Urinary albumin levels (Figure 4) Although elevations of urinary albumin levels both in ƒàg/ml and ƒàg/gcr, were noted in the IDDM patients compared with controls, statistical Fig. 2. Urinary NAG and ƒà2-microglobulin levels in patients with non-diabetic ketoacidosis. Urinary NAG levels were abnormally high in 22 of 83 (26.5%) patients with non-diabetic ketoacidosis. Urinary ƒà2-microglobulin levels were also increased to above the normal range. Shaded zone represents the normal range of the values. analysis revealed no significant difference. However, 5 of 29 (17.2%) IDDM patients were found to have higher urinary albumin concentra tions beyond 30ƒÀg/ml which Mogesen et al. [4] suggested as the level indicative of later clinical proteinuria development. These five patients had a mean duration of the disease of 6.0 }4.1 years and a mean HbAlc level of 9.5 }2.0%, neither of which were statistically different from those of the remaining IDDM patients with urinary albumin concentrations lower than 30ƒÀg/ml. Japanese Journal of Nephrology Vol. 34, No. 1,

4 60 Tadashi Asami, et al. Fig, 3. Urinary ƒà2-microglobulin levels in patients with IDDM. The values were expressed in ƒàg/l of urine and ƒàg/gram of urinary creatinine. ƒà 2-microglobulin levels in IDDM were elevated compared with those in controls. However, statistical analysis revealed a significant p value only in the FVM-urine. No significant differences were noted between the values in the FVM-urine and in the OPC-urine. For the abbreviations used, see the legend in Figure 1. Fig. 4. Urinary microalbumin levels in patients with IDDM. The values are expressed in ƒàg/ml of urine and ƒàg/mg of urinary creatinine. Urinary microalbumin levels in IDDM were elevated compared with those in controls. However, the differences were statistically not significant. 60 Japanese Journal of Nephrology Vol. 34, No. 1, 1992

5 Renal tubular and glomerular dysfunction in IDDM 61 Fig. 5. Correlation between urinary NAG and microalbumin in patients with IDDM. A significant correlation was noted between the levels of urinary NAG and microalbumin both in the FVM-urine and in the OPC-urine. For the abbreviations, see the legend in Figure 1. Relationship among urinary NAG, BMG, and albumin levels Urinary NAG and albumin levels showed a significant correlation both in the FVM-urine (r=0.734, p ƒ0.001) and in the OPC-urine (r=0.496, p ƒ0.01) (Figure 5). A weak but signifi cant correlation was noted between BMG/gCr. and albumin/gcr. (r=0.426, p ƒ0.05). Although other relations were analyzed, the p values were not statistically significant: albumin and BMG; not significant (r=0.083 in FVM-urine and r= in OPC-urine), NAG and BMG; not significant Discussion (r=0.06 in FVM-urine and r=0.238 in OPCurine). In this study we observed a significant eleva tion of urinary NAG both in FVM-and OPCurine, although urinary albumin levels did not show any difference compared with the controls. An increase in urinary BMG was also observed, however, it was not as prominent as NAG. These data partially conflict with those presented by Mogensen [5], who showed that BMG excretion did not increase until urinary albumin exceed 1,000 micrograms per minute. A possible explanation for this may be the difference in age of the subjects at examination, because predictive studies of diabetic nephropathy, based on early microalbuminuria [3,4,13], have been conducted mainly on adult patients in contrast to ours. Since urinary NAG and BMG are known to increase in various renal tubular disorders, and there was no significant correlation between urine and serum NAG values, the elevation NAG is considered of urinary to be caused by the increased excretion of enzymes from lysosomes in tubular cells activated by excessive loading at glucose beyond the reabsorption capacity. Both increased urinary NAG and BMG levels were demonstrated in IDDM patients with glucosuria, but were not found in patients with normal urine. In addition, ketone bodies, examined in patients with non diabetic ketoacidosis, were demonstrated to cause elevations of both urinary NAG and BMG levels. Although the precise mechanism is unknown, these findings suggest that some IDDM patients may have tubular cells more vulnerable to the excessive loading of urinary glucose or ketone bodies than those who do not develp renal complications. Of interest is the statistically significant correla Japanese Journal of Nephrology Vol. 34, No. 1,

6 62 Tadashi Asami, et al. tion between urinary NAG and albumin noted in both FVM-and OPC-urine, and between BMG and albumin noted in both FVM-and OPC-urine, suggesting that albumin excretion rates track quite closely with markers of tubular handling of such as NAG and BMG. In an attempt to find addi tional factors contributing to renal tubular disorders, other clinical and laboratory data were examined. However, the duration of the disease, HbAlc levels, and urinary glucose concentrations were not statistically related to urinary NAG or BMG excretion, and none of the five IDDM patients, having mild micro-albuminuria, showed any difference compared to those without microalbuminuria. Viewing several reports, the presumed relation between the good control and the prevention of diabetic nephropathy remains controversial, and the cumulative incidence of diabetic nephropathy has been reported in only about 35 percent of all IDDM patients [14,15]. These reports suggest that multiple factors, such as blood hyperviscosity [16, 17], hypertension [13,17], glomerular hyperfiltra tion [18], blood rheology changes [16], or even inherited predisposition to hypertension [19,20], may be involved in the development of diabetic nephropathy. In conclusion, we propose that microalbu minuria, predictive of later development of overt diabetic nephropathy, may be preceded by eleva tions in urinary NAG and BMG levels in children with IDDM. Since only limited data are now available, a long-term follow-up study of the 29 IDDM patients is needed to understand the significance in diabetic nephropathy. Mailing address: Tadashi Asami MD Department of Pediatrics, School of Medicine, Niigata University, Asahimachi-dori 757, Niigata, Japan. References 1. Kimmelstiel P, Wilson C: Intercapillary lesions in glomeruli of the kidney. Am J Pathol 12:83-97, Parving HH, Oxenboll B, Svendsen PA, Christiansen JS, Andersen AR: Early detection of patients at risk of developing diabetic nephropathy: a longitudinal study of urinary albumin excretion. Acta Endocrinol (Copenh) 100: , Viberti GC, Hill RD, Jarrett RJ, Argyropoulos A, Mahmud Y, Keen H: Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet 1: Mogensen CE, Christensen CK: Predicting diabetic nephropathy in insulin-dependent patients. New Engl J Med 311:89-93, Mogensen CE: Microalbuminuria as a predictor of clinical diabetic nephropathy. Kidney Int 31: , Ritchie S, Waugh D: The pathology of Armanni Ebstein diabetic nephropathy. Am J Pathol 33: , Whiting PH, Ross IS, Borthwick L: Serum and urine N-acetyl-ƒÀ-D-glucosaminidase in diabetics on diagnosis and subsequent treatment, and stable insulin dependent diabetics. Clin Chim Acta 92: , Ellis EN, Brouhard BH, Lagrone L, Travis LB: Urinary excretion of N-acetyl-ƒÀ-D-glucosaminidase in children with type I diabetes mellitus. Diabetes Care 6: , Milteny M, Korner A, Tulassay T, Szabo A: Tubular dysfunction in type I diabetes mellitus. Arch Dis Child 60: , Watanabe Y, Kunoi K, Maki Y, Nakamura Y, Fusishima M: Contribution of glycemic control to the levels of urinary N-acetyl-ƒÀ-D-glucosaminidase (NAG), total protein, ƒà2-microglobulin and serum NAG in type 1 (insulin-dependent) diabetes mellitus without macroalbuminuria. Clin Nephrol 28: , Asami T, Watanabe S, Sakai K: Urinary ƒà-d-nacetylglucosaminidase (NAG) and urinary low molecular proteins in renal tubular disorders: Differentiation of renal tubular injury and dysfunction. Acta Medica et Biologica 30: , Harano Y, Shigeta Y: Sensitive and simplified method for the differential determination of keton bodies. Clin Chim Acta 134: , Mathiesen ER, Oxenboll B, Johansen K, Svendsen PA, Deckert T: Incipient nephropathy in Type I (insulin dependent) diabetes. Diabetologia 26: , Andersen AR, Christiansen JS, Andersen JK, Kreiner S, Decker T: Diabetic nephropathy in Type I (insulin dependent) diabetes:an epidemiological study. Diabetologia 25: , Krolewski AS, Quarran JH, Cristlieb AR, bunsick EJ, Kahn CR: The changing natural history of nephropathy in type I diabetes. Am J Med 78: , Solerte SB, Fioravanti M: Hemodynamic alteration in long-term insulin-dependent diabetic patients with overt nephropathy: role of blood hyperviscosity and plasma protein changes. Clin Nephrol 28: , Parving HH, Smidt UM, Frisberg B, Bonnevie Nielsen B, Andersen AR: A prospective study of glomerula filtration rate and arterial blood pressure in insulin-dependent diabetics with diabetic nephropathy. Diabetologia 20: , Rosenstock CE, Raskin P: Early diabetic nephropathy assessment and potential interventions. Diab Care 9: , Vivberti GC, Keen H, Wiseman MJ: Raised arterial 62 Japanese Journal of Nephrology Vol. 34, No. 1, 1992

7 Renal tubular and glomerular dysfunction in IDDM 63 pressure in parents of proteinuric insulin dependent diabetics. Br Med J 295: , Krolewski AS, Canessa M, Warram JH, Laffel LMB, Christlieb AR, Knowler W, Rand LI: Predisposition to hypertension and susceptibility to renal diseases in insulin-dependent diabetes mellitus. N Eng JMed 318: , 1987 Japanese Journal of Nephrology Vol. 34, No. 1,

Tubular dysfunction and microalbuminuria in insulin dependent diabetes

Archives of Disease in Childhood, 1988, 63, 244-249 Tubular dysfunction and microalbuminuria in insulin dependent diabetes C WALTON,* H J BODANSKY,* J K WALES,* M A FORBES,t AND E H COOPERt *University