Pediatr Nephrol (2004) 19:1361–1366DOI 10.1007/s00467-004-1610-1

O R I G I N A L A R T I C L E

Peter Holmquist · Sture Sj�blad · Ole Torffvit

Pore size and charge selectivity of the glomerular membraneat the time of diagnosis of diabetes

Received: 3 December 2003 / Revised: 21 June 2004 / Accepted: 22 June 2004 / Published online: 22 October 2004� IPNA 2004

Abstract The urinary albumin excretion rate is in-creased at the time of diagnosis of diabetes. We investi-gated whether this is caused by change in pore size orcharge selectivity in the glomerular basement membrane.Urine excretion of immunoglobulins (IgG2, IgG4), gly-cosaminoglycans (GAG), and albumin was analyzed dur-ing the first 20 days after diagnosis of diabetes in childrenaged 4–15 years; 36 diabetic and 24 age-matched appar-ently healthy children were included. The excretion ofalbumin was significantly increased on day 1 in the dia-betic children. Between day 1 and 20 the excretion ofIgG2 and IgG4 decreased significantly from normal to alevel below normal. GAG excretion was not affected. TheGAG/creatinine index (GAGCI) was normal. IgG2CI wassignificantly below normal on days 4–20. IgG4CI wasbelow normal on days 2–20. The albumin creatinine indexdecreased significantly from day 1 to normal levels onday 4–20. A charge selectivity index, expressed as theratio between the neutrally charged IgG2 and the nega-tively charged IgG4, was significantly below the normallevel on days 16 and 20. In conclusion, an increased al-bumin excretion rate in urine did not seem to be caused bya change in charge selectivity. Other explanations such aschange in the small pore radius or tubular reabsorption aresuggested.

Keywords Albuminuria · Diabetes · Immunoglobulins ·Glycosaminoglycans · Selectivity index

Introduction

The underlying mechanism causing plasma protein to leakinto the urine is not yet fully understood [1]. Hemody-namic changes in hypertrophied kidneys caused by hy-perglycemia at the time of diagnosis has been found toincrease urinary albumin excretion through enlargementof glomeruli and increased glomerular filtration surfacearea [2, 3, 4]. In addition, a reduced proximal tubularreabsorption has been found in patients with microalbu-minuria [5]. Studies also point to a deranged glomerularbasement membrane (GBM) with alterations in pore sizeand charge selectivity as a reason for microalbuminuria[6, 7, 8, 9]. The mean glomerular pore Stoke radius is55 mm [6]. An increase in pore size permits albumin andimmunoglobulins to pass the GBM [6]. In patients withmicroalbuminuria a reduced negative charge over theGBM has been found to permit not only the negativelycharged albumin but even more of the negatively chargedIgG4 to pass, in contrast to neutrally charged IgG2.Normally both albumin and IgG4 are repelled at thenegatively charged pore entrance [7, 10, 11]. The surfaceover the pores is covered with negatively charged gly-cosaminoglycans (GAG). A reduced synthesis of GAGdue to hyperglycemia or a thickening of the GBM isthought to be responsible for the reduced negative chargeover the GBM [7, 8, 12]. A decreased negative chargeover the GBM in microalbuminuric patients selectivelyincreases the IgG4 excretion and creates a low IgG1/IgG4selectivity index [5, 8, 10]. The tubular reabsorption isalso charge dependent, thus it may be difficult to differ-entiate between glomerular filtration and tubular reab-sorption changes as a cause of proteinuria [5]. Tubulardysfunction has been found to increase the excretion oflow molecular weight proteins and persists over a longtime in newly diagnosed diabetes [13, 14]. Ishibashi [5]has shown that under normal conditions tubular reab-sorption of albumin is greater than that of IgG1, which isgreater than that of IgG4. In diabetes there is a greaterreduction in the reabsorption of albumin than IgG1. IgG4is not affected in proximal tubular epithelial cell damage.

P. Holmquist ()) · S. Sj�bladDepartment of Pediatrics,University Hospital,221 85 Lund, Swedene-mail: Peter.Holmquist@skane.seTel.: +46-46171000Fax: +46-46145459

O. TorffvitDepartment of Medicine,University Hospital Lund,Sweden

A selectively increased IgG1 excretion creates a highIgG1/IgG4 selectivity index in patients with deranged tu-bular function [5]. After insulin therapy and in spite ofnormal glucose metabolism and albumin excretion, aprolonged tubular derangement has been suggested innewly diagnosed children [2, 15]. IgG2 and IgG1 have thesame Stoke radius of 55 mm and both are neutrallycharged. Thus, IgG2 may be expected to behave likeIgG4. Microalbuminuria is well known at the time ofdiagnosis of diabetes in children, but less is known aboutthe changes responsible [1].

Our hypothesis is that the only natural human modelfor studying the effects of diabetes on the kidney inpreviously healthy subjects is to study this at the time ofdiagnosis of type 1 diabetes. If an increased albuminexcretion rate was caused by a change in the charge se-lectivity of the GBM this would be treated in a differentway than if it was caused by a change in pore size. Oneway to treat might be to restore the charge by low mo-lecular heparin or GAG. Thus our aim was to studywhether the increased urinary albumin excretion rate atthe time of diagnosis of diabetes is caused by a change inpore size or charge selectivity in the GBM. To that endwe measured urinary excretion rates of IgG2, IgG4, andalbumin as markers since their size and charge are dif-ferent. The urinary excretion of GAG was studied as asubstitute for measuring the synthesis of GAG.

Materials and methods

Subjects

Subjects participated in an ongoing study of diabetic children in thelocal recruiting area of the Department of Pediatrics, UniversityHospital in Lund, constituting approximately 225,000 inhabitantsof whom 50,000 are under the age of 15 years. Inclusion criteria forjoining this part of the study were diabetes mellitus, initial diag-nosis and treatment at our department, age 4–16 years, Caucasianorigin, and no other ongoing disease. There were 36 children in-

cluded. As controls, 24 healthy school children of similar ageparticipated. All participants and parents gave their informed con-sent. The study was approved by the local ethics committee.

Metabolic treatment

The study period started on the 1st day (day 1) after diagnosis (day0) and ended after 20 days of treatment when the metabolic de-rangement was corrected and the patients were normoglycemic(day 20). All children were initially treated with continuous intra-venous insulin at 0.05–0.20 U/kg per hour together with sa-line solution (130 mmol Na, 4 mmol K, 2 mmol Ca, 1 mmol Mg,30 mmol acetate, 110 mmol Cl) at 4–10 ml/kg per hour. During thefirst 12 h of treatment the goal for the blood glucose level was10 mmol/l. When the blood glucose was below 10 mmol/l the salinesolution was changed to 5% glucose with 40 mmol/l of Na andK. Within 36 h the intravenous insulin treatment was ended andsubcutaneous insulin injections started. Fast-acting insulin was in-troduced as four divided doses during the subsequent 3–5 days.Thereafter, medium-acting insulin was given starting with anovernight dose. At discharge, after 3 weeks most children hadcombined doses of fast- and medium-acting insulin in the morningand afternoon and an overnight dose of medium-acting insulin.Older children were even treated with rapid-acting insulin atlunchtime.

Methods

On day 1 and day 20 fasting venous blood samples were takenbetween 7.00 and 9.00 a.m. Overnight timed urine collections wereanalyzed for GAG by a method previously described with a de-tection limit of 1.5 mg/l [16]. Albumin [17], IgG2, and IgG4 [10]were analyzed with ELISA techniques previously described. Serumand urinary concentrations of creatinine were analyzed by an en-zymatic method (creatinine hydrolase, EKTA Chem-analyser, In-strument Kodak, N.Y., USA). Serum creatinine reference values forchildren <5 years are 35–70 mol/l, 5–10 years 40–85 mol/l, and 10–15 years 50–90 mol/l. Glomerular filtration rate (GFR) was mea-sured using iohexol (Omnipaque 300 mg/ml non-ionic contrastmedium) [3] on day 1 and day 20 in a non-fasting state. Age-relatedreference values using capillary measurements at 3 and 4 h after anintravenous injection of iohexol ranged from 75 to 125 ml/min.Ketonuria was measured on the day of diagnosis (day 0) with urineKetodiabur dipsticks (Boehringer Mannheim, Mannheim, Ger-many). Body mass index (BMI) was calculated as kilograms per

Table 1 Baseline data in subjects with diabetes on day 1 and day 20 and control children

Diabetic children day 1 Control children Diabetic children day 20

(n=36) (n=24) (n=36)

Age (years) 10 (4–16) 10 (7–12)Gender (M/F) 18/18 11/13BMI (kg/m2) 15.6 (12.6–31.6)* 16.7 (13.8–26.8) 17.8 (14.8–31.6)

(n=23)Blood glucose, mean per day (mmol/l) 10.4 (6.8–16.7)* 5.6 (4.4–8.9)

(n=35)Iohexol clearance (ml/min per 1.73 m2) 129 (93–223) 130 (87–268)

(n=29) (n =27)Serum creatinine (�mol/l)HbA1c (%) 9.9 (5.6–15.3)

(n=35)pH 7.37 (7.06–7.44)

(n=35)Base excess (mmol/l) �0.2 (�26 to 5.2)

(n=35)

* P<0.001 for day 1 versus day 20 in diabetic children

1362

square meter. HbA1c was measured on the day of diagnosis (day 0)with high-speed equipment (DCA 2000, Bayer, Germany) or cationexchange chromatography on high-performance liquid chromatog-raphy (Auto-A, Kyoto-Diaiichi, Kagaku, Kyoto, Japan) both with anormal value of 4%–6%. For blood glucose, Hemocue (�ngel-holm, Sweden) and Glucometer Elite (Bayer Diagnostica, Zurich,Switzerland) were used and daily mean blood glucose calculatedfrom a minimum of five measurements.

Statistics

Results are given as the median and range. Spearman’s correlationcoefficient and Mann Whitney U test were used. For differences innumber between groups we used chi-squared with Fisher’s exacttest. Patients were tested using a general linear model for the effect.When significant differences after log10 transformation were de-tected for differences of repeated measures on several days theywere further tested with Wilcoxon’s test for the P value on eachday. Significance was taken as P <0.05 (two-tailed). The analysis ofthe data was performed using the Statistic Package for SocialSciences (SPSS, Chicago, USA).

Calculations

Excretion rates and clearances were calculated in relation to1.73 m2 body surface area (weight0.425�height0.725�71.84/100) toadjust for size and gender. The charge selectivity index was cal-culated using the ratio between urinary concentrations of IgG2 andIgG4. The urinary albumin/creatinine ratio, albumin creatinine in-dex (ACI), was used to estimate the degree of albuminuria [18]. Asimilar index was calculated for IgG2 (IgG2CI), IgG4 (IgG4CI),and GAG (GAGCI).

Results

Table 1 shows baseline data with a significant differencefor BMI and median blood glucose on day 1 versus day 20in children with diabetes. Table 2 shows timed overnighturine protein excretion. On day 1 the excretion of albuminwas significantly increased in diabetic children. From day4 the albumin excretion was significantly lower than onday 1, but was not different from non-diabetic children.The excretion of IgG2 and IgG4 was significantly lowerthan for non-diabetic children (days 4–20 and 2–20 re-spectively). The excretion of GAG was not significantlydifferent over time or versus non-diabetic children. Thesubgroup above normal (4 �g/min per 1.73 m2) did nothave changed handling of the proteins studied (data notshown). A significant increase in the ACI was found ondays 1 and 2 compared with the non-diabetic children(Fig. 1). A significantly reduced IgG2CI and IgG4CI wasfound on days 4–20 and days 2–20 respectively comparedwith the non-diabetic children (Figs. 2, 3). A significantlylow selectivity index (IgG2/IgG4) is shown on days 16and 20 compared with the non-diabetic children (Fig. 4).No significant change for GAGCI was found (Fig. 5).

Possible effects of dehydration

In order to study the effects of dehydration caused by thediabetic state we compared patients above and below the T

able

2U

rine

excr

etio

nra

tes

per

1.73

m2

body

surf

ace

area

inni

ght

urin

eco

llec

tion

in36

diab

etic

and

24co

ntro

lch

ildr

en.

Uri

nary

excr

etio

nsda

y1

toda

y20

indi

abet

icch

ildr

ena

Day

saf

ter

diag

nosi

s1

24

516

20C

ontr

olch

ildr

en

IgG

4(n

g/m

inpe

r1.

73m

2)

533

301*

2,

*528

2*3,

*620

7*3,

*637

6*2,

*448

2*3,

*494

7(1

19–5

6,00

3)(3

.7–2

,219

.3)

(52.

4–1,

836)

(64.

1–2,

912)

(48.

1–6,

006)

(58.

5-9,

806)

(111

.1–8

,207

)(n

=33

)(n

=34

)(n

=33

)(n

=34

)(n

=34

)(n

=19

)Ig

G2

(ng/

min

per

1.73

m2)

1,29

165

256

1*3,

*641

1*3,

*537

6cf

435*

3,

*62,

141

(156

–213

,109

)(3

.7–8

8,41

1)(5

2.4–

33,5

37)

(71.

4–23

,493

)(4

8.1–

6,00

6)(5

8.5–

10,8

975)

(159

.5–2

4,31

4)(n

=33

)(n

=34

)(n

=33

)(n

=34

)(n

=34

)(n

=19

)A

lbum

in7.

0*1

4.8

3.7*

43.

9*4

2.5*

42.

5*6

4.1

(�g/

min

per

1.73

m2)

(0.4

–60.

5)(0

.2–6

3.6)

(0.9

–88.

6)(0

.4–8

9.6)

(0.2

–79.

5)(0

.2–2

3.9)

(0.5

–10.

1)(n

=35

)(n

=32

)(n

=33

)(n

=32

)(n

=33

)(n

=33

)(n

=19

)G

AG

39.6

(2.7

–137

.2)

35.4

36.1

37.8

35.6

38.1

39.2

(�g/

min

per

1.73

m2)

(0.8

–478

.6)

(0.6

–88.

8)(6

.6–1

02.2

)(7

.8–8

2.1)

(4.4

–66.

1)(5

.4–5

9.6)

(n=

35)

(n=

30)

(n=

31)

(n=

32)

(n=

33)

(n=

33)

(n=

19)

*1P

<0.

05,

*2P

<0.

01,

*3P

<0.

001

for

diab

etic

vs.

cont

rol

chil

dren

;*4

P<

0.05

,*5

P<

0.01

,*6

P<

0.00

1fo

rda

y1

vs.

days

2,4,

5,16

,an

d20

indi

abet

icch

ildr

ena

Dat

aar

egi

ven

asm

edia

nan

dra

nge

1363

median BMI (15.55, range 12.6–31.6 kg/m2), HbA1c (9.9,range 3.6–15.3%), blood glucose at admission (22.0,range 9.1–49.0 mmol/l) and the mean during the 1st dayof admission (10.4, range 6.8–16.7 mmol/l), serum cre-atinine (44, range 29–75 �mol/l), and serum sodium (136,range 121–147 mmol/l). For all urinary parameters wefound no effect of dehydration except for low BMI versushigh BMI, i.e., dehydrated versus normally hydrated pa-tients, albumin excretion rate corrected for surface area11.49 (2.96–60.53) versus 5.41 (0.35–20.42) �g/min per1.73 m2 (P<0.05), ACI 1.84 (0.65–14.56) versus 0.94(0.05–3.9) �g/mmol (P<0.01), and GCI 7.81 (2.84–17.74)versus 5.81 (0.31–10.8) �g/mmol (P<0.05). We found nodifference for HbA1c and serum creatinine. For high bloodglucose at entry versus low blood glucose, i.e., dehydratedversus normally hydrated patients, the GAG excretionrate corrected for body surface area was 35.0 (17.3–68.1)

versus 46.9 (2.7–137.2) �g/min per 1.73 m2 (P<0.05). Forhigh blood glucose mean level on the 1st day of admis-sion versus low blood glucose, i.e., dehydrated vs. nor-mally hydrated patients, the GAG excretion rate correctedfor body surface area was 36.1 (20.3–49.6) versus 48.1(2.7–137.2) �g/min per 1.73 m2 (P<0.05). For high serumsodium versus low serum sodium, i.e., dehydrated versusnormally hydrated patients, the IgG2/IgG4 ratio was 2.36(0.7–32.2) versus 1.47 (0.35–94.95) (P<0.05).

Discussion

We found a high excretion of albumin on day 1 thatnormalized on day 4. The excretion of GAG was un-changed. The excretion of IgG2 and IgG4 was not in-

Fig. 1 Urinary albumin creatinine index (ACI) (�g/mmol) (median)in diabetic children (solid line) and control children (stippled line).* P <0.05, *** P <0.001 vs. control children

Fig. 2 Urinary IgG2 creatinine index (IgG2CI) (�g/mmol) (medi-an) in diabetic children (solid line). Median of control children(2.73) not shown. * P <0.05, ** P <0.01, *** P <0.001 vs. controlchildren

Fig. 3 Urinary IgG4 creatinine index (IgG4CI) (�g/mmol) (medi-an) in diabetic children (solid line). Median of control children(2.39) not shown. * P <0.05, ** P <0.01, *** P <0.001 vs. controlchildren

Fig. 4 Charge selectivity index (IgG2/IgG4) in diabetic children(solid line) and control children (stippled line). ** P <0.01 vs.control children

1364

creased on day 1 but was significantly decreased on days4–20 compared with the non-diabetic children. The IgG2/IgG4 selectivity index was normal at entry but signifi-cantly lower on days 16 and 20 compared with the non-diabetic children. At admission, the diabetic children hadhigh blood glucose and the greatest changes should thusbe found on day 1. A high excretion of albumin was in-deed found. This may be due to increased filtrationthrough GBM and/or decreased tubular reabsorption.Albumin is filtered in small amounts through small-sizedpores of the GBM, whereas immunoglobulins are filteredthrough the larger pores [6]. An increased albumin ex-cretion through GBM may thus be due to an increasedpore size or decreased negative charge of the GBM. In thepresent study, urinary excretion of immunoglobulins wasnot affected on day 1. As both IgG2 and IgG4 had lowexcretion rates it seems that larger pores were not in-volved. A low excretion rate of IgG2 and IgG4 may becaused by an increased tubular reabsorption caused byhyperfunctioning large kidneys induced by the hyper-glycemia [2]. As the urinary volumes were increased atadmission [15] this might be of significance for tubularreabsorption. In the present report we have not given themeasured urinary volumes as this has been presented in aprevious separate report [15]. In that study we were notable to explain the changes in tubular function by changein urinary volumes [15], although we did not measuretubular reabsorption rates.

A low excretion of GAG has previously been found tocorrelate with a reduced negative charge over the GBM[10]. GAG was unaffected during the observation time.Whether the excretion of GAG can serve as a reliablemarker for glomerular charge selectivity is unknown.Negatively charged sugar moieties, local pH, sulfatases,and heparanase may also be of interest. With a loss ofnegative charge, IgG4 should pass through the pores inequal amounts to IgG2 instead of being repelled. On day1 no change in charge selectivity seemed to affect the

greater pores as the IgG2/IgG4 ratio was unchanged. Thisis in contrast to microalbuminuric type 1 diabetic patientswho have a reduced selective index that improves whenblood glucose is normalized [19]. On the subsequent daysIgG2 and IgG4 excretion was significantly decreasedcompared with day 1 and with the non-diabetic children.On day 20, when glucose metabolism was normal, theneutral-charged IgG2 was decreased more than the neg-atively charged IgG4. A reduced negative charge over theGBM would lead to increased filtration of both IgG4 andalbumin. Decreased IgG2 and IgG4 excretion could hy-pothetically be caused by a reduced pore size or an in-creased tubular reabsorption. Large kidneys and in par-ticular increased tubular mass may explain the change inreabsorption [2]. If the consequence is increased tubularreabsorption this might lead to decreased excretion ofIgG2 but leave IgG4 unchanged. A tubular dysfunctionthat persists over a long period has been previously de-scribed in diabetic children [13, 14, 15]. In the presentstudy we did not measure other proteins like b2-micro-globulin that are reabsorbed by the proximal tubules.Reversible tubular proteinuria has been suggested toprecede microalbuminuria [14] in one study and to be ofno predictive value in another [20]. At present we have noappropriate control subjects to indicate whether our re-sults are specific to diabetes or not. In the present studywe have tried to evaluate the effect of dehydration onurinary excretion of our different markers. We consideredlow BMI, high HbA1c or serum creatinine, high bloodglucose at entry or during the 1st day at hospital, and highserum sodium as a sign of dehydration. Thus, we foundthat dehydration was associated with increased excretionof albumin but a decrease in GAG and the IgG2/IgG4ratio. Although the consequences of type 1 diabetes can-not be perfectly mimicked, studies of children with non-diabetic dehydration with or without acidosis and hyper-osmolar states are necessary.

In conclusion, we suggest that the albuminuria at thetime of diagnosis of diabetes does not reflect a change incharge but may be caused by a change in selective poresize or decreased tubular reabsorption. An increased tu-bular reabsorption of IgG2 is at present speculative.

Acknowledgements The study was supported by grants fromAlm�rs Foundation, Skane County Council Foundation, SwedishDiabetes Federation, and University of Lund. The help of �saPettersson, Renal Research Laboratory, and the staff of the Pedi-atric Department in Lund and Inger Marie Jensen is greatly ap-preciated.

References

1. Jalanko H (2003) Pathogenesis of proteinuria: lessons learnedfrom nephrin and podocin. Pediatr Nephrol 18:487–491

2. Sandahl Christiansen J, Gammelgaard J, Tronier B, AabySvendsen P, Parving H-H (1982) Kidney function and size indiabetics before and during initial insulin treatment. Kidney Int21:683–688

Fig. 5 Urinary glycosaminoglycan creatinine index (GCI) (�g/mmol) in diabetic children (solid line) and control children (stip-pled line). No difference vs. control children was noted

1365

3. Krutzen E, Bck SE, Nilsson-Ehle P (1984) Plasma clearanceof a new contrast agent, iohexol: a method for the assessment ofglomerular filtration rate. J Lab Clin Med 104:955–961

4. Throssell D, Harris KPG, Bevington A, Furness PN, Howie AJ,Walls J (1996) Renal effects of metabolic acidosis in the nor-mal rat. Nephron 73:450–455

5. Ishibashi F (1993) Glomerular clearance and tubular reab-sorption of IgG1 and IgG4 in microalbuminuric patients withnon-insulin-dependent diabetes mellitus (NIDDM). DiabetesRes Clin Pract 22:45–51

6. Tencer J, Torffvit O, Thysell H, Rippe B, Grubb A (1998)Proteinuria selectivity index based upon a2-macroglobulin orIgM is superior to the IgG based index in differentiatingglomerular diseases. Kidney Int 54:2098–2105

7. Goode NP, Shires M, Crellin DM, Aparicio SR, Davison AM(1995) Alterations of glomerular basement membrane chargeand structure in diabetic nephropathy. Diabetologia 38:1455–1465

8. Brenner BM, Hostetter TH, Humes HD (1978) Glomerularpermselectivity: barrier function based on discrimination ofmolecular size and charge. Am J Physiol 234:455–460

9. Deckert TD, Feldt-Rasmussen B, Djurup R, Deckert M (1988)Glomerular size and charge selectivity in insulin-dependentdiabetes mellitus. Kidney Int 33:100–106

10. Torffvit O, Rippe B (1999) Size and charge selectivity of theglomerular filter in patients with insulin-dependent diabetesmellitus: urinary immunglobulins and glycosaminoglycans.Nephron 83:301–307

11. Tencer J, Torffvit O, Thysell, H, Rippe B, Grubb A (1999)Urine IgG2/IgG4-ratio indicates the significance of the glom-erular capillary wall for the macromolecular transport inglomerular diseases. Nephrol Dial Transplant 14:1425–1429

12. Tamsma JT, Born J van den, Bruijn JA, Assman KJM, WeeningJJ, Berden JHM, Wieslander J, Schrama E, Hermans J,Veerkamp JH, Lemkes HHPJ, Woude FJ van der (1994) Ex-pression of glomerular extracellular matrix components in hu-

man diabetic nephropathy: decrease of heparan sulphate in theglomerular basement membrane. Diabetologia 37:313–320

13. Widstam-Attorp U, Berg U (1992) Urinary protein excretionand renal function in young people with diabetes mellitus.Nephrol Dial Transplant 7:487–492

14. Ginevri F, Piccotti E, Alinovi R, DeToni T, Biagini C, ChiggeriGM, Gusmano R (1993) Reversible tubular proteinuria pre-cedes microalbuminuria and correlates with the metabolic sta-tus in diabetic children. Pediatr Nephrol 7:23–26

15. Holmquist P, Torffvit O, Jørgensen PE, Tørring N, Nexø E,Sj�blad S (2001) Early urinary changes in Tamm-Horsfallprotein and epidermal growth factor in diabetic children.Pediatr Nephrol 16:488–492

16. Tencer J, Torffvit O, Grubb A, Bj�rnsson S, Thysell H, Rippe B(1997) Decreased excretion of urine glucosaminoglycans asmarker in renal amyloidosis. Nephrol Dial Transplant 12:1161–1166

17. Torffvit O, Wieslander J (1986) A simplified enzyme-linkedimmunosorbent assay for urinary albumin. Scand J Clin LabInvest 46:545–548

18. Dyson EH, Will EJ, Davison AM, O’Malley AH, Shepard HT,Jones RG (1992) Use of the urinary protein creatinine index toassess proteinuria in renal transplant patients. Nephrol DialTransplant 7:450–452

19. Bangstad HJ, Kofoed-Enevoldsen A, Dahl-Jørgensen K,Hanssen KF (1992) Glomerular charge selectivity and the in-fluence of improved blood glucose control in type 1 (insulin-dependent) diabetic patients with microalbuminuria. Diabeto-logia 35:1165–1169

20. Schultz CJ, Dalton RN, Neit HAW, Konopelska-Bahu T,Dunger DB on behalf of the Oxford Regional Prospective StudyGroup (2001) Markers of renal tubular dysfunction measuredannually do not predict risk of microalbuminuria in the firstfew years after the diagnosis of type I diabetes. Diabetologia44:224–229

1366