ORIGINAL ARTICLE

Urinary transforming growth factor-b1, collagen IV and the effect ofinsulin in children at diagnosis of diabetes mellitus

PETER HOLMQUIST1 & OLE TORFFVIT2

1Department of Paediatrics and 2Institute of Clinical Sciences, Department of Renal Medicine, University Hospital Lund,

Sweden

AbstractObjective. This study investigated whether metabolic derangement at diagnosis of diabetes mellitus affects the function ofthe basement membrane and the excretion of several components and whether insulin treatment can normalize this. It wasdesigned to evaluate urinary excretion rates of transforming growth factor-b1 (TGF-b1), the carboxy-terminal domain ofcollagen IV (NC1) and albumin in children during the first 20 days of treatment after diagnosis of type 1 diabetes. Materialand methods. Thirty-four newly diagnosed diabetic children between 4 and 16 years of age and 26 healthy children ofmatching age were studied with timed overnight urine collections. Urine was collected during the first 20 days of treatment.Results. Urinary excretion of albumin and TGF-b1 in diabetic children were significantly increased at entry but normalizedduring 20 days of treatment with insulin compared with control children. In contrast, the non-significant high NC1excretion at diagnosis did not change but became significantly increased after 20 days of insulin treatment. Overall, thekidney size was within normal limits and unaffected by treatment. The largest kidneys had less NC1 excretion (R��0.67,pB0.05, n�13) and a lower glomerular filtration rate (R��0.77, pB0.01, n�10) than the smallest kidneys. After20 days of treatment TGF-b1 excretion had decreased in children with kidney size�8.5 cm. Conclusion. Correction of themetabolic derangement with insulin decreased excretion of albumin and TGF-b1, but had no effect on kidney size and urineNC1 excretion, presumably because the observation period was too short.

Key Words: Albuminuria, children, collagen IV, type 1 diabetes, transforming growth factor-b

Introduction

Urine cytokines and growth factors are involved in

the development of glomerular and tubular hyper-

trophy and basement membrane thickening, char-

acteristics of diabetic nephropathy [1�4]. An

important cytokine in this context is transforming

growth factor-b1 (TGF-b1) [5]. This is up-regulated

by hyperglycaemia within 24 h of onset of diabetes in

the rat model and attenuated by insulin [6]. TGF-b1

is implicated in the pathogenesis of diabetic glomer-

ulosclerosis and tubulointerstitial fibrosis [7,8]. Dia-

betic patients with an increased urinary TGF-b1

excretion rate are prone to developing nephropathy

[8�10]. TGF-b1 stimulates collagen formation in the

extracellular matrix [1,7,9,11] and proteoglycan

synthesis [12]. One of the end-products is collagen

IV [1]. A high urine collagen IV excretion rate in

diabetic patients has been described as being asso-

ciated with renal function and size [7,13,14].

High blood glucose stimulates and angiotensin-

converting enzyme (ACE) inhibition decreases

TGF-b1 levels [9,11,15]. A direct effect of insulin

on the TGF-b1 excretion rate has been suggested

[14,16]. Few studies on urinary TGF-b1 excretion

are available in newly diagnosed diabetic patients

[17], especially in children [16,18,19]. In rat models

ACE inhibition [20] and monoclonal anti-TGF-bantibodies [21] affect TGF-b1 and collagen IV. The

combination fully arrests the renal injury of diabetic

nephropathy [22].

The hypothesis in this study was that correction of

the metabolic derangement with insulin affects the

function of the basement membrane and several of

its components and that insulin treatment may

normalize this. In humans studies of the kidney

Correspondence: P. Holmquist, Department of Paediatrics, University Hospital, SE-221 85 Lund, Sweden. Tel: �46 46 171000. Fax: �46 46 145459.

E-mail: Peter.Holmquist@skane.se

Scandinavian Journal of Urology and Nephrology, 2009; 43: 142�147

(Received 13 February 2008; accepted 23 September 2008)

ISSN 0036-5599 print/ISSN 1651-2065 online # 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)

DOI: 10.1080/00365590802502111

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have to be done indirectly. Thus, the study was

designed to evaluate urinary excretion rates of TGF-

b1, the carboxy-terminal domain of collagen IV

(NC1), glycosaminoglycans (GAG), Tamm-Horsfall

protein (THP) and albumin in children during the

first 20 days of treatment after diagnosis of type 1

diabetes.

Material and methods

Subjects

The recruiting area of the Department of Paedia-

trics, University Hospital in Lund, has approxi-

mately 250 000 inhabitants, of whom 50 000 are

under the age of 15 years. Included in this study

were children with initial diagnosis and subsequent

treatment at this department. Thirty-four children

were between 4 and 16 years of age. As control

subjects, 26 healthy school children of similar age

participated. All participants were of Caucasian

origin. Children and parents gave their informed

consent, and the local ethics committee approved

the study.

Metabolic treatment

The study started at diagnosis of diabetes and ended

after 20 days of treatment at the hospital. During

this time metabolic derangement was corrected and

the blood glucose normalized. After admission, all

children were treated with intravenous insulin at

0.05�0.20 U/kg/h together with saline solution

(130 mmol Na, 4 mmol K, 2 mmol Ca, 1 mmol

Mg, 30 mmol Ac, 110 mmol Cl) at 4�10 ml/kg/h.

Treatment during the first 12 h aimed at blood

glucose 10 mmol/l. When blood glucose was stable

(B10 mmol/l) the saline solution was changed to

5% glucose and 40 mmol/l of sodium and potassium

added. Within 36 h the intravenous was changed to

subcutaneous insulin injections. Fast-acting insulin

was given as four divided doses during the subse-

quent 3�5 days. Thereafter, medium-acting insulin

was started with an overnight dose. At discharge

after 3 weeks most children had combined doses of

fast- and medium-acting insulin in the morning and

afternoon, and medium-acting insulin was given at

night. Older children were treated with rapid-acting

insulin at lunchtime.

Methods

On days 1 and 20 venous blood tests were taken in a

resting position and fasting between 07.00 and

09.00 h. Timed overnight urine collections were

analysed. Albumin [23], THP [24] and NC1 [13]

were analysed with an enzyme-linked immunosor-

bent assay (ELISA) technique as described pre-

viously. GAG was analysed as described previously

[25]. Biologically active TGF-b1 was analysed with a

commercially available solid-phase sandwich ELISA

(Emax Immunoassay System; Promega Corp., Ma-

dison, WI, USA). Serum and urinary concentrations

of creatinine were analysed by an enzymic method

(creatinine-hydrolase; EKTA Chem-analyser Instru-

ment, Kodak, New York, USA). Serum creatinine

reference values for childrenB5 years�35�70 mol/l,

5�10 years�40�85 mol/l and 10�15 years of age�50�90 mol/l. Ketonuria was measured on the day of

diagnosis (day 0) with urine Ketodiabur Dipsticks

(Boehringer Mannheim, Mannheim, Germany).

Glycosylated haemoglobin (HbA1c) was measured

on the day of diagnosis with high-speed equipment

(DCA 2000; Bayer, Germany) or cation-exchange

chromatography on high-performance liquid chro-

matography (HPLC) (Auto-A; Kyoto-Diaiichi, Ka-

gaku Co., Kyoto, Japan), both with a normal value of

4�6%. The methods were adjusted to give similar

results. Hemocue (Angelholm, Sweden) and Gluc-

ometer Elite (Bayer Diagnostica, Zurich, Switzer-

land) were used to measure blood glucose. Blood

glucose on days 1 and 20 was calculated as the mean

of a minimum of five measurements a day.

A trained paediatric radiologist measured kidney

size with ultrasonography (Toshiba SSA-270A). The

kidney size was taken as the mean from three

measurements of the length in both kidneys. They

were judged according to renal ultrasound measure-

ments described in control children aged 9�10

years�9.2 cm (SD 0.9) and 10�11 years�9.2 cm

(SD 0.8) [26]. According to these results kidney size

is between 8 and 10 cm for children between 4 and

16 years of age. Glomerular filtration rate (GFR)

was measured as the clearance of an intravenous

injection of 5 ml of iohexol (Omnipaque 300 mg I/

ml non-ionic contrast medium) on days 1 and 20 in

a non-fasting state [27]. Age-related reference values

using capillary measurements at 3 and 4 h ranged

from 75 to 125 ml/min/1.73 m2.

Statistics

Results are given as the median and range. Spear-

man’s correlation coefficient and Mann�Whitney U

test were used. For differences between and within

groups on different days chi-square with Fisher’s

exact test was used. Differences between different

days were tested with Wilcoxon’s paired test. Sig-

nificance was taken as pB0.05 (two-tailed). The

data were analysed using the Statistical Package for

Social Sciences (SPSS, Chicago, IL, USA).

TGF-b1, collagen and insulin in diabetes 143

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Calculations

Body mass index (BMI) was calculated as kg/m2.

Excretion rates were calculated in relation to

1.73 m2 body surface area (�weight0.425�height0.725�71.84/100) to adjust for size and gender

or as the ratio between the substances measured and

urine creatinine.

Results

At diagnosis base deficit was �0.2 (�26 to 5)

mmol/l, pH 7.37 (7.06�7.44) and blood glucose

21.9 (9.1�49) mmol/l. Twenty-one out of 34 chil-

dren had ketonuria at entry. Urinary excretion rates

of albumin, TGF-b1, THP, GAG and NC1 were

similar in children with and without ketonuria and

not affected by gender (data not shown). Table I

shows the baseline and day 20 data for all diabetic

and control children. At entry urinary TGF-b1 and

albumin were significantly higher in diabetic than in

control children. On day 20 albumin and TGF-b1

had normalized. There was no significant difference

between NC1 excretion at entry and day 20, and on

day 20 the excretion was significant higher than in

control children. After 20 days BMI was increased,

insulin dose decreased from 1.4 (0.0�3.5) to 0.6

(0.2�1.0) units/kg (p�0.000) and the median of the

daily mean blood glucose decreased. THP and GAG

excretion were unchanged during the treatment

period.

Kidney size was measured in 14 patients at entry

and repeated in five patients on day 20. In addition,

two patients not measured at entry were tested on

day 20. Thus, in total seven patients were measured

on day 20. The kidney size was normal in compar-

ison with a normal reference material with a kidney

size of 8�10 cm for children between 4 and 16 years

of age [26]. In the present study, the children had a

median age of 10 years and a median kidney size of

9 cm. No correlation was found between kidney size

on day 1 (0.509, n�14) and day 20 (0.382, n�7)

and age. There was no change in renal size or GFR

during the 20 days. At entry patients with largest

kidneys had a low NC1 excretion rate (R��0.77,

p�0.002, n�13) (Figure 1) and decreased GFR

(R��0.77, p�0.009, n�10) (Figure 2). In con-

trast, on day 20 patients with large kidneys had a

high excretion of albumin (R�0.86, p�0.014) and

Figure 1. At diagnosis, diabetes patients with the largest kidneys

had low urinary excretion of the C-terminal domain of collagen IV

(NC1)/min/1.73 m2 (R��0.77, p�0.002, n�13).

Table I. Data on diabetic children on days 1 and 20, and control children.

Diabetic day 1 Diabetic day 20 Control

(n�34) (n�31) (n�26)

Age (years) 10 (4�16) 10 (7�12)

Gender (M/F) 17/17 15/16 8/13

BMI 16 (13�23) 17 (13�23)** 17 (14�27)

HbA1c (%) 10.0 (5.6�15.3)

Mean blood glucose (mmol/L) 11.0 (6.8�16.7) 5.7 (4.4�8.9)***

S-Creatinine (mmol/l) 44 (29�75) 45 (32�73)

GFR (ml/min/1.73 m2) 132 (93�223) 130 (87�187)

Kidney size (cm) 9.0 (7.3�10.5) (n�14) 8.7 (7.7�10.3) (n�7)

Albumin/creatinine (mg/mmol) 1.1 (0.05�14.6)$$ 0.45 (0.04�4.9)** 0.55 (0.03�26.6)

TGF-b/creatinine (ng/mmol) 0.08 (0.01�73.1)$ 0.03 (0.01�32.0) 0.02 (0.01�3.1)

NC1/creatinine (ng/mmol) 1.5 (0.2�6.3) 2.0 (0.4�25.6)$ 0.8 (0.5�3.4)

THP/creatinine (mg/mmol) 5.54 (0.09�23.7) 3.21 (0.18�65.3) 9.24 (0.5�22.1)

GAG/creatinine (mg/mmol) 6.5 (0.3�17.7) 5.8 (1.2�69.5) 8.2 (4.5�13.4)

Data are given as median (range).

BMI�body mass index; HbA1c; glycosylated haemoglobin; GFR�glomerular filtration rate of iohexol; TGF�transforming growth factor;

NC1�C-terminal domain of collagen IV; THP�Tamm-Horsfall protein; GAG�glycosaminoglycans.

***pB0.001, **pB0.01 vs day 1; $$pB0.01, $pB0.05, vs control.

144 P. Holmquist & O. Torffvit

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a larger insulin dose (R�0.81, p�0.027) than

patients with smallest kidneys.

At entry, no association was found between NC1

excretion and GFR or TGF-b1, nor between TGF-

b1 and kidney size and GFR. However, if urine data

from controls were included, a correlation was

revealed between urine NC1 and TGF-b1 excretions

on day 1 (R�0.39, p�0.006, n�49) but not on

day 20 (R�0.27, p�0.072, n�46). No associations

were found between GFR, albumin, GAG, THP,

NC1 or TGF-b1 excretions and HbA1c, or mean

blood glucose. Urine NC1 was associated with GAG

(R�0.39, p�0.026, n�33), which was associated

with THP (R�0.49, p�0.004, n�34), which was

associated with NC1 (R�0.52, p�0.002, n�33).

On day 20, a high insulin dose per kilogram body

weight was associated with increased urinary excre-

tion of TGF-b1 (R�0.36, p�0.046). On day 20,

TGF-b1 was associated with renal size (R��0.76,

p�0.049, n�7). Five out of seven patients with

kidney size above 8.5 cm had no excretion of

TGF-b1.

Discussion

At diagnosis of diabetes mellitus, increased urinary

excretion rates of albumin, TGF-b1 and NC1 were

found compared with control children. Treatment

for 20 days normalized albumin and TGF-b1 but not

NC1 excretion rates. A high blood glucose stimu-

lates hypertrophy of tubular cells [4,11], and in-

creased urinary TGF-b1 excretion [3,9,11] and

collagen IV production [4,9]. Metabolic control,

measured as HbA1c, may [17,28] or may not

[16,18,19] correlate with urinary TGF-b1 excretion.

In this study, no association was found between

HbA1c, blood glucose and TGF-b1 excretion at

entry. Sustained high urinary TGF-b1 excretion

has been found in children at the onset of diabetes

[16] and in prepubertal and postpubertal children

[19]. However, the excretion of TGF-b1 may also be

low [18]. An elevated TGF-b1 excretion may be the

result of an inflammatory reaction or an immunolo-

gical response mediated by TGF-b1 itself [17]. In

the kidneys TGF-b1 is filtered across the glomerular

capillaries or secreted from epithelial cells in the

tubular system [7].

Insulin increases TGF-b1 production [14,16] but

may decrease urinary excretion [11,28]. Treatment

with insulin decreased urinary excretion of albumin

and TGF-b1, but did not significantly affect NC1

excretion and kidney size. Whether this is due to

insulin itself or to normalization of glycaemia or any

other effect of insulin is unclear. The normalization

of high TGF-b1 excretion in the present study may

reflect an effect of insulin treatment [11,28] and an

improved metabolic control [17]. An association

between insulin dose and TGF-b1 excretion was

actually found on day 20. Insulin affects both renal

size and GFR [29]. Renal size decreases with

achievement of good metabolic control [30] and

persistent large kidneys are predictive of the devel-

opment of renal insufficiency [31]. In the present

study the median renal size for children between 4

and 16 years of age was within normal limits of 8�10 cm [26] and did not change during treatment.

Those children with the largest kidneys on day 1 had

reduced GFR and excreted less NC1. A low GFR

due to poor metabolic control may be compensated

by an increase in renal size (tubular mass). Since

kidney size was measured twice in only five patients

and in another two patients only on day 20 these are

uncertain data regarding changes in or related to this

parameter. The association between NC1 and GAG

and THP found in the present study indicates that

NC1 production may be increased not only in the

glomerular basement membrane but even in the

tubular membranes. However, those with the largest

kidneys at entry had a decreased NC1 excretion.

This is in contrast to a positive correlation between

NC1, GFR and kidney size previously found in an

adult population of patients with a long duration of

diabetes [32]. As the adult population was in a stable

metabolic status the negative association in the

present study must be caused by a deranged meta-

bolic status at entry characterized by glucosuria and

insulin deficiency [17]. A decreased excretion of

THP is in keeping with an experimental study in the

diabetic rat showing decreased production in large

kidneys [33]. Similarly, the synthesis of NC1 and

Figure 2. At entry patients with largest kidneys had decreased

glomerular filtration rate (GFR) (R��0.77, p�0.009, n�10).

TGF-b1, collagen and insulin in diabetes 145

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GAG may be expected to decrease, as decreased

excretion of TGF-b1 was seen in large kidneys.

Diabetic patients without microalbuminuria have

increased collagen production associated with TGF-

b1 excretion [14]. In contrast, adult patients without

microalbuminuria using the present assay for NC1

have a normal excretion rate of NC1 that increases

with microalbuminuria and then decreases with

overt nephropathy [13]. Results in previous studies

on collagen IV depend on the assay and the antigen

used. In one study u-collagen IV increased as renal

function decreased, but no difference was found in

normoalbuminuric and microalbuminuric patients

[34]. The study by Watanabe et al., using the

collagen 7S domain, found high u-collagen IV in

patients with micro�macroalbuminuria [35]. In the

present study no association was found between

TGF-b1 and NC1 urinary excretion in the diabetic

patients, but with the control group included an

association was found on day 1. The results indicate

that other factors than TGF-b1 re responsible for the

persistently high NC1 excretion.

Over time the relationship between synthesis and

breakdown of collagens may alter and adjust the

thickness of the basement membranes [4]. At

diagnosis the large filtrating capillary area seen in

prepubertal children may compensate for a mesan-

gial expansion [8,29] and explain the capacity to

excrete more NC1 and increase GFR even in

patients with small but still normal sized kidneys.

Furthermore, neutralization of TGF-b1 production

in a mouse model reverses mesangial expansion,

inhibits further decrease in GFR, but does not affect

albuminuria [9]. In the present study, albumin

excretion normalized during treatment, but those

with the largest kidneys continued to excrete more

albumin. These children also needed more insulin

and could represent a group of patients with more

deranged metabolic control at entry. In line with

many studies, this investigation found no association

between GFR and albumin, NC1 or TGF-b1 excre-

tion [2,3,8,17,19]. Serum creatinine and GFR were

unchanged during treatment.

Hyperfiltration has long been suggested to predict

the development of diabetic nephropathy [36�39].

Kidney hyperfiltration [18] and hypertrophy [19]

stimulate TGF-b1 production, which promotes

growth [11]. Angiotensin II blockade reduces renal

TGF-b1 production, which may be of significance as

TGF-b1 adjusts homeostasis and renal hyperfiltra-

tion through vasoconstriction properties [11,15].

After 20 days of treatment TGF-b1 was associated

with renal size in the present study. This finding is,

however, of limited value since there were few

patients and only five out of seven patients had no

excretion of TGF-b1.

In conclusion, insulin treatment at diagnosis of

diabetes normalized increased excretion of both

albumin and TGF-b1. Thus, increased albumin

and TGF-b1 was probably caused by deranged

metabolic control. In contrast, insulin had no effect

on NC1. Longitudinal studies should be carried out

to clarify whether sustained high urinary excretion of

NC1 could be a marker of later nephropathy.

Acknowledgements

The help from Asa Pettersson, Renal Research

Laboratory, and the staff of the Paediatric Depart-

ment in Lund is highly appreciated.

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TGF-b1, collagen and insulin in diabetes 147

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