Abstract

Background. There is accumulating evidence that C-peptide exerts beneficial renal effects in type-1 diabetes by reducing glomerular hyperfiltration, albuminuria and glomerular hypertrophy in the early stage of nephropathy. The aim of this study was to clarify further the effects of C-peptide on renal structural changes in type-1 diabetic rats.

Methods. The effects of C-peptide or placebo on glomerular volume, mesangial expansion, glomerular basement membrane thickness, albuminuria and glomerular filtration rate (GFR) were studied in three groups of rats: a non-diabetic group (N, n = 9) and two groups that, during 8 weeks of diabetes, were left untreated for 4 weeks and then given a subcutaneous infusion of either placebo (D, n = 11) or C-peptide (DCp, n = 11) during the next 4 weeks. Furthermore, GFR was studied after 4 weeks of diabetes in an additional diabetic group (D-early, n = 9) and in an age-matched non-diabetic group (N-early, n = 9).

Results. After 4 weeks, GFR in the D-early group was 102% higher than in the N-early group. GFR after 8 weeks did not differ between the study groups. The D group presented with a 33% larger glomerular volume than the N group (P<0.001), while glomerular volume in the DCp group was similar to that in the N-group. Total mesangial and mesangial matrix fractions were increased by 46% (P<0.001) and 133% (P<0.001), respectively, in the D group. The corresponding values in the DCp group did not differ from those for the non-diabetic animals. Neither the thickness of the glomerular basement membrane nor the level of albuminuria differed significantly between the study groups.

Conclusions. C-peptide administration in replacement dose to streptozotocin-diabetic rats serves to limit or prevent the glomerular hypertrophy and the mesangial matrix expansion seen in the post-hyperfiltration phase of early diabetic nephropathy.

Introduction

Patients with type-1 diabetes lack both insulin and C-peptide. The latter is synthesized in and released from the pancreatic β-cells in equimolar amounts with insulin. In recent years, it has been shown that, contrary to previous views, C-peptide has biological effects of its own. Thus, studies in type-1 diabetic patients with incipient nephropathy have revealed beneficial effects of C-peptide on glomerular hyperfiltration, a proposed risk factor for the development of diabetic nephropathy, and on urinary albumin excretion [1,2]. Furthermore, both an acute and a 2 week infusion of C-peptide to streptozotocin-diabetic rats have resulted in prevention of hyperfiltration and in reduced albuminuria [3,4]. Recent findings indicate that C-peptide and the angiotensin-converting enzyme inhibitor captopril may be equally effective in lowering glomerular hyperfiltration in type-1 diabetic rats [5].

The exact mechanism behind the beneficial effects of C-peptide on renal function in type-1 diabetes is not fully understood. Specific binding of C-peptide to cell membranes has been demonstrated for various cell types, including rat renal tubular and mesangial cells, and there are indications that the binding site is a G-protein-coupled receptor [6]. Binding leads to an increased intracellular Ca2+ concentration and subsequently to stimulation of Na+,K+-ATPase [7] and endothelial nitric oxide synthase (eNOS) activities [8]. However, the relationship between these events and the beneficial effects on renal function and glomerular morphology remain to be clarified.

The morphological changes in diabetic nephropathy are characterized by mesangial matrix expansion and glomerular basement membrane (GBM) thickening [9]. Since little information is available on renal morphology during C-peptide treatment in diabetes, the main objective of the present study was to investigate the effects of C-peptide on glomerular volume, mesangial expansion, GBM thickening, albuminuria and glomerular filtration rate (GFR) in streptozotocin diabetic rats.

Materials and methods

Eight-week-old male Wistar rats (Møllegaard, Copenhagen, Denmark) with an initial weight of ∼250 g were divided into three groups and studied for 8 weeks: non-diabetic placebo-treated rats (N group, n = 9), diabetic placebo-treated rats (D group, n = 11) and diabetic rats treated with rat C-peptide II (Genosys Biotechnologies, UK) (DCp group, n = 11). In two additional groups, designated normal early group (N-early, n = 9) and diabetic early group (D-early, n = 7), the GFR was studied after 4 weeks for comparison with the three groups above. All animals had free access to tap water and standardized chow (R36, Lactamin, Ewos, Södertälje, Sweden) throughout the study period. Diabetes was induced by intravenous injection of streptozotocin 60 mg/kg body weight. Treatment with C-peptide (50 pmol/kg/min) dissolved in isotonic saline in the DCp group or with saline alone in the other two main study groups was initiated 4 weeks after onset of diabetes and administered as a continuous subcutaneous infusion for 4 weeks by an osmotic pump (type 2002, Alzet, USA) placed in the subcutaneous tissue of the neck. The study protocol was reviewed and approved by the institutional animal ethics committee.

Metabolic cages

Once a week, the rats were put in metabolic cages (Techniplast Gazzada 3701MO-000, Buguggiate, Italy) for 24 h. Body weight, daily intake of water and food, and excretion of urine and faeces were measured individually. Urine samples were collected for analyses of albumin, sodium and potassium excretion and osmolality. Blood samples for blood glucose measurements were taken from the tip of the tail.

Determination of GFR

GFR was measured in the N-early and D-early groups after 4 weeks of diabetes. In the other study groups, GFR was measured after 8 weeks of diabetes, i.e. after 4 weeks without and 4 weeks with treatment with C-peptide or placebo. The rats were anaesthetized by an intraperitoneal injection of Inactin® (sodium 5-sec-butyl-5-ethyl-2-thiobarbiturate; RBI Natick, MA), 70 mg/kg body weight, and placed on a heating pad to maintain body temperature at ∼37.5°C. Spontaneous breathing was facilitated by a cannula inserted into the trachea. The femoral vein was cannulated for infusions. The femoral artery was cannulated for blood sampling and for measurements of the arterial blood pressure. The bladder was catheterized by a suprapubic approach. Thereafter, an infusion of isotonic saline containing [3H]inulin (Pharmacia AB, Uppsala, Sweden) 4 mg/ml was started. After a bolus dose of 1 ml (5 μCi), the infusion rate was maintained at 5 ml/h/kg body weight. At steady state, after ∼45 min, three urine samples were collected at 20 min intervals for analyses of urine volume, osmolality and concentrations of sodium, potassium and [3H]inulin. At the midpoint of each of the three 20 min urine collection periods, plasma samples (∼60 μl) for analyses of [3H]inulin were obtained to allow calculation of the GFR. When the GFR measurements had been completed, a blood sample for determination of the C-peptide concentration was taken. To ensure adequate renal perfusion, the sample volume was replaced by an equal volume of isotonic saline.

Fixation of the kidney

The left kidney, the renal vessels and aorta were exposed via a subcostal incision. Thereafter, the femoral artery catheter was adjusted to the level of the left renal artery and the aorta subsequently was ligated proximally and distally to the left renal artery. The left kidney was then perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at a pressure adjusted to the mean arterial blood pressure recorded just before the perfusion was started. For electron microscopy, cortical tissue blocks were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4).

Analyses

Urine volumes were measured gravimetrically. Urinary sodium and potassium concentrations were determined by flame photometry (IL 543; Instrumentation Lab., Milano, Italy) and urine osmolality by a freezing point depression method (Model 3 MO; Advanced Instruments, MA). Urine albumin concentration was measured by nephelometry. Blood glucose concentrations were analysed by means of Accutrend® (Boehringer Mannheim GmbH, Mannheim, Germany) when obtained from the rat's tail and by a glucose oxidase method (Glucose Analyzer, Yellow Spring Instruments, USA) when obtained at the end of the final experiment. [3H]Inulin in plasma and urine was determined by liquid scintillation counting (PW 4700, Philips, The Netherlands). The sample (1 μl of urine or 10 μl of plasma) was mixed in 1 ml of water; thereafter 3 ml of scintillation fluid (Pico-Flour 40™, CiAB, Chemical Instruments AB, Lidingö, Sweden) was added. A radioimmunoassay technique (Linco Research Inc., USA) was used to measure the plasma concentrations of rat C-peptide.

Light microscopy

The mean glomerular volume was estimated by Cavalieri's principle as described in an earlier study [3].

Electron miscroscopy

For electron microscopy, cortical tissue was post-fixed in 1% osmium tetroxide and embedded in Epon by standard procedures. Ultrathin sections were stained with uranylacetate and lead citrate and studied in a JEM 100S electron microscope (Jeol, Tokyo, Japan). From each rat, 4–5 glomeruli were analysed. The reference space of the glomerular tuft was defined as in Osterby and Gundersen [10]. At 3000×, sets of 8–14 micrographs per glomerulus were taken in a systematic random manner by moving the specimen stage between predetermined points. The mesangial volume fraction and the mesangial matrix volume fraction were analysed by point counting, using a superimposed square lattice grid with ∼3 µm between the points at tissue level. The basement membrane thickness was estimated using the orthogonal intercept method of Jensen et al. [11] on a separate set of 6–9 micrographs taken from each glomerulus at 7500×. The final magnification was corrected using a grating grid with 2160 lines per mm.

Statistical methods

Differences in metabolic data, glomerular volume, renal size, GFR, mesangial volume and GBM thickness were evaluated by ANOVA followed by Tukey's post hoc test. Albuminuria (after the values had been logarithmically transformed) was compared with the basal state using ANOVA for repeated measurements within each group followed by Tukey's post hoc test. Statistical significance is defined as a P-value <0.05. Data in text, figures and tables are presented as the mean±SEM.

Results

Data from the metabolic cages

Before streptozotocin treatment, there were no statistically significant differences between the study groups in body weight, water intake, urine volume, food intake, faeces excretion, urine osmolality or urinary sodium and potassium excretion. Table 1 shows the results from the metabolic cages recorded at 4 and 8 weeks after induction of diabetes. At 8 weeks, the DCp group differed significantly from the normal group in all variables measured except for urine albumin excretion and urine osmolality. There were no significant differences in these variables between the D and DCp groups. The average blood glucose levels during weeks 1–4 (before C-peptide or placebo treatment) and during weeks 5–8 (during treatment) are shown in Table 1. The C-peptide treatment did not affect the glucose levels. In the DCp group, the plasma C-peptide concentration was 1.6±0.2 nmol/l, not differing significantly from that in the non-diabetic group (1.7±0.2 nmol/l; Table 1). In contrast, the diabetic group not receiving C-peptide showed a C-peptide level of only 0.1±0 nmol/l, as expected after streptozotocin treatment (Table 1).

Table 1.

Data from the metabolic cages and C-peptide levels

Group N
 
 D
 
 DCp
 
 
Time Week 4 Week 8 Week 4 Week 8 Week 4 Week 8 
Body weight (g) 316±7 362±7 257±7c 250±7c 250±6c 241±7c 
Water intake (ml/day) 20±2 21±2 167±9c 150±8c 156±10c 130±12c 
Urine volume (ml/day) 12±1 13±2 151±9c 134±8c 140±11c 120±12c 
Food intake (g/day) 14±1 12±1 33±1c 30±1c 31±1c 27±2c 
Faeces (g/day) 6±1 6±1 17±1c 16±1c 16±2c 14±1b 
U-osmolality (mOsm/kg) 1210±131 1087±87 889±16b 887±15a 903±33b 917±25 
U-Na (mmol/day) 1.5±0.1 1.3±0.1 2.9±0.2c 3.1±0.2c 3.1±0.3c 2.9±0.2c 
U-K (mmol/day 1.9±0.1 1.9±0.1 3.5±0.3b 3.0±0.2b 3.1±0.3a 2.7±0.2a 
U-albumin (µg/day) 109±23 239±67 546±133a 591±212 441±126 698±113 
Blood glucose (mmol/l) 5.7±0.2 5.7±0.2 24±0.8c 24.5±0.4c 25.4±0.6c 24.8±0.4c 
Plasma C-peptide (nmol/l)  1.7±0.2  0.1±0c  1.6±0.2 
Group N
 
 D
 
 DCp
 
 
Time Week 4 Week 8 Week 4 Week 8 Week 4 Week 8 
Body weight (g) 316±7 362±7 257±7c 250±7c 250±6c 241±7c 
Water intake (ml/day) 20±2 21±2 167±9c 150±8c 156±10c 130±12c 
Urine volume (ml/day) 12±1 13±2 151±9c 134±8c 140±11c 120±12c 
Food intake (g/day) 14±1 12±1 33±1c 30±1c 31±1c 27±2c 
Faeces (g/day) 6±1 6±1 17±1c 16±1c 16±2c 14±1b 
U-osmolality (mOsm/kg) 1210±131 1087±87 889±16b 887±15a 903±33b 917±25 
U-Na (mmol/day) 1.5±0.1 1.3±0.1 2.9±0.2c 3.1±0.2c 3.1±0.3c 2.9±0.2c 
U-K (mmol/day 1.9±0.1 1.9±0.1 3.5±0.3b 3.0±0.2b 3.1±0.3a 2.7±0.2a 
U-albumin (µg/day) 109±23 239±67 546±133a 591±212 441±126 698±113 
Blood glucose (mmol/l) 5.7±0.2 5.7±0.2 24±0.8c 24.5±0.4c 25.4±0.6c 24.8±0.4c 
Plasma C-peptide (nmol/l)  1.7±0.2  0.1±0c  1.6±0.2 

Data from the metabolic cages 4 and 8 weeks after diabetes onset except for blood glucose, which is an average of the first 4 and last 4 weeks, respectively, and C-peptide, which is measured at the end of the study. Non-diabetic placebo-treated group (N, n = 9), diabetic placebo-treated group (D, n = 11), diabetic C-peptide-treated rats (DCp, n = 11). ANOVA followed by Tukey's post hoc test was used for the statistical analyses. The superscripts a, b and c indicate significant differences from the N group at the same time point: aP<0.05, bP<0.01 and cP<0.001.

Glomerular filtration rate

The results indicate that after 4 weeks of diabetes, GFR was on average 102% greater in the diabetic compared with the non-diabetic group (Figure 1): D-early 5.23±0.78 ml/min and N-early 2.59±0.37 ml/min (P<0.001). Results for the other groups show that after 8 weeks of diabetes, there was no longer a statistically significant increase in GFR, although GFR still tended to be higher in the diabetic untreated group (P<0.051). Thus, GFR in the D group was 3.39±0.24 ml/min and in the N group 2.47±0.08 ml/min. GFR in the DCp group was 2.77±0.31 ml/min, not significantly different from the N group.

Fig. 1.

Glomerular filtration rate (GFR) measured as the clearance of inulin. In the first two groups, i.e. the normal early (N-early, n = 9) and the diabetic early (D-early, n = 7) groups, GFR was measured 4 weeks after diabetes onset without any treatment. In the main study groups, i.e. the normal placebo-treated group (N group, n = 9), the diabetic placebo-treated group (D group, n = 11) and the diabetic C-peptide-treated group (DCp, n = 11), GFR was measured 8 weeks after diabetes onset, thus, 4 weeks after start of treatment. Asterisks indicate statistical difference compared with the N-early group after 4 weeks and the N group after 8 weeks: ***P<0.001.

Fig. 1.

Glomerular filtration rate (GFR) measured as the clearance of inulin. In the first two groups, i.e. the normal early (N-early, n = 9) and the diabetic early (D-early, n = 7) groups, GFR was measured 4 weeks after diabetes onset without any treatment. In the main study groups, i.e. the normal placebo-treated group (N group, n = 9), the diabetic placebo-treated group (D group, n = 11) and the diabetic C-peptide-treated group (DCp, n = 11), GFR was measured 8 weeks after diabetes onset, thus, 4 weeks after start of treatment. Asterisks indicate statistical difference compared with the N-early group after 4 weeks and the N group after 8 weeks: ***P<0.001.

Glomerular volume

The average glomerular volume was 1.34±0.03 × 106 µm3 in the D group, or 33% greater than in the N group (1.01±0.05 × 106 µm3, P<0.001) (Figure 2). The corresponding value for the DCp group (1.09±0.02 × 106 µm3) was similar to that of the non-diabetic animals and 20% lower than that of the D group (P<0.001).

Fig. 2.

Glomerular volume, mesangial volume fraction, mesangial matrix volume fraction and glomerular basement membrane thickness after 8 weeks (4 weeks after treatment start) in each study group: the normal placebo-treated group (N group, n = 9), the diabetic placebo-treated group (D group, n = 11) and the diabetic C-peptide-treated group (DCp, n = 11). ANOVA followed by Tukey's post hoc test was used for the statistical analysis. Asterisks indicate statistically significant differences from the N group: ***P<0.001.

Fig. 2.

Glomerular volume, mesangial volume fraction, mesangial matrix volume fraction and glomerular basement membrane thickness after 8 weeks (4 weeks after treatment start) in each study group: the normal placebo-treated group (N group, n = 9), the diabetic placebo-treated group (D group, n = 11) and the diabetic C-peptide-treated group (DCp, n = 11). ANOVA followed by Tukey's post hoc test was used for the statistical analysis. Asterisks indicate statistically significant differences from the N group: ***P<0.001.

Mesangial volume, mesangial matrix volume and interstitial tissue

The mesangial volume expressed as a fraction of the glomerular volume was 12.3±0.7% in the N group, 18.0±0.9% in the D group and 14.2±0.7% in the DCp group (Figures 2 and 3). Thus, the D group showed a mesangial volume fraction that was 46% larger than in the N group (P<0.001) and 27% larger than in the DCp group (P<0.01). The DCp group did not differ significantly from the normal group.

Fig. 3.

PAS-stained glomeruli of normal placebo treated rat (N group, A), diabetic placebo-treated rat (D group, B) and C-peptide-treated rat (DCp group, C). Note the widened mesangial areas (arrows) in the D rat (B) containing PAS-positive mesangial matrix. This matrix expansion is partially prevented in the DCp rat (C).

Fig. 3.

PAS-stained glomeruli of normal placebo treated rat (N group, A), diabetic placebo-treated rat (D group, B) and C-peptide-treated rat (DCp group, C). Note the widened mesangial areas (arrows) in the D rat (B) containing PAS-positive mesangial matrix. This matrix expansion is partially prevented in the DCp rat (C).

The relationships for the mesangial matrix volume fraction were similar to those for the total mesangial volume fraction. Thus, the mesangial matrix volume expressed as a fraction of the total glomerular volume was 3.0±0.3% in the N group, 7.0±0.5% in the D group and 4.4±0.6% in the DCp group (Figure 2). The D group showed a mesangial matrix fraction that was more than twice that in the N group (P<0.001) and 59% larger than in the DCp group (P<0.01). The DCp group did not differ significantly from the N group. The mesangial cell volume fraction (data not shown) of the glomerular volume did not differ significantly between the study groups. There were no signs of interstitial expansion, fibrosis or tubular atrophy in any of the study groups.

Glomerular basement membrane thickness

The thickness of the GBM did not differ significantly between the study groups. Thus, GBM thickness was 147±2 nm in the N group, 156±6 nm in the D group and 153±2 nm in the DCp group (Figure 2).

Albuminuria

The urinary albumin excretion rate in the healthy N group after 4 and 8 weeks was 109±23 and 239±67 µg/day, respectively (Table 1). In the D group, the albumin excretion was 546±133 µg/day (P<0.05 vs the N group) after 4 weeks and 591±212 µg/day after 8 weeks (NS vs the N group). The DCp group showed an albumin excretion of 441±126 and 698±113 µg/day after 4 and 8 weeks, respectively (both NS vs the normal group).

Discussion

Evidence is accumulating that C-peptide is a bioactive peptide that can exert beneficial effects on the kidney in type-1 diabetes, resulting in prevention or retardation of diabetic nephropathy [1–5]. Previous studies have established that C-peptide can limit glomerular hypertrophy in type-1 diabetic rats [3]. The glomerular morphology in diabetic nephropathy is characterized primarily by mesangial expansion and GBM thickening. The effects of C-peptide on the specific renal structural components in type-1 diabetes have not been examined. Consequently, this study was undertaken to explore further the effects of C-peptide on diabetes-induced renal morphological changes.

GFR was measured after 4 weeks in diabetic (D-early) and non-diabetic rats (N-early). Characteristic glomerular hyperfiltration was found in the diabetic group at this early stage. After 8 weeks, GFR in the placebo-treated group of diabetic rats (D group) was no longer statistically significantly increased compared with the non-diabetic group (N group), indicating that after 8 weeks of diabetes the hyperfiltration phase was over (Figure 1). This is the expected course of nephropathy in diabetes and at this stage the morphological changes usually become more established [12]. The absence of significant glomerular hyperfiltration in the diabetic rats after 8 weeks explains why C-peptide, in this study and in contrast to our earlier studies in diabetic rats [3–5], showed no statistically significant effect on the GFR.

Glomerular hyperfiltration and glomerular hypertrophy usually occur simultaneously, but the hypertrophy can persist after the GFR has been normalized [13]. Accordingly, after 8 weeks and a substantial decrease in GFR, the D group still had significant glomerular hypertrophy. The glomerular volume in the D group was 33% larger than in the N group. In the DCp group, an increase in glomerular volume was prevented by the continuous infusion of physiological doses of C-peptide (Figure 2). This is in keeping with earlier findings in streptozotocin-diabetic rats [3]. In the present study, the increased glomerular size is explained in part by a 46% larger (P<0.001) mesangial volume fraction in the D group compared with the N group (Figure 2). Specifically, electron microscopy of the glomeruli reveals that the mesangial expansion is due to an increased matrix volume (Figure 2) rather than an increased cell volume. In contrast, the C-peptide-treated group showed no or minimal mesangial expansion. This is a finding of interest since matrix accumulation is related to clinical manifestations of diabetic nephropathy and may cause occlusion of the nephrons [14]. In this context, it is noted that a recent study in streptozotocin-diabetic mice has demonstrated that diabetes-induced upregulation of transforming growth factor-β (TGF-β) in glomeruli is prevented by C-peptide administration. Moreover, in vitro studies of mouse podocytes show that C-peptide dose-dependently inhibits TGF-β-induced upregulation of collagen IV [15]. These observations may provide insight into the mechanism whereby C-peptide contributes to diminished mesangial matrix expansion in the diabetic state. The present findings are in keeping with the observation that after pancreas transplantation in humans, mesangial matrix expansion is reversed [16]. This amelioration has been ascribed to improved glycaemic control. However, it has also been suggested that the finding may be attributed to restoration of C-peptide levels after transplantation [17]. Furthermore, improved graft survival rates and diminished urinary albumin excretion have been observed after successful islet transplantation [17], again results that may be supported by earlier findings regarding C-peptide and albumin excretion [1,2].

The mechanism underlying the mesangial matrix accumulation in type-1 diabetes is likely to be multifactorial, including an imbalance between production and degradation of the matrix components collagen IV and fibronectin [18]. Growth factors such as platelet-derived growth factor, TGF-β and vascular endothelial growth factor are likely to contribute to this process. Non-enzymatic glycosylation and formation of advanced glycation end-products tend to increase matrix protein deposition and render the glycated matrix component less susceptible to proteolysis [18]. To what extent C-peptide affects these variables is not known. GBM thickening is another hallmark of diabetic nephropathy, although it is not as closely related to the clinical findings as mesangial expansion [14]. In this study, GBM thickness was not significantly increased in the diabetic placebo-treated group compared with the non-diabetic group (Figure 2), and in the DCp group it did not differ significantly from the other study groups. It is conceivable that a longer study period is required in order to elicit GBM thickening in Wistar rats; the time point for its occurrence varies greatly in the literature (from 8 weeks to 6 months [19]).

Interstitial expansion, fibrosis and tubular atrophy have been proposed as important factors in predicting subsequent progression to renal failure. However, in this material, there were no such changes in any of the study groups. Thus, at this early stage of experimental diabetes, glomerular changes are the only assessable indicators of renal damage. This supports the view that the interstitial changes in diabetic nephropathy are associated with a more advanced stage of glomerulopathy [20] than studied in the present report.

In contrast to previous observations in both animals and humans [2–4], the albumin excretion rate in the diabetic groups was not significantly increased compared with the normal group, nor was there an effect of C-peptide on this variable. A possible explanation for these findings may be the large dispersion in the urinary albumin excretion data. In addition, the absence of C-peptide treatment during the first 4 weeks of diabetes may have caused damage to the filtration barrier that was too severe to be reversed in only 4 weeks of subsequent C-peptide therapy. Furthermore, in earlier animal studies of C-peptide effects in diabetes, Sprague–Dawley rats rather than Wistar rats were used and the two strains may differ in the relationship between the morphological changes and albuminuria.

In summary, administration of C-peptide to physiological levels for 4 weeks results in almost complete prevention of the glomerular hypertrophy and mesangial matrix expansion that otherwise occur in streptozotocin-diabetic Wistar rats.

Conflict of interest statement. B.L.J., J.W. and K.E. own shares in Creative Peptides Sweden Inc., Stockholm, and J.W. and K.E. are employed by this company.

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Author notes

1Department of Nephrology, Danderyd Hospital, Departments of 2Nephrology, 3Pathology and 4Clinical Physiology, Karolinska University Hospital, Stockholm and 5Department of Cell Biology, Biomedicum, Uppsala University, Sweden

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