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Department of Nephrology, Leiden University Medical Center, Leiden, The
Netherlands
Department of Endocrinology, Leiden University Medical Center, Leiden, The
Netherlands
Department of Clinical Chemistry, Leiden University Medical Center,
Leiden, The Netherlands
§
Department of Hematology, Leiden University Medical Center, Leiden, The
Netherlands
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Fifth Medical Clinic, Mannheim Hospital, University of Heidelberg,
Germany.
Correspondence to Dr. Johan W. van der Pijl, Department of Nephrology, C3-P26, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: +31 71 5262218; Fax: +31 71 5248118; E-mail: j.w.van_der_pijl{at}nephrology.medfac.leidenuniv.nl
Abstract
Abstract. In diabetic nephropathy, heparan sulfate glycosaminoglycan side chains are reduced in glomerular basement membranes proportionally to the degree of proteinuria. Recently, it was demonstrated that additional therapy with danaparoid sodium, a mixture of sulfated glycosaminoglycans with mainly heparan sulfate, lowered proteinuria in type 1 diabetes patients with diabetic nephropathy. A randomized placebo-controlled parallel study was performed with 750 anti-Xa units of danaparoid sodium once daily in type 2 diabetes patients with severe proteinuria. The aim of the study was to evaluate the possible effects of danaparoid sodium on proteinuria, endothelial dysfunction, and hard exudates in the retina and to determine the safety/tolerability of this drug. Twenty-two patients completed the study, and one patient had to stop prematurely after 6 wk of danaparoid sodium treatment because of urticaria at the injection sites. Apart from a small decrease of hemoglobin and minor skin hematomas at the injection site in five patients in the danaparoid sodium group, no other safety parameters showed any clinically or statistically significant difference between and within groups. The relative change in time of both the urinary albumin and protein excretion rate corrected for creatinine did not differ between both treatment arms (P = 0.2 and 0.49, respectively). No retinal complications or changes of hard exudates occurred. von Willebrand factor was elevated in both groups, but was not influenced by either treatment modality. Contrary to the beneficial effects that occurred in type 1 diabetes patients with diabetic nephropathy, treatment for 8 wk with 750 anti-Xa units of danaparoid sodium gave no reduction of proteinuria, hard exudates, and von Willebrand factor.
In diabetic nephropathy, heparan sulfate is reduced in basement membranes of glomeruli (1), in basement membranes of extrarenal small vessels (2), and also in nonendothelial lining basement membranes of the skin (3). The importance of heparan sulfate in the glomerular basement membrane for maintaining the glomerular charge barrier has been underscored by studies in which heparinitase (4) or a monoclonal antibody directed against heparan sulfate glycosaminoglycan (GAG) side chain (5) led to albuminuria in experimental animals.
In addition, heparan sulfate glycosaminoglycan side chains may have biologic functions that could possibly be imitated with therapy of similar molecules like heparin (6). Indeed, supplementation of GAG prevented the development and halted the progression of diabetic nephropathy in rats with streptozotocin-induced diabetes mellitus (7). Treatment with glycosaminoglycans for human micro- and macroalbuminuric diabetic nephropathy has been shown to be feasible and beneficial in diabetes mellitus type 1 (8,9,10,11). Moreover, we observed concomitantly a regression of retinal hard exudates (12). Controlled studies in patients with type 2 diabetes mellitus, however, are limited and show conflicting results (13,14). We performed this prospective study to evaluate whether treatment with danaparoid sodium exerts beneficial effects on the albumin excretion rate as a primary parameter in type 2 diabetes patients with severe proteinuria and renal insufficiency. As secondary end points, we looked at the endothelial dysfunction as estimated by plasma levels of von Willebrand factor, and at the retinopathy by evaluating the severity of hard exudates.
Materials and Methods
Patients
The study was designed as a phase II, randomized, double-blind,
placebo-controlled parallel study with a duration of 8 wk of treatment and 4
wk of follow-up. The medical ethics committee of the Leiden University Medical
Center approved the protocol of the study, and all patients gave informed
consent. Eligible patients were randomly allocated to treatment once daily
with either 750 anti-Xa units of danaparoid sodium (Orgaran®) or 0.6 ml of
saline with sulfite as placebo (both kindly provided by NV Organon, Oss, The
Netherlands). Danaparoid sodium is a mixture of sulfated GAG, consisting of
84% heparan sulfate, 12% dermatan sulfate, and 4% chondroitin sulfate,
isolated from porcine intestinal mucosa. Clearance takes place mainly by the
kidneys. The patients were trained to self-administer the study medication in
an abdominal skinfold, avoiding the injection of protamine-containing insulin
preparations at the same site. The date and time of each injection were
recorded daily and checked at the end of each treatment period, together with
the number of returned unused ampoules.
For the sample size calculations, we used the results of our previous study to estimate a difference of 40% relative change between placebo and real treatment with a mean SD of 30% (11). Thus, nine patients would be required in each group to give a power of 80% in detecting a significant difference in albumin excretion rate at the 5% significance level.
The study population consisted of 23 patients with diabetes mellitus type 2 and diabetic nephropathy—defined by an albumin excretion rate (AER) >300 mg/24 h in sterile urine and the presence of diabetic retinopathy. The use of antihypertensive medication was only allowed if given at a stable dosage at least 6 wk before start of the study. We used the same exclusion criteria as in our previous study (11).
In diabetic nephropathy, both proteinuria and macroalbuminuria are associated with a steady decline of renal function. Taking the relative short duration of treatment into account, we decided to use proteinuria and albuminuria as surrogate markers. Every 2 wk, urinary AER, urinary protein excretion rate (PER), and endogenous creatinine clearances were measured twice and the means were compared with baseline. AER and PER were expressed per mmol of creatinine to correct for incomplete urinary collections. von Willebrand factor antigen (vWF) in plasma was determined as a marker for endothelial dysfunction (15).
Fundus photography was performed at inclusion and at 8 wk primarily as a safety parameter to monitor potential bleeding complications and secondarily to evaluate putative effects on hard exudates. Seven-field, red-free photographs of both eyes were obtained with a Zeiss camera after mydriasis. To evaluate the course of signs of retinal leakage, a 45° photograph centered around the fovea was projected and all hard exudates were traced. Hard exudates were semiquantitatively graded, ranging from 1+ (minimal) to 5+ (extensive circinate). Photographs were studied masked for pre- or posttreatment assessment and without knowledge of treatment assignment.
Analytical Methods
A general medical history was obtained together with a medical examination,
and the following study and safety/tolerability parameters were tested
regularly. Electrolytes, urea, creatinine, protein, albumin, liver enzymes,
and urinary protein were assessed on a Hitachi 747 or 911 autoanalyzer
(Boehringer Mannheim, Mannheim, Germany). Urinary albumin was assessed using
an immunonephelometric autoanalyzer (Array Protein System, Beckman
Instruments, Brea, CA) with specific antibodies. The Technicon H-1 system
(Bayer Diagnostics, Munich, Germany) was used for the analysis of hematologic
parameters. Prothrombin time was determined using the Recombiplastin reagent
(Ortho-Clinical Diagnostics, Beerse, Belgium), and activated partial
thromboplastin time was determined using the Cephotest reagent (Nyegaard,
Oslo, Norway), both on an Electra 1000C coagulometer (Medical Laboratory
Automation, Pleasantville, NY). Antithrombin and anti-Xa activity were
assessed on an STA analyzer (Boehringer Mannheim) with Coamatic Antithrombin
and Coatest LMWHeparin as reagents (Chromogenix,
Mölndal, Sweden). von Willebrand factor antigen
was assessed in plasma with an enzyme-linked immunosorbent assay, using
polyclonal antibodies (normal values: 0.5 to 1.5 IU/ml). HbA1c was
assessed on HPLC after hemolysis.
Statistical Analyses
Delta values (relative change) for AER/creat and PER/creat were calculated
as follows: 100% x (nth week treatment value —
pretreatment value)/(pretreatment value). The mean changes of the parameters
during the two treatment periods were analyzed using a mixed-model ANOVA with
random patient factor to account for the correlations between the repeated
measurements, and fixed treatment and time factors. Nonparametric
Mann-Whitney, Friedman, and 
2 tests were used where
appropriate. Results are given as medians with interquartile ranges unless
otherwise indicated. Significance was accepted at P < 0.05.
Results
Tables 1 and 2 show that typical features of type 2 diabetes patients with nephropathy such as hypertension, high vWF values, diabetic retinopathy, decreased renal function, and obesity were present in the study population. Background diabetic retinopathy was found in the patients, and nine of them had previously been treated with photocoagulation. One patient had microscopic hematuria and underwent a kidney biopsy showing typical diabetic glomerulopathy. The other patients fulfilled the criteria for the clinical diagnosis of overt diabetic nephropathy based on retinopathy, proteinuria, and a high von Willebrand factor.
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According to the checklists, all patients had used the prescribed 56 ampoules except for one patient who had to stop after 6 wk of danaparoid sodium treatment because of local urticaria and hematomas. A significant increase in plasma anti-Xa levels (maximum 0.28 U/ml) was only found for the patients on danaparoid sodium (P < 0.0001). Small hematomas at the injection sites occurred in five patients on danaparoid sodium and in one patient on placebo. The hemoglobin levels decreased within the danaparoid sodium group after 8 wk (the median fell with 0.2 mmol/L, P < 0.02). In each group, one patient needed treatment in the follow-up period with diuretics because of a nephrotic syndrome. Both groups had comparable blood glucose levels at every time point. Table 3 shows that the 8 wk HbA1c values were slightly increased compared with baseline (danaparoid sodium group P < 0.01 and placebo P = 0.05), but danaparoid and placebo treatment groups did not differ at 0 and 8 wk (P = 0.71 and 0.66, respectively). BP control was comparable between both groups (P = 0.55) (Table 3 and Figure 1).
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Figure 2 shows the results for the AER corrected for creatinine (similar data and figure for the PER not shown). Both primary parameters AER/creat and PER/creat did not change over time between the two treatment modalities (P = 0.2 and 0.49, respectively). We could not detect any subgroup of patients responding with a consistent reduction of proteinuria. von Willebrand factor antigen was elevated and showed equal results for both groups at baseline (P = 0.76). We found no differences with regard to the vWF values and the treatment modality on the consecutive time points. Renal function assessed by creatinine clearance also did not change over time.
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With regard to the diabetic retinopathy, no signs of progression or bleeding complications were found. We excluded from further analysis two eyes from two patients since these eyes needed photocoagulation because of macula edema and neovascularization (Table 2). The fundus photographs with evaluable hard exudates (n = 16 eyes on danaparoid sodium and n = 10 eyes on placebo) did not show any changes at 8 wk (P = 1.00 and 0.16, respectively) (Table 3).
Discussion
We found in this prospective controlled study no favorable effects on proteinuria, vWF values, and hard exudates in patients with diabetes mellitus type 2 after treatment with danaparoid sodium despite adequate compliance and dosage as assessed by the measurable anti-Xa levels. It is remarkable that the effect of danaparoid sodium therapy on renal and retinal leakage of protein was paralleled, notwithstanding the fact that the evaluation of hard exudates was not a primary end point. We have observed in type 1 diabetes patients also such a parallel, albeit in the opposite direction, with a reduction of both hard exudates and proteinuria (11,12).
The exact mechanism by which glycosaminoglycans interfere with the process leading to diabetic nephropathy remains to be determined. It is tempting to speculate that the differentiated response on the same therapy suggests that the diabetic nephropathy of type 1 diabetes mellitus varies from that seen in type 2 diabetes mellitus. It has been found that glomerular changes are less pronounced in type 2 diabetes patients with microalbuminuria than in patients with type 1 diabetes mellitus (16,17). Theoretically, nondiabetic renal disease such as glomerulonephritis instead of diabetic glomerulopathy might have been present, and thus may have confounded the study results. The prevalence of nondiabetic renal disease has been reported to vary between 9 and 66% in type 2 diabetes patients (18), but when both retinopathy and proteinuria are present, the diabetic glomerulopathy is the most likely diagnosis (18,19). Fioretto and coworkers have shown that the findings of diabetic retinopathy and high vWF values help to identify typical diabetic glomerular renal lesions (20). The present study was performed in patients with proteinuria, retinopathy, and high vWF values, and thus apt to have typical diabetic nephropathy—perhaps already too advanced to expect an effect after 8 wk of glycosaminoglycan therapy.
Only two other controlled studies have been reported in type 2 diabetes with conflicting results (13,14). Nielsen et al. conducted a placebo-controlled study with low molecular weight heparin during 3 wk in 44 patients with varying rates of albuminuria, and similar to our results no changes were found in the transcapillary escape and the urinary excretion rate of albumin (14). A reduction of albuminuria in 12 patients, of whom only three were moderately macroalbuminuric, has been reported during a 4-mo therapy with an oral formulation of glycosaminoglycans (13). It should be noted that the metabolic and BP control were better regulated in this study in the GAG-treated patients compared with the patients on placebo with consequently putative effects on albuminuria. On the other hand, a treatment duration of 3 wk (Nielsen et al.) and 8 wk (this study) might in fact not be enough to obtain a measurable effect in type 2 diabetes patients. However, this is at variance with the findings of several studies in type 1 diabetes mellitus: Not only 3 mo of therapy, but also much shorter periods down to 3 wk of treatment with various glycosaminoglycans appeared to be effective in reducing albuminuria in both micro- and macroalbuminuria (8,9,10,11). We think that a long-term study—with a duration of at least 8 to 12 mo of GAG therapy based on the studies of Gambaro et al. (7,21)—is warranted, taking into account the time to establish the renal lesions, before it can be concluded definitely that therapy with glycosaminoglycans is not of any benefit for patients with type 2 diabetes and overt diabetic nephropathy.
The reduced expression of heparan sulfate glycosaminoglycan side chains, one of the findings that gave rise to the glycosaminoglycan treatment, has also been reported for renal biopsies in patients with type 2 diabetes mellitus (1). This finding does not preclude that the various mechanisms that influence and interact with the heparan sulfate metabolism, such as hyperglycemia and cytokine and growth factor production, evolve differently and in a differentiated way in type 1 versus type 2 diabetic nephropathy (6).
We postulated that a treatment regimen with glycosaminoglycans might influence favorably the endothelial dysfunction. Previously, other investigators have already reported that endothelial dysfunction, as assessed by high von Willebrand factor plasma values, can be found in type 2 diabetes patients with diabetic nephropathy (15), which was confirmed by our findings (22). Heparins and heparan sulfate exert both antiinflammatory actions and antiproliferative effects. Furthermore, glycosaminoglycans also inhibit the synthesis and release of endothelin-1 by endothelial cells (23). Myrup et al. found a clear effect of glycosaminoglycans on the AER in microalbuminuric type 1 diabetes patients, but no effect on either vWF or transcapillary escape rate of albumin (8,24). In this study, we found no beneficial effect of the treatment with danaparoid sodium on the vWF levels.
In conclusion, contrary to the positive outcome in type 1 diabetes patients with diabetic nephropathy and retinopathy, treatment with 750 anti-Xa units of danaparoid sodium once daily for 8 wk gave no reduction of proteinuria, hard exudates, or von Willebrand factor. Additional studies are needed to determine whether danaparoid sodium treatment should be initiated at an earlier stage of the disease, for a longer period of time, or whether danaparoid sodium in type 2 diabetes mellitus is ineffective.
Acknowledgments
Acknowledgments
We are indebted to Mieke van Aartrijk, Jan Brachter, Jan Egberts, Sarah Marshall, and Koos Zwinderman for their help and contributions.
Footnotes
American Society of Nephrology
References
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  J. F.M. Lensen, A. L.W.M.M. Rops, T. J.M. Wijnhoven, T. Hafmans, W. F.J. Feitz, E. Oosterwijk, B. Banas, R. J.M. Bindels, L. P.W.J. van den Heuvel, J. van der Vlag, et al. Localization and Functional Characterization of Glycosaminoglycan Domains in the Normal Human Kidney as Revealed by Phage Display-Derived Single Chain Antibodies J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1279 - 1288. [Abstract] [Full Text] [PDF]
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G. Gambaro, I. Kinalska, A. Oksa, P. Pont'uch, M. Hertlova, J. Olsovsky, J. Manitius, D. Fedele, S. Czekalski, J. Perusicova, et al. Oral Sulodexide Reduces Albuminuria in Microalbuminuric and Macroalbuminuric Type 1 and Type 2 Diabetic Patients: The Di.N.A.S. Randomized Trial J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1615 - 1625. [Abstract] [Full Text] [PDF]
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, danaparoid sodium; [UNK], placebo treatment. Error bars
give the 95% confidence interval of the mean. Over time there is no change
between both groups (P = 0.55).