Review paper
Preclinical and clinical evidence of nephro- and cardiovascular protective effects of glycosaminoglycans

Introduction

Despite advances in dialysis techniques, pharmacological treatment, and patient rehabilitation programmes, mortality and morbidity rates of end-stage renal disease (ESRD) are still high and mainly related to cardiovascular diseases (CVD) [1, 2]. Diabetic nephropathy is still the leading cause of ESRD [3] and an important cause of morbidity and mortality in patients with either type 1 or type 2 diabetes mellitus, both directly and as a risk factor for cardiovascular disease [4]. The microangiopathic complications of diabetes increase with longer duration of diabetes and worse glycaemic control [5]. In fact, the presence of albuminuria or proteinuria is a well known risk factor for coronary heart disease [6, 7]. Although recent evidence shows that an early multi-pharmacological approach is able to slow the progression of diabetic nephropathy to ESRD [8], the disease rarely stops and slightly regresses just in a few selected and optimally treated patients [9]. So, a better understanding of the pathophysiology of chronic kidney disease is needed in order to develop new efficacious treatments for this pandemic disease.
The aim of this review is to evaluate the available literature data supporting the possible role of glycosaminoglycans (GAGs) in renovascular pathology and their possible usefulness in the treatment of patients affected by diabetic nephropathy.

Role of glycosaminoglycans in renal physiology

The glomerular filtration barrier consists of fenestrated glomerular endothelium, podocyte foot processes interconnected by slit diaphragms, and intervening glomerular basement membrane (GBM). Its characterization as both a size and charge-selective barrier emerged from studies conducted decades ago. Podocyte cytoskeleton and its connections with specific glycans and proteins constitute the basis of slit diaphragm and cell-extracellular matrix interactions. Anionic sites in GBM consist of GAGs rich in heparin sulphate (HS) and their removal by enzymatic digestion resulted in increased permeability [10-12].
Glycosaminoglycans are long, unbranched mucopolysaccharides that consist of repeating disaccharide units. Apart from hyaluronan, which is uniquely synthesized without a protein core and is "spun out" by enzymes at cell surfaces directly into the extracellular space, the other GAGs are usually added to protein cores in the Golgi apparatus to yield proteoglycans [13].
Glomerular basement membrane charge is imparted by the sulphated GAG side chains of proteoglycans (HS proteoglycans [HSPGs] and, less, hyaluronic acid) and to a lesser extent by carboxyl and sialyl groups of glycoproteins. These negatively charged molecules play an important role in establishing a charge-selective barrier that restricts the passage of negatively charged molecules, such as albumin and other proteins [14].

Role of glycosaminoglycans in renal pathologies: experimental data

Nowadays, the charge selectivity phenomenon has received renewed attention with the identification of mechanisms of synthesis of barrier-related molecules [15, 16]. In particular, GBM HS proteoglycans (and more specifically on perlecan, collagen XVIII, and agrin) are considered primary charge barrier components. Segmented loss of GBM HS has been reported in membranous glomerulo­nephritis, lupus nephritis, minimal change disease and diabetic nephropathy in humans [17, 18] and rat models of Adriamycin and Heymann nephritis [19]. Van der Born et al. observed that streptozotocin-induced dia­betic rats with diabetic nephropathy experienced a significant decrease in glomerular HS/4-hydroxyproline ratio (showing increased collagen and relatively decreased GBM HS content) compared with control rats, and that was associated with selective proteinuria and glomerular hyperfiltration [20]. In another experimental model, non-diabetic mice knock-out for the Ext1 gene encoding a subunit of HS co-polymerase develop proteinuria that is less impressive than that expected from the available knowledge on renal physiology, suggesting that the cessation of polymerisation of podocyte-secreted HS that affects glomerular ultrafiltration charge may not be a serious cause of albuminuria, and there may be other roles of these molecules, including podocyte behaviour or morphology, as indicated in this study [21].
However, whether charge selectivity is actually important for glomerular function for the extent of proteinuria is still a matter of debate [22]. Recent in vivo manipulations of glomerular HS proteoglycans put in perspective (but did not exclude) a role for either molecules themselves or their anionic charge, which is altered greatly by loss of HS but caused insignificant albu­minuria, in glomerular filtration [16]. On the other hand, mice without HS attachment sites on perlecan revealed normal glomerular structure and no evidence of renal disease, except slightly increased susceptibility to protein-overload albuminuria [23, 24]. Collagen XVIII mutants had mild mesangial expansion with slightly elevated serum creatinine levels [25] and podocyte-specific agrin knock-out mice had a significant GBM charge defect but normal renal function [26]. Perlecan HS and perlecan-HS/agrin double mutant rats experience significant charge reduction on GBM, but no renal dysfunction or proteinuria [27]. Lastly, the removal of HS in rats did not result in acute proteinuria [28].
Moreover, in renal biopsies of different human primary proteinuric diseases, pronounced alteration in tubulointerstitial HS proteoglycans is evident and strongly related to the inflammatory processes [29]. In fact, GAGs have a role in modulation of inflammation in tissues. Heparin sulphate proteo­glycans can bind the leukocyte adhesion molecule L-selectin and chemokines, suggesting their role in inflammation [29-32]. Mouse and human glomerular endothelial cells activated by tumour necrosis factor (TNF)- or interleukin 1- showed increased expression of inflammatory N- and 6-O-sulphated HS domains and these are impor­tant in leukocyte trafficking and inflammation [33].
Heparin sulphate is an important constituent of subendothelial extracellular matrix and basement membrane structure and vascular HS is decreased in atherosclerosis, diabetes and during inflammation. It is also affected by lipoproteins, and lipoprotein-modulated perlecan may play an important role in vascular smooth muscle growth, and thus in atherosclerosis [34]. Celie et al. showed the role of microvascular BM HS in inflammatory responses, in human renal allograft biopsies [35]. Heparin sulphate and GAGs play a major role in adhesion of leukocytes to glomerular cells, and that is important for proliferative glomerulopathies, inflammation and angiogenesis. Under dynamic flow conditions addition of HS, heparin and tinzaparin and removal of HS on mouse glomerular endothelial cells the number of rolling and adhering leukocytes decreases about 2-3-fold and the rolling velocity doubles [36]. Heparin sulphate also binds to cell surface receptors and is involved in the modulation of inflammation. CD44/HS actions are well studied in inflamed synovial membrane macrophages, and it is involved in the regulation of growth factors during inflammation and wound healing [37, 38].

Experimental data supporting the potential use of glycosaminoglycans in proteinuric diseases

Besides HSPGs, heparanase may have a role in renal diseases; together with the changes in glomerular cell-GBM interactions and loss of HS, increased heparanase release might cause the release of HS-bound factors and HS fragments in the glomeruli or changes in intracellular signalling by binding of heparanase to glomerular cells [39]. In diabetic nephropathy, the HS content of the GBM is decreased and that causes protein leak into the urinary space [40]; the increased amount of heparanase enzyme in response to hyperglycaemia may be one explanation. On the other hand, heparanase upregulation by high glucose is prevented by insulin and/or heparin in endothelial cell cultures [41]. In humans, increased heparanase level is associated with reduced HS, as observed in renal biopsies of diabetic patients with nephropathy [42], whereas in renal biopsies of different human primary proteinuric diseases pronounced alterations in tubulointerstitial HSPGs were evident and strongly related to inflammatory processes [29]. This is probably related to more relevant involvement of renal glomeruli in diabetic nephropathy than in other proteinuric diseases.
Evidence from in vitro and diabetic animal studies reveal that the administration of heparin increases synthesis of HS [43], and other anionic glycoproteins can effectively prevent the bioche­mical alterations that promote albuminuria [44]. Enoxaparin, a low-molecular weight heparin, was also tested on patients with diabetic and non-diabetic glomerulopathies. The proteinuria-decreasing effect of this heparin was found not to be related to the renin-angiotensin system, and its glomerular filter-related effect was suggested [45].
Angiotensin II (AT-II) receptor blockers are renin-angiotensin system (RAS) modulators with very well known antiproteinuric activity [46]. Angiotensin II inhibits HSPG expression in human podocytes [47] and heparins modulate AT-II signalling in glomerular cells [48], inhibiting aldosterone synthesis [49] and lowering proteinuria in diabetic patients [45], but this effect is less pronounced in other forms of proteinuric renal diseases and its relation to haemodynamic changes produced by RAS is not proven in clinical trials [45]. Of course, heparins are not easily administrable for chronic treatments.
In this context, heparinoids were considered as potentially useful antiproteinuric drugs that could have synergistic effects with an RAS modulator [50]. In particular, sulodexide, a soluble, highly purified preparation of low-molecular weight GAGs composed of fast-moving heparin (80%) and dermatan sulphate (20%) derived from porcine intestine, appeared to be a promising treatment for diabetic proteinuria partially resistant to RAS blocking agents [51]. It prevents HS degradation, reconstruction of HS content of GBM, and in vivo inhibition of heparanase [40].
In fact, sulodexide is concentrated in renal parenchyma for a long time after administration [52] and in preliminary trials it has been supposed that it reduces albuminuria acting in vivo as a heparanase inhibitor that reaches the glomerular capillary wall and prevents HS degradation, thus allowing reconstruction of HS content and restoration of glomerular basement membrane ionic permselectivity [40]. Its antiproteinuric effect appears to be mainly related to the basal proteinuria and to the treatment duration [53], independently from its antithrombotic and profibrinolytic activity.
Studies in mouse articular chondrocytes after lipopolysaccharide stimulation also showed anti-inflammatory and anti-apoptotic actions of GAGs [54]. Moreover, sulodexide seems to have powerful anti-inflammatory activity in experimental models [55]. In a model of cultured human umbilical endothelial cells exposed to high glucose concentration, sulodexide suppresses cellular inflammation and prevents glucose cytotoxicity [56]; it is able to reverse the glucose-related cell release of free oxygen radicals, monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6), and the inactivation of the cell repair mechanism induced by exposure to glucose. However, these anti-inflammatory effects have not been demonstrated in humans yet. Therefore, in rats with streptozotocin-induced diabetes, sulodexide exerts direct endothelial protective effects [57] that could also be involved in kidney protection. However, the anti-inflammatory role of GAGs and heparin was already known in humans for decades, and especially in patients with allergy and asthma [58].

Clinical evidence of antiproteinuric effects of glycosaminoglycans

The antiproteinuric effects of GAGs, and especially of sulodexide, have been known for nearly two decades [59], and many clinical studies confirm its potential usefulness in treating nephropathies and especially in diabetic nephro­pathy.
In particular, the largest and more methodo­logically correct clinical trial was the one carried out by Gambaro et al.: the Diabetic nephropathy and Albuminuria Study (Di.N.A.S.) involved 223 patients with type 1 and 2 diabetes with both macro- and micro-albuminuria [60]. In this trial, 200 mg/day sulodexide treatment for 4 months was associated with 46% decrease in albumin excretion rate from baseline in diabetics who were not receiving concomitant angiotensin-converting enzyme (ACE) inhibitor treatment, and urinary albumin excretion was maintained even 2 months after drug intake interruption. The reduction in albuminuria was dose-dependent in this study, i.e. 100 mg/day sulodexide caused a mean decrease in albumin excretion rate of about 17% from baseline among the patients who were not receiving concomitant ACE inhibitor medication. The difference of albumin excretion vs. placebo was 62% in all diabetics with 200 mg/day sulodexide. The antiproteinuric effect of sulodexide appeared to be independent from the baseline blood pressure level and from the use of ACE inhibitors, meaning that even in patients already receiving ACE inhibitors, sulodexide was able to decrease the albumin excretion rate to approximately the same extent as in patients without ACE inhibitors (respectively 40% and 46% from baseline after 4 months with 200 mg/day sulodexide) [60].
However, this supposed additive effect of sulodexide was not confirmed either for ACE inhibitors when used at the maximum recommended dosage or for angiotensin receptor blockers (ARBs) also when used at the maximum recommended dosage. In fact, although in a pilot study on 149 microalbuminuric type 2 diabetic patients a trend for an increased rate of therapeutic success (return to normoalbuminuria or a decrease in albumin : creatinine ratio [ACR] of at least 50% from the baseline value) was observed for sulodexide, 200 mg/day for 6 months, compared to placebo (33.3% vs. 15.4% of the patients achieving the efficacy endpoint respectively; p = 0.075) [75], in the two related subsequent large trials respectively on microalbuminuric and macroalbuminuric type 2 diabetic patients the favourable trend of the preliminary pilot study for the additive effect of sulodexide in patients already treated with the maximum recommended dosages of ACE inhibitors or ARBs was not confirmed, even if the detailed results of these two studies are not known yet [76].
Other clinical trials focused on the mechanism of the antiproteinuric activity of sulodexide. Sulikowska et al. studied whether the albuminuria-lowering effect of sulodexide comes from its renovascular or tubular effects [61]. Dopamine infusion causes efferent arteriolar dilatation and increases creatinine clearance in normal people [62]. They tested dopamine-induced glomerular filtration response testing and urinary N-acetyl--D-gluco­saminidase measurements to test proximal tubular integrity, besides albuminuria, on type 1 diabetic patients. Patients were divided into placebo and daily 100 mg sulodexide groups for 120 days and dopamine testing was performed only on patients taking sulodexide. Sulodexide caused a decrease in albumin excretion from 126.1 ±15.41 to 96.3 ±13.7 mg/day in the treatment group compared with a decrease from 106.8 ±21.4 to 126.8 ±29.6 mg/day in controls. N-acetyl--D-glucosaminidase measure­ments also changed from 5.1 ±0.62 to 4.7 ±0.40 U/gCre in the sulodexide group and from 5.9 ±0.87 to 6.3 ±1.35 U/gCre in the control group with placebo. The response increase in creatinine clearance to dopamine infusion was from 13.2 ±2.1 to 15.44 ±1.9% (+16.9% increase) in patients taking this drug. The conclusion was that sulodexide affects intrarenal vascular reactivity and also improves N-acetyl--D-glucosaminidase tests, indicating amelioration of tubular damage [61].
Whatever the main mechanism may be, the role of sulodexide as an antiproteinuric agent is suggested by the fundamental Di.N.A.S. trial and a large number of smaller studies on both type 1 and 2 diabetic patients (Table I) [53, 60, 61, 63-75]. Although these smaller studies often had an open design and a short duration, and did not involve homogeneous patient categories, nevertheless they contributed to the evidence of a clinically favourable antiproteinuric effect of sulodexide.

Other effects of glycosaminoglycans on cardiorenal physiology

The potential cardiovascular effects of sulodexide and GAGs are summarized in Table II.
Interactions with HS modify and contribute to various protein actions and intercellular signalling by cytokines and growth factors, and some proteins share binding sites with HS [78]. Extracellular matrix of blood vessel walls also contain considerable amounts of proteoglycans and systemic hypertension may change the content of subendothelial matrix of vessels: this may also contribute to increased peripheral vascular resistance, as shown in animal models [79].
As stated above, at least a part of the renal histological degradation observed in diabetes is related to inflammatory processes, and sulodexide showed anti-inflammatory activity in different animal models [55]. A pilot study recently conducted on 11 healthy men concluded that it may also decrease transforming growth factor 1 (TGF-1) release [80].
The haemorheological and lipid lowering actions of GAGs have also been known for the last two decades. In fact, sulodexide decreases triglycerides [81], increases Apo-A1 and HDL-C levels [82] and blood viscosity [83], decreases D-dimer and fibrinogen levels [84, 85], and releases tissue plasminogen activator [86, 87]. Sulodexide has a dual effect on coagulation: antithrombin catalysis by fast moving heparin component and heparin cofactor II catalysis by dermatan sulphate component [88]. It causes inhibition of thrombus formation and growth with less systemic anticoagulation than comparable antithrombotic doses of heparins [89] and may also reduce oxidative stress slightly expressed by malonylaldehyde and superoxide dismutase in diabetics [72]. Therefore, in rats with streptozotocin-induced diabetes, sulodexide exerts direct endothelial protective effects, improving acetylcholine-induced relaxation of isolated aorta and mesangial arteries and reducing the number of circulating endothelial cells [57].

Conclusions

A relatively large body of literature supports the antiproteinuric and nephroprotective effects of GAGs and sulodexide, especially in diabetic nephropathy. These could derive from their effect on vascular permeability and inflammation, and on endothelium protection and haemorheology improvement. Sulodexide has the advantage of being an oral medication with few and usually mild side effects of intestinal type (i.e. diarrhoea, nausea, dyspepsia). Its antiproteinuric and nephroprotective role in therapy may be played in patients not tolerating ARBs and ACE inhibitors or in patients who are resistant to dosages of ARBs and ACE inhibitors which are not being given at the maximum recommended level, for safety or other reasons. In fact two recent clinical trials were not able to confirm in patients already receiving the maximal recommended dosages of ARBs or ACE inhibitors the previous statistically significant additional effects of sulodexide observed by Gambaro et al. [60] also in patients being treated with unspecified dosages of ACE inhibitors. Certainly, more clinical research is needed to understand which factors influence the drug efficacy and, consequently, which patients could a priori obtain the best effect from this treatment. Therefore, further clinical trials are currently ongoing with sulodexide as an antiproteinuric agent.

References
 1. Levin A. Clinical epidemiology of cardiovascular disease in chronic kidney disease prior to dialysis. Semin Dial 2003; 16: 101-5.  
2. Sarnak MJ, Levey AS. Epidemiology of cardiac disease in dialysis patients. Semin Dial 1999; 12: 69-76.  
3. Radbill B, Murphy B, LeRoith D. Rationale and strategies for early detection and management of diabetic kidney disease. Mayo Clin Proc 2008; 83: 1373-81.  
4. Martínez Castelao A. Advances in diabetes mellitus, diabetic nephropathy, metabolic syndrome and cardio-vascular-renal risk. Nefrologia 2008; 28 Suppl 5: 79-84.  
5. Rogowicz A, Litwinowicz M, Pilacinski S, Zozulinska D, Wierusz-Wysocka B. Does early insulin treatment decrease the risk of microangiopathy in non-obese adults with diabetes. Arch Med Sci 2007; 3: 129-35.  
6. Perkovic V, Verdon C, Ninomiya T, et al. The relationship between proteinuria and coronary risk: a systematic review and meta-analysis. PLoS Med 2008; 5: e207.  
7. Barylski M, Banach M, Mikhailidis DP, Pawlicki L, Kowal­ski J. Decreased kidney function as a risk factor for cardiovascular events in subjects with metabolic syndrome – a pilot study. Arch Med Sci 2008; 4: 417-23.
8.  Weir MR. Microalbuminuria and cardiovascular disease. Clin J Am Soc Nephrol 2007; 2: 581-90.  
9. Cortinovis M, Cattaneo D, Perico N, Remuzzi G. Investigational drugs for diabetic nephropathy. Expert Opin Investig Drugs 2008; 17: 1487-500.
10. Kanwar YS, Farquhar MG. Presence of heparan sulphate in the glomerular basement membranes. Proc Natl Acad Sci U S A 1979; 76: 1303-7.
11. Kanwar YS, Farquhar MG. Isolation of glycosaminoglycans (heparin sulphate) from glomerular basement membranes. Proc Natl Acad Sci U S A 1979; 76: 4493.
12. Kanwar YS, Linker A, Farquhar MG. Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans heparin sulphate) by enzyme digestion. J Cell Biol 1980; 86: 688-93.
13. Raman R, Sasisekharan V, Sasisekharan R. Structural insights into biological roles of protein-glycosaminoglycan interactions. Chem Biol 2005; 12: 267-77.
14. Deen WM, Lazzara MJ, Myers BD. Structural determinants of glomerular permeability. Am J Physiol Renal Physiol 2001; 281: F579-F596.
15. Harvey SJ, Miner JH. Revisiting the glomerular charge barrier in the molecular era. Curr Opin Nephrol Hypertens 2008; 17: 393-8.
16. Miner JH. Glomerular filtration: the charge debate charges ahead. Kidney Int 2008; 74: 259-61.
17. van der Born J, van del Havuel L, Bakker M, et al. Distribution of GBM heparin sulphate proteoglycan core protein and side chanins in human glomerular disease. Kidney Int 1993; 43: 454-63.
18. Tamsma J, van der Born J, Bruijn J, et al. Expression of glomerular extracellular matrix components in human diabetic nephropathy: decrease of heparin sulphate in the glomerular basal membrane. Diabetologia 1994; 37: 313-20.
19. Raats C, Luca M, Bakker M, et al. Reduction in glomerular heparin sulphate correlates with complement deposition and albuminuria in active Heymann nephritis. J Am Soc Nephrol 1999; 10: 1689-99.
20. van den Born J, van Kraats AA, Bakker MA, et al. Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparin sulphate. Diabetologia 1995; 38: 161-72.
21. Chen S, Wassenhove-McCarthy DJ, Yamaguchi Y, et al. Loss of heparin sulfate glycosaminoglycan assembly in podocytes does not lead to proteinuria. Kidney Int 2008; 74: 289-99.
22. Comper WD, Glascow EF. Charge selectivity in kidney ultrafiltration. Kidney Int 1995; 47: 1242-51.
23. Rossi M, Morita H, Sormunen R, et al. Heparan sulphate chains of perlecan are indispensible in the lens capsule but not in the kidney. EMBO J 2003; 22: 236-45.
24. Morita H, Yoshimura A, Inui K, et al. Heparan sulphate of perlecan is involved in glomerular filtration. J Am Soc Nephrol 2005; 16: 1703-10.
25. Utriainen A, Sormunen R, Kettunen M, et al. Structurally altered besement membranes and hydrocephalus in a type XVIII collagen deficient mouse line. Hum Mol Genet 2004; 13: 2089-99.
26. Harvey S, Burgess R, Miner J. Podocyte derived agrin is responsible for glomerular basement membrane anionic charge [Abstract]. J Am Soc Nephrol 2005; 16: 1A.
27. Goldberg S, Harvey SJ, Cunningham J, Tryggvason K, Miner JH. Glomerular filtration is normal in the absence of both agrin and perlecan-heparan sulphate from the glomerular basement membrane. Nephrol Dial Transplant 2009; 24: 2044-51.
28. Winjhoven TJ, Lensen JF, Wismans RG, et al. In vivo degradation of heparan sulphates in the glomerular basement membrane does not result in proteinuria. J Am Soc Nephrol 2007; 18: 823-32.
29. Celie JW, Reijmers RM, Slot EM, et al. Tubulointerstitial heparan sulfate proteoglycan changes in human renal diseases correlate with leukocyte influx and proteinuria. Am J Physiol Renal Physiol 2008; 294: F253-63.
30. Kawashima H, Watanabe N, Hirose M, et al. Collagen XVIII a basement membrane heparin sulphate proteoglycan, interacts with L-selectin and monocyte chemoattractant protein-1. J Biol Chem 2003; 278: 13069-76.
31. Parish CR. Role of heparin sulphate I in inflammation. Nat Rev Immunol 2006; 6: 633-43.
32. Wang L, Fuster M, Sriramarao P, Esko JD. Endothelial heparin sulphate deficiency impairs L-selectin – and chemokine-mediated neutrophile trafficking during inflammatory responses. Nat Immunol 2005; 6: 902-10.
33. Rops AL, van der Hoven MJ, Baselmans MM, et al. Heparan sulphate domains on cultured activated glomerular endothelial cells mediate leukocyte trafficking. Kidney Int 2008; 73: 52-62.
34. Pillarisetti S. Lipoprotein modulation of seubendothelial heparin sulphate proteoglycans (perlecan) and athero­genicity. Trends Cardiovasc Med 2000; 10: 60-5.
35. Celie JW, Rutjes NW, Keuning ED, et al. Subendothelial heparan sulphate proteoglycans become major L-selectin and monocyte chemoattractant protein-1 ligands upon renal ischemia/reperfusion. Am J Pathol 2007; 170: 1865-78.
36. Rops AL, Jacobs CW, Linssen PC, et al. Heparan sulphate on activated glomerular endothelial cells and exogenious heparinoids influence the rolling and adhesion of leuckocytes. Nephrol Dial Transplant 2007; 22: 1070-7.
37. Jones M, Tussey M, Athanasou N, Jackson DG. Heparan sulphate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action. J Biol Chem 2000; 275: 7964-74.
38. Taylor KR, Gallo RL. Glycosaminoglycans and their proteoglycans; host-associated molecular aptterns for initiation and modulation of inflammation. FASEB J 2006; 20: 9-22.
39. van der Hoven MJ, Rops AL, Vladovsky I, Levidiotis V, Berden JH, van der Vlag J. Heparanase in glomerular diseases. Kidney Int 2007; 72: 543-8.
40. Lewis EJ, Xu X. Abnormal glomerular permeability characteristics in diabetic nephropathy, implications for the therapeutic use of low-molecular weight heparin. Diabetes Care 2008; 31 Suppl 2: S202-7.
41. Han J, Woytowitch AE, Mandal AK, Hiebert LM. Heparanase upregulation in high glucose-treated endothelial cells is prevented by insulin and heparin. Exp Biol Med (Maywood) 2007; 232: 927-34.
42. Wijnhoven TJ, van den Hoven MJ, Ding H, et al. Heparanase induces a differential loss of heparan sulphate domains in overt diabetic nephropathy. Diabetologia 2008; 51: 372-82.
43. Jensen T. Pathogenesis of diabetic vascular disease, evidence for the role of reduced heparin sulphate proteoglycan. Diabetes 1997; 46 Suppl 2: S98-100.
44. Wijnhoven TJ, Lensen JF, Rops AL, et al. Anti-proteinuric effects of glycosaminoglycan-based drugs. Curr Opin Mol Ther 2007; 9: 364-77.
45. Benck U, Haeckel S, Clorius JH, van der Woude FJ. Proteinuria-lowering effect of heparin therapy in diabetic nephropathy without affecting the renin-angiotensin-aldosterone system. Clin J Am Soc Nephrol 2007; 2: 58-67.
46. Ravera M, Re M, Weiss U, Deferrari L, Deferrari G. Emerging therapeutic strategies in diabetic nephropathy. J Nephrol 2007; 20 Suppl 12: S23-32.
47. Brinkkoetter PT, Holtgrefe S, van der Woude FJ, Yard BA. Angiotensin II type-1 receptor mediated changes in heparan sulphate proteoglycans in human SV40 transformed podocytes. J Am Soc Nephrol 2004; 15: 33-40.
48. Köppel H, Yard BA, Christ M, Wehling M, van der Woude FJ. Modulation of angiotensin-II mediated signalling by heparan sulphate glycosaminoglycans. Nephrol Dial Transplant 2003; 18: 2240-7.
49. Oster JR, Singer I, Fishman LM. Heparin induced aldosterone suppression and hyperkalemia. Am J Med 1995; 98: 575-86.
50. Goh SY, Jasik M, Cooper ME. Agents in development for the treatment of diabetic nephropathy. Expert Opin Emerg Drugs 2008; 13: 447-63.
51. Weiss R, Niecestro R, Raz I. The role of sulodexide in the treatment of diabetic nephropathy. Drugs 2007; 67: 2681-96.
52. Ruggeri A, Guizzardi S, Franchi M, Morocutti M, Mastacchi R. Pharmacokinetics and distribution of a fluoresceinated glycosaminoglycan, sulodexide, in rats. Part II: Organ distribution in rats. Arzneimittelforschung 1985; 35: 1517-9.
53. Shestakova MV, Chugunova LA, Vorontsov AV, Dedov II. The efficacy of sulodexide – a low-molecular heparin – in the therapy of diabetic nephropathy. Ter Arkh 1997; 69: 34-7.
54. Campo GM, Avenoso A, Campo S, et al. Glycosami­noglycans modulate inflammation and apoptosis in LPS-treated chondrocytes. J Cell Biochem 2009; 106: 83-92.
55. Karoń J, Połubinska A, Antoniewicz AA, Sumińska-Jasińska K, Breborowicz A. Anti-inflammatory effect of sulodexide during acute peritonitis in rats. Blood Purif 2007; 25: 510-4.
56. Ciszewicz M, Polubinska A, Antoniewicz A, Suminska-Jasinska K, Breborowicz A. Sulodexide suppresses inflammation in human endothelial cells and prevents glucose cytotoxicity. Transl Res 2009; 153: 118-23.
57. Kristová V, Lísková S, Sotníková R, Vojtko R, Kurtan­ský A. Sulodexide improves endothelial dysfunction in streptozotocin-induced diabetes in rats. Physiol Res 2008; 57: 491-4.
58. Lever R, Page C. Glycosaminoglycans, airways inflam­mation and bronchial hyperresponsiveness. Pulm Pharmacol Ther 2001; 14: 249-54.
59. Tamsma JT, van der Woude FJ, Lemkes HH. Effect of sulphated glycosaminoglycans on proteinuria in patients with overt diabetic (type 1) nephropathy. Nephrol Dial Transplant 1996; 11: 182-5.
60. Gambaro G, Kinalska Oksa A, Pont’uch P, et al. Oral sulodexide reduces albuminuria in microalbuminuric and macroalbuminuric and type 1 and type 2 diabetic patients; the Di.N.A.S. randomized trial. J Am Soc Nephr 2002; 13: 1615-25.
61. Sulikowska B, Olejniczak H, Muszynska M, et al. Effect of sulodexide on albuminuria, NAG excretion and glomerular filtration response to dopamine in diabetic patients. Am J Nephrol 2006; 26: 621-8.
62. Sulikowska B, Nieweglowski T, Manitius J, Lisiak-Szydlowska W, Rutkowski B. Effect of 12 month therapy with omega-3 polyunsaturated acids on glomerular filtration response to dopamine in IgA nephropathy. Am J Nephrol 2004; 24: 474-82.
63. Szelachowska M, Poplawska A, Topolska J, Kinalska I, Grimaldi M. A pilot study of the effect of the glyco­saminoglycan sulodexide on microalbuminuria in type I diabetic patients. Curr Med Res Opin 1997; 13: 539-45.
64. Sorrenti G, Grimaldi M, Canova N, Palazzini E, Melchionda N. Glycosaminoglycans as a possible tool for micro- and macroalbuminuria in diabetic patients. A pilot study. J Int Med Res 1997; 25: 81-6.
65. Solini A, Vergnani L, Ricci F, Crepaldi G. Glycosamino­glycans delay the progression of nephropathy in NIDDM. Diabetes Care 1997; 20: 819-23.
66. Skrha J, Perusicová J, Pont’uch P, Oksa A. Glycosami­noglycan sulodexide decreases albuminuria in diabetic patients. Diabetes Res Clin Pract 1997; 38: 25-31.
67. Dedov I, Shestakova M, Vorontzov A, Palazzini E. A randomized, controlled study of sulodexide therapy for the treatment of diabetic nephropathy. Nephrol Dial Transplant 1997; 12: 2295-300.
68. Poplawska A, Szelachowska M, Topolska J, Wysocka-Solowie B, Kinalska I. Effect of glycosaminoglycans on urinary albumin excretion in insulin-dependent diabetic patients with micro- or macroalbuminuria. Diabetes Res Clin Pract 1997; 38: 109-14.
69. Perusicová J, Skrha J. The effect of sulodexide, a glycosaminoglycan, on albuminuria in diabetic patients. Vnitr Lek 1997; 43: 748-52.
70. Zalevskaia AG, Astamirova KhS, Karpova IA, Popova SG. A trial of the use of the low-molecular heparin sulodexide in the therapy of diabetic nephropathy. Ter Arkh 1998; 70: 71-4.
71. Rasovskii BL, Tarasov AV, Trel’skaia NIu, Severina TI, Chernykh EF. Sulodexide in the treatment of diabetic nephropathy. Klin Med (Mosk) 1998; 76: 40-42.
72. Skrha J, Perusicová J, Kvasnicka J, Hilgertová J. The effect of glycosaminoglycan sulodexide on oxidative stress and fibrinolysis in diabetes mellitus. Sb Lek 1998; 99: 103-9.
73. Oksa A, Pontuch P, Kratochvilova H. The effect of glycosaminoglycan sulodexide on albuminuria in patients with diabetes mellitus. Bratisl Lek Listy 1999; 100: 486-9.
74. Achour A, Kacem M, Dibej K, Skhiri H, Bouraoui S, El May M. One year course of oral sulodexide in the management of diabetic nephropathy. J Nephrol 2005; 18: 568-74.
75. Heerspink HL, Greene T, Lewis JB, et al.; Collaborative Study Group. Effects of sulodexide in patients with type 2 diabetes and persistent albuminuria. Nephrol Dial Transplant 2008; 23: 1946-54.
76. Lambers Heerspink HJ, Fowler MJ, Volgi J, et al.; Collaborative Study Group. Rationale for and study design of the sulodexide trials in Type 2 diabetic, hypertensive patients with microalbuminuria or overt nephropathy. Diabet Med 2007; 24: 1290-5.
77. Vilayur E, Harris DC. Emerging therapies for chronic kidney disease: what is their role? Nat Rev Nephrol 2009; 5: 375-83.
78. Kreguger J, Spillmann D, Li J, Lindahl U. Interactions between heparan sulphate and proteins: the concept of specificity. J Cell Biol 2006; 174: 323-7.
79. Reynertson RH, Parmley RT, Rodén L, Oparil S. Proteoglycans and hypertension 1. A biochemical and ultrastructural study of arta glycosaminoglycans in spontaneously hypertensive rats. Coll Relat Res 1986; 6: 77-101.
80. Borawski J, Dubowski M, Pawlak K, Mysliwiec M. Effect of sulodexide on plasma transforming growth factor-beta1 in healthy volunteers. Clin Appl Thromb Hemost 2010; 16: 60-5.
81. Pisano L, Moronesi F, Falco F, et al. The use of sulodexide in the treatment of peripheral vasculopathy accompanying metabolic diseases. Controlled study in hyperlipidemic and diabetic subjects. Thromb Res 1986; 41: 23-31.
82. Crepaldi G, Fellin R, Calabro` A, et al. Double-blind multicenter trial on a new medium molecular weight glycosaminoglycan. Current therapeutic effects and perspectives for clinical use. Atherosclerosis 1990; 81: 233-43.
83. Calabro` A, Rossi A, Baiocchi MR, Coscetti G, Fellin R, Crepaldi G. Effect of sulodexide on hemorheological parameters in a group of patients with peripheral atherosclerotic vascular disease. Ric Clin Lab 1985; 15 Suppl 1: 455-63.
84. Kim SB, Kim SH, Lee MS, Chang JW, Lee SK, Park JS. Effects of sulodexide on hemostatic factors, lipid profile and inflammation in chronic peritoneal dialysis patients. Perit Dial Int 2007; 27: 456-60.
85. Coccheri S, Scondotto G, Agnelli G, Palazzini E, Zamboni V; Arterial Arm of the Suavis (Sulodexide Arterial Venous Italian Study Group). Sulodexide in the treatment of intermittent claudication. Results of a randomized, double-blind, multicentre, placebo-controlled study. Eur Heart J 2002; 23: 1057-65.
86. Harenberg J. Review of pharmacodynamics, pharma­cokinetics and therapeutic properties of sulodexide. Med Res Rev 1998; 18: 1-20.
87. Lauver DA, Lucchesi BR. Sulodexide a renewed interest in this glycosaminoglycan. Cardiovasc Drug Rev 2006; 24: 214-26.
88. Cosmi B, Cini M, Legnani C, Pancani C, Calanni F, Coccheri S. Additive thrombin inhibition by fast moving heparin and dermatan sulphate explains the anticoagulant effect of sulodexide, a natural mixture of glycosaminoglycans. Thromb Res 2003; 109: 333-9.
89. Buchanan MR, Liao P, Smith LJ, Ofosu FA. Prevention of thrombus formation and growth by antithrombin III and heparin cofactor-II dependent thrombin inhibitors: importance of heparin cofactor II. Thromb Res 1994; 74: 463-75.