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Anti-inflammatory effects of non-anticoagulant heparinoids in vitro and in a rat renal transplantation
model
Ditmer Talsma S1741241
Supervisor: Jaap van den Born
Experimental Nephrology University Medical Center Groningen
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List of contents
1. Summary 3 2. Samenvatting 4 3. Introduction 5 4. Methods 7
a. ELISA 7 b. Rat kidney transplantation 9 c. Immunohistochemistry 10 d. Quantification of interstitial fibrosis 11 e. Statistics 12
5. Results 12 a. HSPG’s bind L-selectin, CCL2, properdin and factor 12
H in a dose dependent way b. Heparinoids inhibit the interaction of L-selectin,CCL2 and 12
properdin with perlecan c. Inhibition potential of heparinoids, comparing CCL2, 14
L-selectin and properdin d. Heparinoid intervention study in rat renal TX model 15 e. No effects of (non) anticoagulant heparin(oids) 15
on physiological parameters f. No reduction in complement activation by heparin(oid) 16
treatment g. Reduced influx of leukocytes in R-O heparin treated 18
transplanted kidneys 6. Discussion 19 7. Acknowledgements 21 8. References 21 9. Appendix 24
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Summary Introduction –Chronic transplant dysfunction (CTD) is histologically characterized by tissue remodeling including interstitial fibrosis, tubular atrophy, glomerulosclerosis, and transplant vasculopathy, along with a chronic inflammatory component including complement activation and leukocyte infiltration. Currently, there is no treatment to reduce the tissue remodeling in CTD. We earlier showed upregulation of matrix proteoglycans (PGs) in CTD, and now hypothesize that heparinoids, via PG-ligand interaction inhibition can reduce complement activation and leukocyte recruitment and could be a target of intervention. Methods – Heparinoid effectivity to diminish chemokine (CCL2), leukocyte adhesion molecule (L-selectin) and complement (properdin and factor H) binding to immobilized heparan sulfate PG’s was evaluated in an ELISA in vitro approach. The in vivo anti-inflammatory effects of these heparinoids on development of CTD was tested in a rat CTD model. Results – ELISA results showed the binding of L-selectin, CCL-2 and properdin to heparan sulfate PG perlecan. Factor H did not interact with perlecan, however bound with heparin-albumin. The interaction of L-selectin, CCL2 and properdin with perlecan could be dose-dependently inhibited by heparin and two non-anticoagulant heparinoids, namely N-acetyl heparin and RO-heparin. Results showed CCL-2 to be most sensitive for heparinoid inhibition, followed by properdin and L-selectin. In the rat CTD model daily s.c. treatment with the non-anticoagulant RO-heparin reduced tubulo-interstitial inflammation with CD45+ cells by 50% ( p=0.0175). Unexpectedly, RO-heparin increased properdin deposition in the transplanted kidneys. Terminal complement MAC complex formation was non-significantly reduced in all heparin treated groups. Conclusion – Our data show that (non-anticoagulant) heparin(oids) can reduce chemokine, adhesion molecule, and complement binding to heparan sulfate PG’s in vitro, and are effective in reducing inflammation in rat CTD. We therefore suggest (non-anticoagulant) glycomimetics to be a promising therapeutic modality to reduce CTD and possibly other fibrotic diseases.
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Samenvatting
Introductie – Histologisch gezien wordt chronisch transplantaat falen (CTF) gekenmerkt door interstitiële fibrose, tubulaire atrofie, glomerulosclerose en transplanataat vasculopathie in combinatie met een chronische ontstekingscomponent. Op dit moment is er geen effectieve therapie om CTF tegen te gaan. In voorafgaand onderzoek hebben we laten zien dat er een verhoogde expressie is van proteoglycanen (PG) in CTF. Daarom luidt onze hypothese dat door het remmen van de heparan sulfaat proteoglycanen (HSPG) ligand interactie, ontsteking kan worden geremd en dat deze methode mogelijk een goede interventie kan zijn bij CTF. Methode – De capaciteit van heparinoiden om chemokine (CCL2), leukocyt adhesie molecuul (L-selectine) en complement (properdine en factor H) binding aan HSPG’s te remmen werd getest in vitro. De effecten van heparinoiden op de ontwikkeling van CTF in vivo werd getest in een rat CTF model. Resultaten – ELISA resultaten laten zien dat zowel CCL2, L-selectin als properdine binden aan HSPG perlecan. Factor H bind niet met perlecan maar wel met heparine-albumine. De interactie van CCL2, L-selectine en properdine kan concentratie afhankelijk worden geremd door heparine en twee non-anticoagulante heparinoiden, namelijk N-acetyl heparine en R-O heparine. De resultaten lieten zien dat de binding van CCL2 aan HSPG het beste geremd kon worden gevolgd door properdine en L-selectine. In het rat CTF model, zorgden dagelijkse subcutane injecties met een van de (non-)anticoagulante heparin(oiden) voor een reductie in CD45+ leukocyten influx in het tubulo-interstitium van 50% ( p=0.0175). Onverwacht zorgde R-O heparine voor een toegenomen depositie van properdine in de transplantaten. De formatie van het membrane attack complex (MAC) was (niet significant) gereduceerd in de met heparinoiden behandelde groepen. Conclusie – Dit onderzoek laat zien dat (non-)anticoagulante heparin(oiden) effectief zijn in het remmen van CCL2, L-selectine en properdine aan HSPG’s en dat ze zorgen voor een verminderde ontsteking in een rat CTF model. Hiermee laten wij zien dat (non-)anticoagulante glycomimetica een mogelijke aanvulling kunnen zijn op de behandeling van CTF en andere fibrotische aandoeningen.
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Introduction
In the past few decades tremendous improvements have been made in preventing short term loss of kidney allografts, however no corresponding improvements have been made in reducing allograft loss due to long term transplant failure. Advances in surgical and preservation techniques, better immunosuppressant use and improved tissue typing has led to a 1-year graft survival of 90% in 2008 1, unfortunately scientific advances in long term graft survival showed no such improvements 2. A major factor in long term graft failure is chronic transplant dysfunction (CTD), already showing a histological incidence of 70% 2-years after transplantation and is clinically characterized by hypertension, loss of renal function, proteinuria and raised serum triglyceride levels 3-
5. The etiology of CTD is multifactorial and is the result of tissue remodeling in arteries, glomeruli and the tubulo-interstitium leading to transplant arteriopathy (neo-intima formation), glomerulosclerosis, interstitial fibrosis and tubular atrophy. Histological processes causing these lesion are mesangial cell, myofibroblasts and tubular epithelial cell activation, extracellular matrix expansion and chronic inflammation 6,7. Besides immunosuppressive therapy, up to now progressive loss of transplant function is only symptomatically treated by anti-hypertensive, anti-proteinuric treatment and lipid lowering drugs. The involvement of proteoglycans (PG’s) in CTD has been shown by a recent experiment performed by our group revealing an increase in the expression of the PG’s perlecan and versican, in allografted kidney’s in a rat renal transplantation model. They further showed an increased expression of chemokines, CCL2 (MCP-1) and CCL5 (RANTES), and an augmented tubulo-interstitial L-selectin binding of PG’s in rat renal allografts (article in preparation). Furthermore our group has shown that PG’s can bind properdin and factor H, regulatory molecules in the alternative pathway of complement 8,9. This suggests the development of a pro-inflammatory milieu in CTD and PG’s being important mediators in this process. The complement system is part of the innate immunity and consists of several proteins forming an activation cascade which leads to the release of C3a and C5a, important chemotactic proteins, and eventually to the formation of the MAC complex, which is a membrane perforating protein complex. The complement system has 3 pathways, the classical, alternative and lectin pathway and is known to be involved in the development of tubulo-interstitial scarring in multiple progressive renal diseases 10,11. Growing evidence exists that the alternative pathway of complement is activated on tubular cells in proteinuric diseases 12,13 and can therefore be a possible target in CTD treatment. Major regulatory proteins of the alternative pathway are properdin and factor H, resp. an activator and an inhibitor of alternative pathway complement activation. It has been show by our group that PG’s can bind properdin and factor H and that they recognize different epitopes on heparan sulfate 9. This might implicate a role of heparan sulfates in alternative pathway activation regulation. In the initiation of an inflammatory response, the rolling of immune cells, endothelial cell activation, leukocyte firm adhesion and transendothelial migration, cause leukocytes to migrate towards the site inflammation. This process starts with endothelial expression of PG’s forming a receptor-ligand interaction with L-selectin which is
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expressed on the leukocyte cell surface. Subsequent leukocyte rolling causes slowing of the leukocytes. Thereafter chemo-attractants expressed by the endothelial cell is bound by PG’s and presented to their high affinity receptor on the leukocyte cell surface, which causes leukocyte activation leading to integrin expression. Integrins facilitate firm adhesion to the endothelium through their interaction with PG’s and cause transmigration trough the endothelial wall 14-17. In the tubulo-interstitium, leukocytes are guided by a chemokine gradient towards the inflammatory site. PG’s aid in this process by stabilizing the chemokine gradient resulting in more efficient leukocyte migration 14,15,18. PG’s not only influence inflammation but also fibrosis where they are a part of the matrix depositions as shown by our group (article in progress). A direct functional effect of PG’s in fibrosis has been shown by Harmer and colleagues who showed PG’s to be a co-receptor for growth factors (i.e. FGF) 19.
Proteoglycans consist of a protein core structure with covalently attached repetitive disaccharide polymeric chains called glycosaminoglycans (GAG’s). The GAG’s can influence biological systems through their binding affinity with different substrates. Due to the variety in disaccharide make-up of the GAG’s, they are able to bind a large variety of molecules and can, because of their disaccharide patterns, be divided in different groups called keratan sulfate, chondroitin sulfate, dermatan sulfate and heparan sulfate. An important factor in the binding capacity of a GAG is its sulfation pattern 20. High or low sulfation enables the GAG to bind chemokines, adhesion molecules, growth factor and cytokines. When cells get activated (i.e. inflammation) they reduce their expression of HSulf-1, which normally causes 6-O desulfation, resulting in a higher degree of sulfation and subsequently the ability to bind leukocyte L-selectin 21,22.
The main focus of this report will be on heparan sulfate proteoglycans (HSPG’s). HSPG’s can be divided in different subtypes due to their disaccharide pattern and their location in the extracellular compartment. Syndecans and glypicans can be found on the cell surface and agrin, perlecan and collagen XVIII are located in the extracellular matrix. The best known heparan sulfate is heparin, which is a highly sulfated heparan sulfate GAG and is known for almost a century for its anti-coagulant action due to antitrombin III binding and activation. However in recent decades researchers have become aware of the anti-inflammatory actions of heparin 23. In theory due to the higher sulfation pattern of heparin it can bind several inflammatory agents like chemokines and adhesion molecules with a higher affinity compared to cell surface and ECM HSPG’s. When introduced as a free circulating GAG, heparin is therefore able to block the binding of immunological substrates to HSPG’s and subsequently to its receptor. Research has shown interactions of heparin and heparin sulfates with L-selectin, chemokines and growth factors, increasing the plausibility for this theory 16,24.
Now we want to investigate if heparin(oids) are an effective treatment modality in reducing CTD development by altering inflammation and fibrosis. However before testing the anti-inflammatory effects of heparin in the treatment of CTD, some major side effects of heparin have to be overcome. As mentioned earlier, heparin was originally discovered as an anti-coagulant drug and has been used for this purpose in the clinic for a long time. When treating patients with heparin to counteract immunological activation, anticoagulation is a serious side effect and moreover, treatment with heparin can result in heparin-induced thrombocytopenia type II (HIT II) which is an autoimmune response against platelet factor 4, bearing a great risk for thrombosis 25. Therefore in this study we used non-anticoagulant heparinoids, these are heparin derived GAG’s which, due to
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chemical modifications, lost their affinity for antitrombin III resulting in the loss of its anti-coagulation properties. The non-anticoagulant heparinoids used in this study are N-desulfated, N reacetyled heparin (N-acetyl heparin) and periodate-oxidized, borohydride-reduced heparin (R-O heparin). An important aspect is that N-acetyl heparin has a reduced sulfation compared to normal heparin and R-O heparin has not. The anti-inflammatory actions of N-acetyl heparin have been shown in studies in which N-acetyl heparin treatment reduced inflammation induced ischemia-reperfusion damage in rat lungs and in rabbit hearts 26,27. R-O heparins anti-inflammatory actions have been described before by Chen and colleagues, who showed that R-O heparin could inhibit L-selectin binding to heparan sulfates and therefore inhibit neutrophil interaction with human epithelial ovarian cancer cells in vitro 28.
So based on previous data of our group and other heparinoid intervention studies, we hypothesize that HSPGs could be promising therapeutic targets to limit CTD, especially focusing on the potential to inhibit inflammation, mainly complement and cell recruitment cascades. To test this hypothesis, we tested the inhibitory effects of unfractionated heparin, N-acetyl heparin and R-O heparin on CCL2, L-selectin, properdin and factor H binding to HSPG’s. Thereafter in an already performed experiment by our group, in which we intervened with vehicle, unfractionated heparin, and two different non-anticoagulant heparinoids (N-acetyl heparin and R-O heparin) in an established rat model for CTD, we evaluated inflammatory cell influx and complement activation 29. Altogether, our data support the concept that in CTD matrix proteoglycans promote inflammation and complement activation, and represent valuable targets to reduce CTD-related tissue remodeling.
Methods ELISA MCP-1 and L-selectin binding to HSPG Maxisorp 96-well flat bottom microtiter plates (U96 from VWR International, Amsterdam, The Netherlands) were coated overnight in PBS with 5 μg perlecan/ml diluted in PBS (Sigma, Zwijndrecht, TheNetherlands). After washing in PBS, wells were blocked with 5% skimmed milk powder in PBS for 1 h. Thereafter wells were incubated with a 2-step dilution range of either human recombinant MCP-1 (PeproTech, Hamburg, Germany) or human L-selectin IgM chimeric protein (produced as described30,31) starting both at 5 µg/ml. The wells were washed again, and respectively monoclonal mouse anti- human MCP-1 IgG1 (1:1000;eBioscience, Frankfurt, Germany) or HRP labeled rabbit anti-human IgM (1:500;DAKO, Glostrup, Denmark) diluted in 1% skimmed milk powder was added for 1 h. For MCP-1, after a washing step, HRP labeled goat anti-mouse IgG1, 1:500 (Southern biotech, Birmingham, USA) was added. Substrate reaction was done with 3,3’,5,5’-tetramethylbenzidine substrate (Sigma, Zwijndrecht, The Netherlands) for 15 min in the dark, and the reaction was stopped by adding 1.5 N H2SO4. Absorbance was measured at 450 nm in a microplate reader. All incubations were done at room temperature in a volume of 100 μl/well. Inhibition of MCP-1 and L-selectin binding by heparin(oids)
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Maxisorp 96-well flat bottom microtiter plates (U96 from VWR International, Amsterdam, The Netherlands) were coated overnight in PBS with 5 μg perlecan/ml diluted in PBS (Sigma, Zwijndrecht, TheNetherlands). After washing in PBS, wells were blocked with 5% skimmed milk powder in PBS for 1 h. In a separate microtiter plate, 2 μg/ml human L-selectin IgM chimeric protein (produced as described 30,31) or 1,25 μg/ml human recombinant MCP-1 (PeproTech, Hamburg, Germany) was co-incubated with a dilution range of either, unfractionated heparin, N-acetylated heparin or R-O heparin for 30 min, then transferred to the ELISA plate and incubated for 1 h. The wells were washed again, and respectively HRP labeled rabbit anti-human IgM (1:500;DAKO, Glostrup, Denmark) or monoclonal mouse anti- human MCP-1 IgG1 (1:1000;eBioscience, Frankfurt, Germany) diluted in 1% skimmed milk powder was added for 1 h. For MCP-1, after a washing step HRP labeled goat anti-mouse IgG1, 1:500 (Southern biotech, Birmingham, USA) was added. Substrate reaction was done with 3,3’,5,5’-tetramethylbenzidine substrate (Sigma, Zwijndrecht, The Netherlands) for 15 min in the dark, and the reaction was stopped by adding 1.5 N H2SO4. Absorbance was measured at 450 nm in a microplate reader. All incubations were done at room temperature in a volume of 100 μl/well. All experiments were performed three times in duplicate. Factor H and properdin binding to HSPG Maxisorp 96-well flat bottom microtiter plates (U96 from VWR International, Amsterdam, The Netherlands) were coated overnight in PBS with 5 μg perlecan/ml or 5 μg heparin-albumin/ml diluted in PBS (Sigma, Zwijndrecht, TheNetherlands). After washing in PBS, wells were blocked with 3% skimmed milk powder in PBS for 1 h. Thereafter wells were incubated with a 2-step dilution range of factor H starting at 5 µg/ml on a perlecan and a heparin-albumin coated plate or with a 2 step dilution range of properdin starting at 5 µg/ml on a perlecan coated plate. The wells were washed again, and respectively HRP labeled mouse anti-CFH (1:500; Abcam, Cambridge, UK) or rabbit anti-human properdin (10 µg/ml) diluted in 1% skimmed milk powder was added for 1 h. For factor H, after a washing step HRP labeled goat anti-mouse, 1:250 (Southern biotech, Birmingham, USA) was added. For properdin, HRP labeled goat anti-rabbit was added (1:2000; DAKO, Glostrup, Denmark). Substrate reaction was done with 3,3’,5,5’-tetramethylbenzidine substrate (Sigma, Zwijndrecht, The Netherlands) for 15 min in the dark, and the reaction was stopped by adding 1.5 N H2SO4. Absorbance was measured at 450 nm in a microplate reader. All incubations were done at room temperature in a volume of 100 μl/well. Inhibition properdin binding by heparin(oids) Maxisorp 96-well flat bottom microtiter plates (U96 from VWR International, Amsterdam, The Netherlands) were coated overnight with 5 μg perlecan/ml diluted in PBS (Sigma, Zwijndrecht, TheNetherlands). After washing in PBS, wells were blocked with 3% skimmed milk powder in PBS for 1 h. In a separate microtiter plate 0,75 μg/ml properdin was co-incubated with a dilution range of either, unfractionated heparin, N-acetylated heparin or R-O heparin for 30 min, and thereafter transferred to the perlecan coated ELISA plate and incubated for 1 h. The wells were washed again, and rabbit anti-human properdin (10 µg/ml) diluted in 1% skimmed milk powder was added for 1 h. After washing HRP labeled goat anti-rabbit was added (1:2000; DAKO, Glostrup, Denmark).
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Substrate reaction was done with 3,3’,5,5’-tetramethylbenzidine substrate (Sigma, Zwijndrecht, The Netherlands) for 15 min in the dark, and the reaction was stopped by adding 1.5 N H2SO4. Absorbance was measured at 450 nm in a microplate reader. All incubations were done at room temperature in a volume of 100 μl/well. Inhibition experiments were performed in three times duplicate. Rat kidney transplantation Rat kidney transplantation experiment was performed in 2010 by Kiran Katta (PhD at the time) and Ditmer Talsma during his master internship in biology. Kidney allotransplantation was performed from female DA donors (n=38) to male WF recipients (n=38) according to standard procedures as described previously 29. Cold and warm ischemia times were 15±3 (mean±SD) and 25 minutes, respectively. After transplantation the recipients were placed in an incubator at 28ºC for approximately 6 hours and caged individually. After transplantation all recipients subcutaneously received Cyclosporine A (5 mg/kg BW/day) for 10 days. The native kidney was removed after 12 to 14 days after transplantation. Animals were weighed every day and observed for signs of decreasing animal welfare reflecting their clinical condition. Upon weight loss of >15% compared to highest measured body weight, animals were sacrificed and regarded as drop outs. Animals were kept in a temperature controlled room, with a 12:12-h light:dark cycle and fed standard rodent chow and water ad libitum.
Heparinoid interventions were performed with regular, unfractionated heparin (Hep; n=9) and two non-anticoagulant heparinoids derived from regular unfractionated heparin: N-desulfated, N-reacetylated heparin (NAc-Hep; n=10) and periodate-oxidized, borohydride-reduced heparin (RO-Hep; n=9). Production and characterization of these heparinoids have been described before 32. The control transplanted group (Con; n=10) received daily vehicle (physiological saline) injections. One day before transplantation, treatment with the respective formulations was started. The above mentioned groups daily received heparin(oids) between 9.00 and 12.00 AM dissolved in physiological salt, injected subcutaneously at 2 mg/kg BW/day until sacrifice. The treatment dose was chosen according to previous studies 33,34 and is in the physiological range normally used for the treatment of thrombotic complications. Total follow up was 65±4 days (mean±SD).
Blood pressure was measured non-invasively with tail cuff method (CODA; Kent Scientific, Torrington, CT). Two weeks before transplantation the rats were trained to undergo blood pressure measurements. Rats were placed individually in metabolic cages to obtain 24h urines, food and water intake measurements. Blood pressure, 24h urine sampling and non-fasting blood sampling by orbital puncture were taken before transplantation (baseline), four and eight weeks after transplantation. Urine was analyzed for urea, creatinine and total protein. Blood was analyzed for urea, creatinine and triglycerides. Analyses were performed on a multi-test analyzer system (Roche Modular; F.Hoffmann-La Roche Ltd, Basel, Switzerland) at the central clinical laboratory of the University Medical Center Groningen. Creatinine clearance was calculated from 24h urinary volume, plasma and urinary creatinine. Kidneys were perfused with saline prior to sacrification. Half of the kidney was fixed in 4% formaldehyde and processed for paraffin embedding and other half was cryopreserved. The local animal ethics committee of the University of Groningen approved all the procedures used in the study
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and the Principles of Laboratory Animal Care (National Institute of Health publication no. 86-23) were followed.
Immunohistochemistry Leukocytes Four µm frozen kidney sections were dried for 30 minutes. Fixation of the samples was achieved by incubating them in acetone for 10 minutes. Endogenous peroxidase blocking was done by incubating the sections for 30 minutes in 0,03% H2O2. After washing sections were incubated for 60 minutes at RT with mouse anti-rat CD45 antibody (clone OX-1) 35,36 diluted 1:2 in PBS. Sequential incubation was done with goat anti-mouse IgG1 HRP labeled antibody (SouthernBiotech, Birmingham, USA) diluted 1:100 in 1% NRS in PBS, for 30 minutes at RT. Sections were fluorescently labeled using Tyramide TRITC, diluted 1:50 in amplification buffer for 10 minutes. Thereafter sections were digitalized and 30 snapshots were randomly taken at 200x magnification from the cortical regions. Quantification was done by using the MacBiophotonics ImageJ program (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). Data are expressed as % positive stained surface areas. Neutrophils Four µm frozen kidney sections were dried for 30 minutes. Fixation of the samples was achieved by incubating them in acetone for 10 minutes. Thereafter sections were incubated for 60 minutes at RT with mouse anti-rat granulocyte antibody (HIS48, Santa Cruz biotechnology, Santa Cruz, USA) diluted 1:100 in PBS. After washing, endogenous peroxidase was blocked incubating the sections for 30 minutes in 0,03% H2O2. HIS48 staining was detected by incubating the section with goat anti-mouse IgM HRP (Southern Biotech, Birmingham, USA) diluted 1:100 in 1%NRS for 1h at RT. For improved staining, sections were incubated with rabbit anti-goat Ig HRP (DAKO, Glostrup, Denmark) diluted 1:100 in 1%NRS for 1h at RT. HRP activity was developed incubating sections in 3-amino-9-ethyl-carbazole (AEC) for 15 minutes. Thereafter sections were digitalized and 25 snapshots were randomly taken at 100x magnification from the cortical regions. Quantification was done by using the MacBiophotonics ImageJ program (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). Data are expressed as % positive stained surface areas. Properdin Four µm frozen kidney sections were dried for 30 minutes. Fixation of the samples was achieved by incubating them in acetone for 10 minutes. Endogenous peroxidase blocking was done by incubating the sections for 30 minutes in 0,1% H2O2. After washing sections were incubated for 60 minutes at RT with rabbit anti-human properdin diluted 10 μg/ml 37 in PBS. Sequential incubation was done with anti-rabbit poly HRP (envision kit, DAKO, Glostrup, Denmark). HRP activity was developed incubating sections in 3-amino-9-ethyl-carbazole (AEC) for 15 minutes. Thereafter sections were digitalized and 10 snapshots were randomly taken at 100x magnification from the cortical regions. Quantification was done by using the MacBiophotonics ImageJ program (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). Data are expressed as % positive stained surface areas.
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Factor H Four µm frozen kidney sections were dried for 30 minutes. Fixation of the samples was achieved by incubating them in acetone for 10 minutes. Endogenous peroxidase blocking was done by incubating the sections for 30 minutes in 0,1% H2O2. After washing sections were incubated for 60 minutes at RT with goat anti-rat factor H diluted 10 μg/ml 38 in PBS. Sequential incubation was done with rabbit ant-goat HRP (DAKO, Glostrup, Denmark) diluted 1:100 in 1% NRS. HRP activity was developed incubating sections in 3-amino-9-ethyl-carbazole (AEC) for 15 minutes. Thereafter sections were digitalized and 10 snapshots were randomly taken at 100x magnification from the cortical regions. Quantification was done by using the MacBiophotonics ImageJ program (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). Data are expressed as % positive stained surface areas. C3 Four µm frozen kidney sections were dried for 30 minutes. Fixation of the samples was achieved by incubating them in acetone for 10 minutes. Endogenous peroxidase blocking was done by incubating the sections for 30 minutes in 0,1% H2O2. After washing sections were incubated for 60 minutes at RT with rabbit anti-human C3d (DAKO, Glostrup, Denmark) diluted 1:4000 in PBS. Sequential incubation was done with anti-rabbit poly HRP (envision kit, DAKO, Glostrup, Denmark). HRP activity was developed incubating sections in 3-amino-9-ethyl-carbazole (AEC) for 15 minutes. Thereafter sections were digitalized and 10 snapshots were randomly taken at 100x magnification from the cortical regions. Quantification was done by using the MacBiophotonics ImageJ program (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). Data are expressed as % positive stained surface areas. MAC Four µm frozen kidney sections were dried for 30 minutes. Fixation of the samples was achieved by incubating them in acetone for 10 minutes. Endogenous peroxidase blocking was done by incubating the sections for 30 minutes in 0,1% H2O2. After washing sections were incubated for 60 minutes at RT with mouse anti-rat-C5b-9 (Hycult biotech, Uden, The Netherlands) diluted 1:50 in PBS. Sequential incubation was done with rabbit anti-mouse HRP (DAKO, Glostrup, Denmark). HRP activity was developed incubating sections in 3-amino-9-ethyl-carbazole (AEC) for 15 minutes. Thereafter sections were digitalized and 10 snapshots were randomly taken at 100x magnification from the cortical regions. Quantification was done by using the MacBiophotonics ImageJ program (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). Data are expressed as % positive stained surface areas. Quantification of interstitial fibrosis Quantification of interstitial fibrosis was done by staining sections in the following manner. Sections were deparaffinized and incubated in Picro Sirius red solution (0,1g Sirius red in 100 ml picric acid) for 1 hour. Thereafter sections were incubated in 0,01 N
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HCl for 2 minutes, dehydrated and covered using DEPEX (Klinipath BV, Duiven, Netherlands) mounting medium. Per kidney 25 photomicrographs were randomly taken at 200x magnification and digitally analyzed using MacBiophotonics ImageJ program (Rasband, W.S., ImageJ, U.S. National Institute of Health, Bethesda, Maryland, USA). The amount of interstitial fibrosis was expressed as % positive stained surface area. Statistics The inhibitory effects of heparinoids in vitro were analyzed by a two-way ANOVA. Differences in leukocyte infiltration, complement activation and fibrosis in rat renal tissues were statistically analyzed using a t-test. P<0.05 was considered statistically significant.
Results
HSPG’s bind L-selectin, CCL2, properdin and factor H in a dose dependent way CCL2, L-selectin, properdin and factor H binding to the heparan sulfate PG perlecan was tested in order to determine the dose used in the heparinoid inhibition experiments. As figure 1 shows, CCL2, L-selectin and properdin showed a dose dependent binding pattern to HSPG perlecan, however factor H did not bind to perlecan, but did bind to heparin-albumin (Fig. 1). The L-selectin dosage used in the heparinoid inhibition experiment was set at 2 µg/ml to achieve an OD of just above 1,0. The CCL2 dosage was set at 1,25 µg/ml. The properdin concentration used in the inhibition experiments was set at 0,75 µg/ml. Heparinoids inhibit the interaction of L-selectin, CCL2 and properdin with perlecan In order to test whether heparin(oids) might compete with the binding of L-selectin, CCL2, and properdin to PGs such as perlecan, a number of inhibition ELISA’s were performed. The concentrations of L-selectin (2 µg/ml), CCL2 (1.25 µg/ml) and properdin (0.75 µg/ml) were chosen as described above. Unfractionated heparin and two different non-anticoagulant heparinoids, namely N-acetylated heparin and RO-heparin, reduced the binding of L-selectin and CCL2 to perlecan in a dose dependent fashion. In the L-selectin inhibition experiments (Fig. 2), IC50 of unfractionated heparin is 2,33 µg/ml. In the CCL2 inhibition experiments, IC50 of unfractionated heparin is 0,251 µg/ml. This strongly suggests that the interaction of PGs with chemokines (such as CCl-2) is more sensitive to glycosaminoglycan intervention compared to L-selectin-PGs interaction. Regarding the non-anticoagulant heparinoids, interestingly, RO-heparin competed with CCL2 very effectively, even ~4x better compared to unfractionated heparin (p<0.001). In the properdin binding inhibition experiment to perlecan, a dose dependent inhibition was seen in all three heparin(oids). A significant stronger inhibition was seen in the heparin group compared to the R-O heparin group and the N-acetyl heparin group (p<0,01). N-acetyl heparin showed the least inhibition potential in this experiment.
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5,00
0
2,50
0
1,25
0
0,62
5
0,31
2
0
1
2
3
4
L-selectin concentration (ug/ml)
OD
450n
m
5,00
0
2,50
0
1,25
0
0,62
5
0,31
2
0
1
2
3
4
CCL2 concentration (ug/ml)
OD
450n
m
5.00
0
2.50
0
1.25
0
0.62
5
0.31
2
0
1
2
3
4
Properdin concentration (ug/ml)
OD
450n
m
5.00
0
2.50
0
1.25
0
0.62
5
0.31
2
0
1
2
3
4
Factor H concentration (ug/ml)
OD
450n
m
5.00
0
2,50
0
1,25
0,6
25
0,31
2
0
1
2
3
4
Factor H concentration (ug/ml)
OD
450n
m
Figure 1, dose dependent binding of L-selectin, CCL2, properdin and factor H to perlecan andheparin-albumin. L-selectin, CCL2, and properdin show dose dependent binding to perlecan (square). Factor Hdid not show binding to perlecan, but did show dose-dependent binding to heparin-albumin (triangle).
14
0,00
16
0,00
63
0,02
50
0,10
00
0,40
00
0
25
50
75
100
**
***
Concentration heparinoids (ug/ml)
% C
CL
2 b
ind
ing
in
hib
itio
n
0,40
0
1,56
0
6,25
0
25,0
0
0
25
50
75
100
**
Heparinoid concentration (ug/ml)
% L
-sele
cti
n b
ind
ing
in
hib
itio
n
0,10
0
0,39
0
1,56
0
6,25
0
25,0
0
0
25
50
75
100
**
**
Heparinoid concentration (ug/ml)
% p
rop
erd
in b
ind
ing
in
hib
itio
n
Figure 2. Heparin(oid) mediated inhibition of CCL2, L-selectin and properdin binding to immobilizedperlecan. Recombinant CCL2, L-selectin, properdin were incubated with either unfractionated heparin, R-O heparinand N-acetyl heparin, where after binding to immobilized perlecan was measured. All heparin(oids) showed adose-dependent inhibition of CCL2, L-selectin and properdin binding. R-O heparin proved to be a stronger CCL2binding inhibitor compared to unfractionated heparin and N-acetyl heparin (p<0,001 compared to heparin), whileunfractionated heparin showed the strongest inhibition potential for L-selectin and properdin binding inhibition (p<0,01compared to R-O heparin). Striking is the ~100-fold lower heparinoid concentration needed to inhibit CCL2 compared toL-selectin and properdin, despite differences in CCL2, L-selectin and properdin molecular weight and concentration.Graphs expressed as mean±SE.
Inhibition potential of heparinoids, comparing CCL2, L-selectin and properdin From the heparinoid inhibition experiment, IC50 heparinoid and the pre-set CCL2, L-selectin and properdin concentrations were recalculated to molar weight and required heparinoid concentrations were calculated to inhibit 1 pmol CCL2, L-selectin or properdin. Striking is the difference in inhibitory capacity of heparinoids between L-selectin and CCL2, in which the latter is inhibited ~700x better by R-O heparin. As shown in the previous experiment, R-O heparin is 4x better in inhibiting the binding of CCL2 compared to heparin and N-acetyl heparin. Heparin is best in inhibiting L-selectin binding by a factor 3 compared to R-O heparin. Heparin is also best in inhibiting
15
properdin compared to R-O heparin and N-acetyl heparin (Table 1). Of the inflammatory molecules tested, CCL2 binding to HSPG’s can be best inhibited heparin(oids) and CCL2 inhibition has therefore probably the best odds of showing an effect in the in vivo study.
Heparinoid intervention study in rat renal TX model
This study included 38 male WF rats that were transplanted with a female DA kidney. During follow-up from 2-9 weeks, pre-term graft loss occurred in 3 rats in the vehicle treated group (n=10), 5 rats in the heparin (n=9), 5 rats in the N-acetyl heparin (n=10) and 2 rats in the RO-heparin group (n=9). Renal graft loss was evidenced by clinical signs such as pilo-erected fur, severe body weight loss, disoriented behaviour and high blood creatinine values. Graft loss among the various heparinoid groups was statistically not significantly different. The rats that had to be sacrificed before the end of the experiment (before 9 weeks after transplantation) were excluded from all histological and biochemical analyses described later. Accordingly, the following groups with mentioned group size were studied: Allografts treated with Vehicle (n=7), with unfractionated heparin (n=4), with N-acetyl heparin (n=5) and with RO-heparin (n=7). In the plasmas of the rats taken at 8 weeks after renal transplantation, four hours after heparin(oid) injection, we measured the activated partial thromboplastin time. In the saline treated transplanted rats this was 75 sec (median value). In the heparin(oid) groups this time was 173 sec in regular heparin group (saline versus regular heparin: p<0.05), 73 sec in the RO-heparin group, and 69 sec in the N-acetyl heparin group (both non-anticoagulant heparinoids being not different from saline treated rats). These data show that both chemically modified heparin preparations indeed were non-anticoagulant, and clearly different from regular heparin, and not from the saline-treated rats. No effects of (non-)anticoagulant heparin(oids) on physiological parameters Treatment with heparin and non-anticoagulant heparinoids had no effect on body weight of the WF recipient rats. During the follow-up, the mean arterial pressure increased gradually in recipient WF rats until the end of the experiment without statistical significances among the groups. Similarly, food and water intake and urine output in all the groups were not affected by the treatment (Table 2, appendix).
Table 1, Comparing inhibitory capacity of the heparinoids for CCL2, L-selectin and properdin Heparinoid, and CCL2, L-selectin and properdin concentrations in the HSPG binding inhibition were recalculated to molar weight and the required amount of heparinoids was calculated to inhibit 1 pmol of CCL2, L-selectin and properdin form binding to immobilized perlecan. Heparinoid values are in pmol.
(Non) anti-coagulant heparin(oid) CCL2 L-selectin Properdin
Heparin 0,087 5,601 0,399
R-O heparin 0,025 17,788 1,339
N-acetyl heparin 0,095 35,978 9,746
16
Vehicle treated groups developed CTD-related renal failure according to previous findings 39, as evidenced by rise in plasma urea and creatinine, rise in urinary protein excretion and blood pressure (Table 2, appendix). The plasma creatinine and urea levels increased in all the groups over time. Although plasma creatinine and urea levels in the RO-heparin group at 8 weeks seems to be higher compared to all other groups, these values were not significantly different. In addition, RO-heparin treated group showed less urinary protein excretion after 8 weeks follow-up compared to untreated rats (Table 2, appendix); however, without reaching the level of statistical significance. Taken together, heparinoids had no effect on renal function and RO-heparin had a tendency to reduce proteinuria. No reduction of complement activation by heparin(oid) treatment As stated in the introduction, complement activation might play an important role in CTD development. Therefore we quantified C3d, MAC complex formation, properdin and factor H. C3d and MAC being markers of complement activation and properdin and factor H being specifically for the alternative pathway activation. Complement activation in CTD was shown by an increased deposition of all complement factors in this study in vehicle treated animals compared to non-transplanted donor kidney’s (not shown). Alternative pathway molecules properdin and factor H showed eminent granular
Figure 5, Expression of C3d, MAC, properdin and factor H in heparin(oid) treated transplanted kidneys Quantification showed properdin expression to be significantly increased in transplanted kidney’s in R-O treated compared to vehicle treated animals (p<0,05). Properdin, factor H and C3d depositions were present in both vascular and cortical tubular compartments, while MAC complex was only seen in cortico-tubular areas. Graphs represent mean±SE
17
glomerular staining in mesangial regions and capillary walls. Granular factor H and properdin depositions were also seen in the cytoplasm and basement membranes of cortical tubules and were diffusely seen interstitial areas. C3d staining was mainly found in glomerular endothelium, and other vessels. Furthermore it was seen in cortical tubular basement membranes. MAC complex depositions were solely seen cortical tubular basement membranes and diffusely on the apical side of tubules. This shows that full complement activation by the production of a MAC complex is seen in the tubular areas
Veh
icle
Hep
arin
R-o
Hep
arin
N-a
cety
l hep
arin
0.0
0.5
1.0
1.5
2.0
2.5*
Me
an
% a
rea
of
CD
45
in
flu
x
Veh
icle
Hep
arin
R-O
hep
arin
Nac
hep
arin
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Me
an
% a
rea
of
HIS
48
in
flu
x
A B
Figure 7, R-O heparin reduces leukocyte influx. A. Quantification of CD45 + cell influx in the kidney showed a reduced influx in the R-O heparin treated group (p=0,0175). Thereafter staining and quantification was done for neutrophils (B) to identify the type of leukocyte reduced. Although we could not find statistical differences, neutrophils seem to be slightly reduced in R-O heparin treated transplants. Graphs represent mean±SE at 9 weeks.
0 50 100 150 2000
3
6
9
Proteinuria mg/24h
% a
rea s
tain
ed
fo
r M
AC
Figure 6, Correlation between MAC formation and proteinuria. Comparing MAC formation and proteinuria in all transplanted kidneys revealed a borderline significant correlation (P=0,0644; R
2=0,1608).
18
but not in the endothelial compartment (Fig. 5). Quantification of the complement factors showed a ~2 fold increase in properdin expression in the all the heparin treated groups compared to the vehicle treated group, R-O heparin even showing a significant increase (p<0.05). No further differences were seen between the groups, although in all heparinoid treated animal MAC formation seem to be reduced (Fig. 5). Because complete complement activation (MAC formation), was solely seen in tubuli and not in the endothelium we tested the correlation of MAC formation with proteinuria. Although not significant, there appeared to be a correlation between MAC formation and proteinuria (P=0,0644; R2=0,1608) (Fig. 6). Reduced influx of leukocytes in R-O heparin treated transplanted kidneys Cortical CD45 staining revealed that the leukocyte influx was significantly decreased by ~50% in RO-heparin group compared to the vehicle treated group (p=0.0175; Fig. 7 A). In order to investigate the subtypes of the leukocytes that are reduced in RO-heparin group, we analyzed the tubulo-interstitial neutrophil influx. Although R-O heparin showed a lower neutrophil influx in the tubulo interstitium, no statistical significance was reached (Fig. 7B). We conclude that RO-heparin reduced the influx of inflammatory cells into the transplanted kidney, most likely neutrophils are involved. Since inflammation might also influence the degree of fibrosis, we also evaluated interstitial fibrosis. In vehicle Tx rats, interstitial fibrosis is increased 5-fold compared to control non-TX rats (not shown). Despite a reduced influx of CD45 positive leukocytes in the R/O heparin treated group, no difference was seen in the deposition of collagen between vehicle treated and heparin treated groups (Fig. 8).
Veh
icle
Hep
arin
R-O
hep
arin
Nac
hep
arin
10
15
20
25
30
Mean
sta
in a
rea (
%)
Figure 8, No reduction in fibrotic depositions in treated animals. Picro Sirius Red staining and quantificantion showed no effect of heparinoid treatment on fibrosis in the transplanted kidney. Graphs represent mean±SE at 9 weeks.
19
Discussion Development of CTD in allograft kidneys is the result of tissue remodeling in all functional entities of the kidney leading to interstitial fibrosis, glomerulosclerosis, tubular atrophy and transplant vasculopathy. In a recent study by our group it was shown that tissue remodeling in CTD is associated with an increased tubulo-interstitial matrix expression of PG’s and chemokines which suggests the formation of a pro-inflammatory milieu. In this report we confirm the binding of CCL2, L-selectin, factor H and properdin to HSPG’s, moreover we show the intervention potential of heparinoids in their in vitro ability to reduce binding of CCL2, L-selectin and properdin to immobilized HSPG’s. The in vivo effects of heparinoids on development of CTD were shown in a rat transplantation model in which the non-anti-coagulant heparinoid R-O heparin reduced leukocyte influx. Combined these findings suggest a therapeutic potential of heparinoids in the treatment of CTD.
At this moment there is no effective treatment to cure CTD. Our data show the potential of heparinoids to inhibit inflammation in a CTD model via a reduction in recruited leukocytes and probably complement MAC formation. It is well known that HSPG’s play a pivotal role in inflammatory leukocyte recruitment, especially due to the interactions of HSPG’s with L-selectin and chemokines as shown by former research done by our group 21,40. Our in vitro data shows the ability of free heparinoids to alter this interaction and reduce L-selectin and chemokine binding to HSPG’s. Therefore we propose the leukocyte influx inhibiting effect of R-O heparin to be due to a reduced interaction of L-selectin with the endothelial HSPG’s causing reduced rolling and due to interference with chemokine presentation causing reduced leukocyte activation and transmigration. These findings are in line with other studies showing the potential of heparin related compounds in reducing inflammation via inhibiting HSPG-chemokine and HSPG-L-selectin interactions 41-43. Combined, literature and our findings show non-anticoagulant heparinoids to be from a therapeutic point of view.
One of the trademarks of the development of CTD is the presence of proteinuria, which is damaging to the kidney. It has been shown that complement plays a role in the damage causing mechanisms in proteinuria by 1), the activation of the alternative pathway due to complement factors present in the ultrafiltrate and 2), the production of complement factors by tubular cells upon activation by filtered serum proteins 12,44. The R-O heparin treated animals showed a borderline significant reduction in proteinuria, associated with a borderline significant reducing effect on the complement activation shown by MAC complex formation. Subsequently a correlation was seen between proteinuria and MAC formation, showing that proteinuria is likely to be involved in complement activation in tubuli in CTD. We suggest that tubulo-epithelial cells lack the complement inhibiting mechanisms seen in endothelial cells, since we have shown in this report that MAC formation in the transplanted kidney is restricted to tubuli. Unexpectedly, an increase was seen in properdin expression in all heparinoid treated groups, especially in the R-O treated animals. We speculate that the heparinoids most likely crosslinked properdin into large oligomeric complexes with increased tissue binding properties, but this has to be proven.
It remains a question why the heparin(oids) turned out to be ineffective with regard to renal function, graft survival and tissue remodeling. In other experimental
20
models, treatment with non-anticoagulant low molecular weight heparin (LMWH) showed prolonged skin, cardiac and renal allograft survival with beneficial outcomes 33,45-47. In addition, LMWH decreases mesangial proliferation and matrix expansion 48,49. Moreover RO-heparin has been described to inhibit FGF2 and VEGF mediated pathways and to reduce tumor growth 50. We suggest that the full HLA-mismatch of our DA to Wistar Furth model, without any immunosuppression after day 10 might explain this. The progressive tissue remodeling in our model might be simply too strong to show (partial) down modulation by heparinoids. Besides to that, it is known that the half-life of unfractionated heparin(oids) as used in our study, is shorter compared to LMW heparinoids. Based on these consideration we propose a future experiment using LMW-heparinoids (including LMW-RO-heparin), in combination with the immunosuppressive agent cyclosporine A.
PG’s are well known for their effect on development of fibrosis via interaction with multiple growth factors and augmenting their binding to high affinity receptors 51. Because GAG’s are pleiotropic, one would expect heparinoids to bind growth factors and interfere with the PG-growth factor axis and subsequently reduce their effect on cell proliferation. The anti-mesangial cell proliferation and anti-ECM expansion properties of heparin have been described in earlier studies by Floege and colleagues. They showed a reduction in bFGF expression in mesangial cells and inhibitied mesangial matrix expension of laminin, Col I and IV, fibronectin and entactin in mesangioproliferative glomerulonephritis upon treatment with heparin 49. However in our intervention study we failed to see an effect of heparinoid treatment on the development of fibrosis. A possible explanation would be that the expression levels of growth factors is too high to be inhibited by the heparinoids used in this study. Alternatively, non-heparin binding growth factors dominate the fibrotic response as seen in the rat CTD model. We however didn’t test the ability of the heparinoids used to inhibit PG-growth factor interactions.
As described in literature, the sulfation pattern is important for the binding capabilities of heparinoids. In N-acetylated heparinoid, N-linked sulfate groups are replaced by acetyl groups. As a consequence, this N-acetylated heparin lost antithrombin binding activity, but also lost some L-selectin binding activity as shown in Figure 3, which might explain lack of anti-inflammatory effect in our study. The chemical treatment on R-O heparin opens some of the disaccharide ring structures. This causes R-O heparin to be more flexible and to lose its affinity for anti-thrombin III. The higher flexibility might enable R-O heparin to be more effective in reducing MCP-1 and L-selectin binding to PG’s compared to unfractionated heparin, which might explain the better inhibition of leukocyte influx.
In recent decades, much effort has been put in designing or identifying biologically active heparin glycomimetics for use as an anti-cancer or anti-inflammatory treatment modality41,52. Our data and other studies show the anti-inflammatory potential of heparin derived molecules as therapeutic agents in renal diseases 33,49. Heparinoid selection for further testing should be strict with the emphasis on selective targeted biological activity and minimum cross reactivity to other biological systems. Other important aspects are the source of the heparinoid (animal derived, semi-synthetic, full synthetic etc.), the costs, pharmacological behaviour and mass producability. Based on our data we speculate that heparin-like glycomimetics might be promising add- on therapeutic modalities in CTD next to immunosuppressants, blood-pressure and lipid
21
lowering medication. Future studies in experimental models and in human renal transplantation setting have to prove efficacy of this assumption.
Acknowledgements I want to thank Jaap van den Born for supervising me and giving me lots of useful comments on scientific interpretation of results, presenting skills and writing. I want to thank Kiran Katta for my introduction in the world of animal experiments and for our cooperation. I want to thank Marian Bulthuis for providing the HIS48 antibody and conjugate. I want to thank the Ronzoni Institute for chemical and biochemical research for providing the heparinoids. And finally I want to thank all my lab colleagues for their support and comments.
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Appendix Table 2, Physiological parameters of recipients at baseline (pre-Tx) and at 4 and 8 weeks post-transplantation.
Wistar Furth rats receiving a Dark Agouti kidney were treated with physiological saline, unfractionated heparin, R-O heparin or N-acetyl heparin from 1 day prior to transplantation until scarification (at 65±4 days). Physiological measures were taken at baseline (prior to transplantation), 4 and 8 weeks. No difference between the treatment groups was seen regarding to survival. Also no statistical differences were seen in physiological parameters, however R-O heparin shows a reduction in proteinuria without reaching statistical difference. Data shown as Median (25%-75% interquartile range).
DA-to-WF allograft
Vehicle (n=7) Unfractionated Heparin (n=4)
RO-Heparin (n=7)
N-acetyl Heparin (n=5)
Body weight (g)
Baseline 290(285-296) 283(274-291) 270(263-284) 271(270-276)
4 weeks after Tx
293(281-313) 299(292-302) 284(281-302) 285(276-290)
8 weeks after Tx
323(298-347) 311(300-320) 298(280-326) 303(299-313)
Food intake (g/24h)
Baseline 9(5-11) 5(5-7) 5(4-7) 5(4-7)
4 weeks after Tx
5(4-8) 7(6-9) 2(1-5) 5(5-11)
8 weeks after Tx
3(3-6) 1,5(1-4) 4(1-7) 2(1-6)
Water intake (ml/24h)
Baseline 17(17-20) 14(12-17) 13(10-16) 12(8-15)
4 weeks after Tx
25(21-27) 29(19-37) 18(16-24) 19(17-23)
8 weeks after Tx
27(24-30) 28(24-31) 22(17-46) 18(9-33)
Urinary output(ml/24h)
Baseline 14(13-17) 15(15-15) 12(11-14) 11(11-12)
4 weeks after Tx
26(18-30) 24(20-28) 15(13-28) 19(19-20)
8 weeks after Tx
33(29-34) 32(28-35) 31(18-44) 30(21-30)
Plasma creatinine (µmol/L)
Baseline 19(18-21) 19(19-21) 15(14-17) 15(15-19)
4 weeks after Tx
73(59-120) 64(52-73) 65(54-168) 81(53-84)
8 weeks after Tx
96(73-139) 72(58-99) 136(70-207) 73(58-122)
Creatinine Clearance (ml/min)
Baseline 3,0(2,8-3,8) 2,6(2,5-3,0) 3,5(2,7-3,7) 3,0(2,3-3,0)
4 weeks after Tx
0,7(0,4-1,1) 1,0(1,0-1,3) 1,0(0,5-1,2) 0,7(0,1-0,8)
8 weeks after Tx
0,6(0,3-1,0) 0,8(0,5-1,2) 0,4(0,2-0,9) 0,9(0,4-1,0)
Plasma urea (mmol/L)
Baseline 6(5-7) 6(5-7) 6(5-6) 6(5-6)
4 weeks after Tx
20(18,3-36) 18(16-20) 20(14-39) 19(15-24)
8 weeks after Tx
28(21-51) 22(18-37) 43(21-64) 27(24-40)
25
Total urinary protein (mg/24h)
Baseline 10(8-11) 7(6-8) 9(7-10) 9(6-9)
4 weeks after Tx
8(7-12) 8(7-11) 9(7-13) 8(7-10)
8 weeks after Tx
56(34-93) 61(57-74) 33(23-43) 94(90-184)
Mean arterial pressure (mmHg)
Baseline 118(111-121) 118(111-121) 118(111-121) 118(111-121)
4 weeks after Tx
110(104-123) 118(106-132) 141(121-148) 146(135-148)
8 weeks after Tx
138(131-165) 143(138-150) 149(135-154) 166(153-171)
