Renal heparan sulfate proteoglycans:A double edged sword

Bij veel nieraandoeningen speelt ontsteking een belangrijke pathofysiologische rol. Processen die hierbij van belang zijn, zijn onder andere de migratie van ontstekingscellen van het bloed naar de weefsels en de activatie van het complement systeem, een eiwitcascade. De afgelopen jaren is het steeds duidelijker geworden dat heparan sulfaten een belangrijke rol vervullen in deze ontstekingsprocessen. Heparan sulfaten zijn eiwitten met daaraan vast saccharideketens. Deze heparan sulfaten bevinden zich op alle cellen en bindweefsels en dus ook op endotheel cellen. In dit proefschrift laten we zien dat dysfunctionele heparan sulfaten op het endotheel leiden tot een verminderde ontstekingsreactie in diabetische nefropathie. Daarnaast laten we zien dat de heparan sulfaten in het endotheliale bindweefsel een belangrijke rol spelen. Behandeling gericht op het remmen van de interactie tussen heparan sulfaten en de immuuncellen in een niertransplantatiemodel leidt tot minder ontsteking maar niet tot een betere nierfunctie. Naast cellulaire ontsteking hebben we ook gekeken naar het remmen van het complement systeem door heparan sulfaten. We hebben laten zien dat heparine, een polysaccharide afgeleid van heparan sulfaten, de lectine route van complement kan remmen. Daarnaast laten we zien dat Salp20, een tekeneiwit, de alternative complement route kan remmen. Deze ontdekkingen zouden de basis kunnen vormen voor nieuwe therapeutische stategiën in de behandeling van nierziekten. Deze bevindingen zijn echter nog pril en er is nog veel onderzoek nodig om te kijken of ze klinische potentie hebben.Inflammation plays an important role in the pathophysiology of many renal diseases. Processes which play an important role in inflammation are, amongst others, migration of inflammatory cells across the endothelial barrier and the complement system, a protein cascade. In the past couple of years it has been shown that heparan sulfates have an important function in these inflammatory processes. Heparan sulfates consist of a protein core with saccharide chains attached to them and are present on all cells and basement membrane, which means also on endothelial cells. In this thesis we show that dysfunctional heparan sulfates on the endothelial cells result in a reduced inflammatory response in diabetic nephropathy. Moreover, we demonstrate that the heparan sulfates on the endothelial basement membranes have an important function in the migration of inflammatory cells. Treatment aiming for the interaction between heparan sulfates and the inflammatory system in a chronic transplant dysfunction model results in a reduced inflammatory response but fails to show improved transplant function. Besides cellular inflammation, we also investigated inhibition of the complement system by heparan sulfates. We showed that heparin, a polysaccharide derived from heparan sulfate, can inhibit the lectin route of complement. We also demonstrate that Salp20, a tick protein, can inhibit the alternative pathway of complement.The discoveries in this thesis can form the basis for new therapeutic entities in the treatment of renal diseases. However our results are only the beginning and more research should be done to confirm the clinical potency of these results

Prevention of Triglyceridemia by (Non-)Anticoagulant Heparin(oids) Does Not Preclude Transplant Vasculopathy and Glomerulosclerosis

Background: In renal transplantation, chronic transplant dysfunction (CTD) is associated with increased PCSK9 and dyslipidemia. PCSK9 is an enzyme that increases plasma cholesterol levels by downregulating LDLR expression. We recently showed increased PCSK9–syndecan-1 interaction in conditions of proteinuria and renal function loss. Treatment with heparin(oids) might be a therapeutic option to improve dyslipidemia and CTD. We investigated the effects of (non-)anticoagulant heparin(oids) on serum lipids, syndecan-1 and PCSK9 levels, and CTD development. Methods: Kidney allotransplantation was performed from female Dark Agouti to male Wistar Furth recipients. Transplanted rats received daily subcutaneous injections of saline, unfractionated heparin, and RO-heparin or NAc-heparin (2 mg heparin(oid)/kg BW) until sacrifice after 9 weeks of treatment. Results: Saline-treated recipients developed hypertension, proteinuria, and loss of creatinine clearance (all p < 0.05 compared to baseline), along with glomerulosclerosis and arterial neo-intima formation. Saline-treated recipients showed significant increase in plasma triglycerides (p < 0.05), borderline increase in non-HDLc/HDLc (p = 0.051), and ∼10-fold increase in serum syndecan-1 (p < 0.05), without significant increase in serum PCSK9 at 8 weeks compared to baseline. Heparin and non-anticoagulant RO-heparin administration in transplanted rats completely prevented an increase in triglycerides compared to saline-treated recipients at 8 weeks (both p < 0.05). Heparin(oids) treatment did not influence serum total cholesterol (TC), plasma syndecan-1 and PCSK9 levels, creatinine clearance, proteinuria, glomerulosclerosis, and arterial neo-intima formation, 8 weeks after transplantation. Combining all groups, increased syndecan-1 shedding was associated with TC (r = 0.5; p = 0.03) and glomerulosclerosis (r = 0.53; p = 0.021), whereas the non-HDLc/HDLc ratio was associated with the neo-intimal score in the transplanted kidneys (r = 0.65; p < 0.001). Conclusion: Prevention of triglyceridemia by (non-)anticoagulant heparin(oids) neither influenced PCSK9/syndecan-1 nor precluded CTD, which however did associate with the shedding of lipoprotein clearance receptor syndecan-1 and the unfavorable cholesterol profile

Basement Membrane Zone Collagens XV and XVIII/Proteoglycans Mediate Leukocyte Influx in Renal Ischemia/Reperfusion

Collagen type XV and XVIII are proteoglycans found in the basement membrane zones of endothelial and epithelial cells, and known for their cryptic anti-angiogenic domains named restin and endostatin, respectively. Mutations or deletions of these collagens are associated with eye, muscle and microvessel phenotypes. We now describe a novel role for these collagens, namely a supportive role in leukocyte recruitment. We subjected mice deficient in collagen XV or collagen XVIII, and their compound mutant, as well as the wild-type control mice to bilateral renal ischemia/reperfusion, and evaluated renal function, tubular injury, and neutrophil and macrophage influx at different time points after ischemia/reperfusion. Five days after ischemia/reperfusion, the collagen XV, collagen XVIII and the compound mutant mice showed diminished serum urea levels compared to wild-type mice (all

Anti-inflammatory effects of non-anticoagulant heparinoids in vitro and in a rat renal transplantation model

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.

The bittersweet taste of tubulo-interstitial glycans

Recently, interesting work was published by Farrar et al. [1] showing the interaction of fucosylated glycoproteins on stressed tubular epithelial cells with collectin-11 leading to complement activation via the lectin route of complement. This elegant work stimulated us to evaluate the dark side (bittersweet taste) of tubulo-interstitial glycans in kidney tissue damage. As will be discussed, glycans not only initiate tubular complement activation but also orchestrate tubulo-interstitial leucocyte recruitment and growth factor responses. In this review we restrict ourselves to tubulo-interstitial damage mainly by proteinuria, ischaemia-reperfusion injury and transplantation, and we discuss the involvement of endothelial and tubular glycans in atypical and Escherichia coli-mediated haemolytic uraemic syndrome. As will be seen, fucosylated, mannosylated, galactosylated and sialylated oligosaccharide structures along with glycosaminoglycans comprise the most important glycans related to kidney injury pathways. Up to now, therapeutic interventions in these glycan-mediated injury pathways are underexplored and warrant further research

Heparin/heparan sulphate interactions with complement-a possible target for reduction of renal function loss?

Current management of end-stage renal failure is based on renal replacement therapy by dialysis or transplantation. Increased occurrence of renal failure in both native and transplanted kidneys indicates a need for novel therapies to stop or limit the progression of the disease. Acute kidney injury and proteinuria are major risk factors in the development of renal failure. In this regard, innate immunity plays an important role in the pathogenesis of renal diseases in both native and transplanted kidneys. The complement system is a major humoral part of innate defense. Next to the well-known complement activators, quite a number of the complement factors react with proteoglycans (PGs) both on cellular membranes and in the extracellular compartment. Therefore, these interactions might serve as targets for intervention. In this review, the current knowledge of interactions between PGs and complement is reviewed, and additionally the options for interference in the progression of renal disease are discussed

Endothelial heparan sulfate deficiency reduces inflammation and fibrosis in murine diabetic nephropathy

Inflammation plays a vital role in the development of diabetic nephropathy, but the underlying regulatory mechanisms are only partially understood. Our previous studies demonstrated that, during acute inflammation, endothelial heparan sulfate (HS) contributes to the adhesion and transendothelial migration of leukocytes into perivascular tissues by direct interaction with L-selectin and the presentation of bound chemokines. In the current study, we aimed to assess the role of endothelial HS on chronic renal inflammation and fibrosis in a diabetic nephropathy mouse model. To reduce sulfation of HS specifically in the endothelium, we generated Ndst1 f/f Tie2Cre + mice in which N-deacetylase/N-sulfotransferase-1 (Ndst1), the gene that initiates HS sulfation modifications in HS biosynthesis, was expressly ablated in endothelium. To induce diabetes, age-matched male Ndst1 f/f Tie2Cre - (wild type) and Ndst1 f/f Tie2Cre + mice on a C57Bl/6J background were injected intraperitoneally with streptozotocin (STZ) (50 mg/kg) on five consecutive days (N = 10-11/group). Urine and plasma were collected. Four weeks after diabetes induction the animals were sacrificed and kidneys were analyzed by immunohistochemistry and qRT-PCR. Compared to healthy controls, diabetic Ndst1 f/f Tie2Cre - mice showed increased glomerular macrophage infiltration, mannose binding lectin complement deposition and glomerulosclerosis, whereas these pathological reactions were prevented significantly in the diabetic Ndst1 f/f Tie2Cre + animals (all three p < 0.01). In addition, the expression of the podocyte damage marker desmin was significantly higher in the Ndst1 f/f Tie2Cre - group compared to the Ndst1 f/f Tie2Cre + animals (p < 0.001), although both groups had comparable numbers of podocytes. In the cortical tubulo-interstitium, similar analyses show decreased interstitial macrophage accumulation in the diabetic Ndst1 f/f Tie2Cre + animals compared to the diabetic Ndst1 f/f Tie2Cre - mice (p < 0.05). Diabetic Ndst1 f/f Tie2Cre + animals also showed reduced interstitial fibrosis as evidenced by reduced density of αSMA-positive myofibroblasts (p < 0.01), diminished collagen III deposition (p < 0.001) and reduced mRNA expression of collagen I (p < 0.001) and fibronectin (p < 0.001). Our studies indicate a pivotal role of endothelial HS in the development of renal inflammation and fibrosis in diabetic nephropathy in mice. These results suggest that HS is a possible target for therapy in diabetic nephropathy

Tubular damage is reduced in double mutant mice deficient of collagen XV and XVIII compared to the WT mice.

<p><i>A–D:</i> Paraffin-embedded sections stained with periodic acid Schiff's (PAS) reagent in sham operated WT(day 1; <i>A</i>), in sham-operated <i>Col15a1^−/−×Col18a1^−/−</i> compound mutant mice (day 1; <i>B</i>) and in WT (<i>C</i>) and <i>Col15a1^−/−×Col18a1^−/−</i> compound mutant mice (<i>D</i>) at day 5 after renal I/R. WT mice showed tubular casts, tubular widening and flattening, and loss of nuclei in tubular cells (black arrows). Such damage was not observed in double collagen mutant mice kidneys. Scale bars 50 µm. <i>E:</i> Percentage of tubular damage was determined at different time points after I/R (see methods) in the <i>Col15a1^−/−</i>, <i>Col18a1^−/−</i> and <i>Col15a1^−/−×Col18a1^−/−</i> double mutant mice compared to the WT controls (*: day 5 p<0.01 double mutant mice compare to WT). Data is presented as mean percentage ± SEM.</p