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

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

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

Serum urea levels in WT, collagen XV and/or XVIII deficient mice after renal I/R.

<p>WT mice showed an increased serum urea levels at day 5 after I/R while all collagen mutant mice (<i>Col15a1^−/−</i> and <i>Col18a1^−/−</i> p<0.05, and <i>Col15a1^−/−×Col18a1^−/−</i> p<0.01) had significantly lower serum urea levels compared to WT at this time point. The results are expresses as serum urea mmol/l ± SEM. *: p<0.05, **: p<0.01.</p

MCP-1-induced monocyte migration is increased in the presence of immobilized heparin-albumin and glycosylated collagen XVIII.

<p><i>A:</i> MCP-1 dose dependently increased the migration of monocytes over a porous membrane. Immobilization of heparin-albumin, mimicking an artificial BM HSPG, promotes monocyte transmigration. Spontaneous migration over albumin-coated membrane in the absence of MCP-1 was set as 1 and the other values were calculated accordingly. The error bars represent SEM. <i>B:</i> Transmigration of monocytes towards MCP-1 (10 ng/ml) was increased in the presence of heparin-albumin and collagen XVIII with long GAG chains. Heparin-albumin increased the monocyte migration significantly compared to albumin coated membrane (p<0.01). N-terminal fragment of short collagen XVIII with long GAG chains promoted transmigration significantly compared to albumin and N-terminal fragment without GAG chain (both p<0.05). Relative to albumin, also the full-length short collagen XVIII promoted MCP-1-induced monocyte transmigration to some extent (not significant). Data is calculated relative to migration over albumin-coated membrane towards 10 ng/ml MCP-1. The error bars represent SEM. *: p<0.05, **: p<0.01.</p

Increased expression of TNF-α and MCP-1 in double mutant mice, lacking both collagen XV and XVIII compared to WT.

<p><i>A–B:</i> Immunofluorescent staining of MCP-1 (red) in WT (<i>A</i>) and double mutant mice (<i>B</i>) at day 5. Double mutant mice showed an increased expression of MCP-1 in peri-tubular capillaries. Scale bars 20 µm. <i>C:</i> mRNA expression of MCP-1 in renal tissue of <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice at day 5 after I/R (***: p<0.001 double mutant mice compared to WT). <i>D:</i> mRNA expression of TNF-α in renal tissue of <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice at day 5 after I/R (**: p<0.01 double mutant mice compared to WT).</p

Tubular cell activation marker VCAM-1 is reduced in double mutant mice, deficient of collagen XV and XVIII compared to the WT mice.

<p><i>A–D:</i> Cryosections stained for VCAM-1 expression in sham operated (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. WT mice showed an upregulation of VCAM-1 in tubular compartment while sham and double collagen deficient mice showed a weak VCAM-1 expression in between of tubuli (peritubular capillaries). Scale bars 50 µm. <i>E:</i> Tubular cell activation was quantified as percentage of VCAM-1 expression in tubular cells at different timepoints after I/R in the <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice (**: at day 5 p<0.01 double mutant mice compared to WT) (see methods). Data is presented as mean percentage ± SEM. <i>F:</i> Quantitative RT-PCR on RNA isolated from renal tissue, day 5 after I/R. VCAM-1 mRNA expression is reduced in all mutant mice, and reached statistical significance in the double KO mice (***: p<0.001).</p

Production and characterization of recombinant mouse Tsp1-C18 and Short-XVIII by Western blotting with anti-all-18 antibody.

<p><i>A:</i> Gel filtration fractions of recombinant Tsp1-C18 with variable degree of glycosylation and non-glycosylated core protein of ∼55 kDa separated by 10% SDS-PAGE. Fractions 29 and 33 with Long and Intermediate GAG chains, respectively, and 39–40 containing core Tsp-C18 were used for binding and migration experiments. <i>B:</i> Expression of full-length Short-XVIII by two representative stable 293-EBNA clones 1 and 2. Apparently non-glycosylated Short-XVIII of ∼180 kDa was detected in cell lysates (L). Conditioned cell culture media (CM) contained highly glycosylated recombinant Short-XVIII migrating as a smear above 200 kDa in 7% SDS-PAGE. Control 293-EBNA cell lysate and CM did not show reactivity for anti-all-18 antibody with short exposure. <i>C:</i> Heparitinase I treatment of intermediate Tsp1-C18 (fractions 31 and 33, MW ∼130 kDa) removed most GAG chains and revealed a core protein of ∼55 kDa. Heparitinase I treatment did not alter the size of low MW Tsp1-C18 (fractions 39–40) suggesting that this band represents non-glycosylated core protein. <i>D:</i> Heparitinase I and chondroitinase ABC treatments of intermediate Tsp1-C18 (fractions 31 and 33) indicate that most of the GAGs within recombinant Tsp1-C18 produced in HEK-293 cells are HS chains.</p

Reduced neutrophil influx after renal I/R in the mutant mice for collagen XV and XVIII.

<p><i>A:</i> Immunofluorescent staining for collagen IV, XV and XVIII (green) and neutrophils (red) in WT and double mutant mice at day 1 after I/R showed presence of collagen IV, XV and XVIII in peritubular capillaries (white arrows) in WT mice and accumulation of neutrophils around these capillaries. Less neutrophil influx (in red) was observed in double mutant mice, also lacking collagen XV and XVIII signals (in green). Nuclei are shown in blue. Scale bars 20 µm. <i>B:</i> Immunofluorescent staining for neutrophils (red) and nuclei (blue) in WT and double mutant sham operated mice at day 1 after I/R showed the absence of neurophils in renal tissues. Scale bars 20 µm. <i>C:</i> Number of neutrophils per HPF (High Power Field) at different timepoints after I/R. At day 1 after reperfusion double mutant mice showed a decreased number of neutrophils compared to WT mice (p<0.05). At day 5 significantly less neutrophils were observed in kidneys of <i>Col15a1^−/−</i> and <i>Col18a1^−/−</i> mice compared to WT (p<0.05) as well as in those of double mutant mice compared to WT (p<0.01). Data is presented as mean ± SEM. *: p<0.05, **: p<0.01.</p