Table 1.
Antibodies used for IHC.
Table 2.
Gene primer sequences primers for qPCR.
Fig 1.
Expression of umbrella cells and tight junctions in human- and porcine bladder biopsies and porcine urothelial cell culture.
Images of biopsied tissue of human bladder, porcine bladder and porcine bladder cell culture. (A) Umbrella cells are visualized in the most apical layer of human and porcine bladder biopsies and porcine bladder cell cultures (CK18). (B) The tight junctions (ZO-1) are seen as a thin belt apically in human and porcine bladder biopsies, in porcine cell cultures it is seen as a network over the urothelial cells (arrows).
Fig 2.
Expression of HA and its receptor in urothelium.
Images of biopsied tissue of human bladder, porcine bladder and porcine bladder cell culture. Staining with HABP, digestion of HA was achieved with hyaluronidase (A2, A4, A6, B2). (A1, A3) HA is most pronounced expressed around the basal layer of the urothelium in human and porcine bladder biopsies (arrows). (A5, B1) HA expression apically and in cell membranes in primary porcine urothelial cell cultures. (C) The CD44-receptor is found around the level of the basal cell layer. HABP, hyaluronic acid binding protein; HA, hyaluronic acid.
Fig 3.
The effect of HA digestion and HA replenishment alone or with CS on barrier function measured by TEER.
(A) A steady increase in TEER is seen in differentiated porcine urothelial cells, corresponding with a tight epithelium and reaching the upper reliable limit of the equipment (3300 Ω cm2) around day 35. In (B) and (C) each point represents a mean ± standard deviation. (B) Hyaluronidase treatment (n = 8) gradually increased TEER, this significantly differed from the untreated group (n = 8) and the PS treated group (n = 8) (p < 0,001). PS decreased TEER. (C) Different treatments (treatment groups n = 8)with HA and/or CS did not affect TEER recovery after PS treatment, full recovery was seen in all groups after 24 hours. TEER, transepithelial electrical resistance; PS, protaminesulfate; HA, hyaluronic acid; CS, chondroitin sulfate; T0, baseline; T3, 3 hours; T5, 5 hours; T7, 7 hours; T12, 12 hours; T24, 24 hours.
Fig 4.
The effect of HA digestion on gene expression level of HA synthesizing genes, other GAG synthesizing genes and barrier markers.
Effect of hyaluronidase treatment compared to PS treatment and untreated samples on gene expression of HA synthesizing genes, other GAG and proteoglycan synthesizing genes, inflammation markers and barrier markers. Expression of genes in hyaluronidase treated and PS treated samples are relative and compared to the untreated samples of that respective timepoint, which is y1 and represents the control line. (A, B) Hyaluronidase treatment increased relative expression of HAS3. Treatment with PS increased relative expression of HAS3 and HAS2. (C, D, E, F) No effect was seen of treatment with hyaluronidase or PS on the expression of other GAG/proteoglycan synthesizing genes (CSGALNACT2, CSGALNACT1, HSPG2, SDC1) (G, F) Hyaluronidase increased the relative expression of IL8, PS increased expression of IL8 and IL6. (I, J, K, L) No effect was seen of PS or Hyaluronidase treatment on barrier markers (TJP1, OCLN, UPK3A, CDH1). PS, protaminesulfate; T3, 3 hours; T5, 5 hours; T7, 7 hours; T12, 12 hours; T24, 24 hours; CP, median crossing point.
Fig 5.
The Effect of clinically applied GAG therapies after cell damage on gene expression level of HA and other GAGs synthesizing genes and inflammation and barrier markers.
Effect of clinically applied GAG therapies on gene expression of HA synthesizing genes, other GAG and proteoglycan synthesizing genes and inflammation and barrier markers. The evaluated GAG therapies are combined HA 1.6% with CS 2%, CS 0.2% and HA 0.16%. The expression is shown relatively to the untreated samples, which are represented by the dotted line. (A) Treatment with all GAG therapies increased the relative expression of almost all GAG producing genes (HAS3, CSGALNACT2, CSGALNACT1, HSPG2, SDC1), HAS2 was only increased after isolated HA treatment. IL6 and IL8 as well as TJP1 and CDH1 increased in relative expression after all GAG therapies. (B) In damaged cells (pre-treated with PS) other GAG/proteoglycan synthesizing genes (CSGALNACT2, CSGALNACT1, HSPG2, SDC1) and barrier markers (TJP1, OCLN, UPK3A and CDH1) were not affected by treatment. (C) For HAS3, HAS2, IL8 and IL6 in damaged cells all GAG therapies lead to an increase. GAG, glycosaminoglycans; PS, protaminesulfate; HA, hyaluronic acid; CS, chondroitin sulfate; T5, 5 hours; T7, 7 hours; T12, 12 hours; T24, 24 hours; CP, median crossing point.