Glycosaminoglycans (GAGs) are molecules that trap water to from a highly impermeable barrier layer on the urothelium. In Interstitial cystitis/Bladder pain syndrome (IC/BPS) this layer is thought to be disrupted. GAG replenishment therapy is used as therapy for IC/BPS, adding exogenous GAGs such as chondroitin sulfate (CS) and hyaluronic acid (HA). The exogenous GAGs are intended to repair the impaired GAG layer. New insights about the functioning of these exogenous GAGs and influence on the urothelial cells are being investigated. [1]
The scope of this study is to examine the effect of GAG therapy on gene expression in damaged urothelial cells for CS and HA synthesizing genes.

Cell Culture
Porcine urothelial cells were cultured in complete keratinocyte serum-free medium (cKSFM; containing KSFM, 1% penicillin/streptomycin, 30ng/mL cholera toxin, 50 µg/mL bovine pituitary extract and 5 ng/mL epidermal growth factor). The cells were seeded in 12-wells plates (75.000 cells per well/3.9cm2/1mL). When confluency was reached (7 days) the medium was supplied with 5% fetal calf serum (FCS) and 2mM calcium chloride (Ca2+Cl) to induce terminal differentiation (7 days). This protocol has previously been applied and controlled on the same primary cell culture with Trans Epithelial Electrical Resistance (TEER) measurements that demonstrated high barrier properties (TEER values of >1000 Ω∙cm2). 

GAG replenishment therapy
All cells, except for the untreated group, were challenged with protaminesulfate 10mg/mL for 1h with a 1:1 ratio with medium to approximate inflamed urothelial and mimic IC/BPS environment.
Afterwards, all the damaged cells were divided into three treatment groups:
1.	CS, 0,2%, Gepan Instill, Pohl-Boskamp GmbH & Co., Hohenlockstedt)
2.	CS 2% & HA 1.6%, IALURIL® Prefill, IBSA, Goodlife
3.	HA 0,16%, Instylan, BMODESTO, Diaco Biofarmaceutici S.R.L. Triest.
Thereafter, cells treated with the GAG therapy in a 1:1 ratio with fresh medium for one hour. Afterwards, all cells were washed three times with Hank’s Balanced Salt Solution (HBSS) and fresh medium was added. Cells were harvested at T3, T5, T7, T12 and T24. In an earlier similar setting undamaged cells were treated with the three GAG therapies and harvest at T3 and T24.

RNA isolation and RT-qPCR
RNA isolation was done using TRIzol reagent (Invitrogen). The RNA was evaluated using a Nanodrop ND-1000 system (ThermoFisher Scientific). RNA was DNase I treated, and cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen). Gene expression was evaluated by SYBR Green qPCR analysis (Roche) on a LightCycler LC480 instrument (Roche). The expression of CS and HA GAG synthesis genes (CSGALNACT1, CSGALNACT2, HAS2 and HAS3) were assayed.

Treating healthy urothelial cells with GAG therapy (HA and/or CS) resulted in a strong (96%) down regulation of HAS2. For HAS3 and CSGALNACT1 approximately 50% reduction was seen compared to controls. CSGALNACT2 was the least affected with a reduction of 20-35%.
HAS2 was upregulated under inflamed conditions, there is a five times higher expression in protamine treated samples in comparison with normal conditions. This response was quick with a peak at T7 hrs and is normalized again at T24hrs. HAS3 expression follows a similar pattern with a 2,5-fold increase in expression compared to controls. The CS synthesizing genes were not evidently influenced by protamine exposure. CSGALNACT1 expression increased at T3hrs for protamine only and with CS treatment afterwards. CSGALNACT2 expression increased after protamine exposure followed by HA treatment. See Figure 1.  
The effect of HA+CS treatment (IALURIL®) after protamine exposure resulted in lower HAS2 expression, except for T5hrs. At T24hrs al GAG therapies led to an increased HAS2 expression compared to control and protamine only. CS treatment stimulates at T3hrs the expression of HAS3 strongly and slightly of CSGALNACT1. The CSGALNACT2 expression is slightly increased by HA treatment at T3hrs.

The downregulation of the GAGs synthesizing genes in healthy urothelial cells after treatment with exogenous GAGs demonstrates that it influences urothelial endogenous GAG synthesis with a negative feedback loop. On the other hand, inflicting damage to the urothelial cells increases the synthesis of hyaluronic acid by increasing HAS2 and HAS3 expression. This is a fast-acting mechanism that normalizes after 24 hrs, with a beneficial effect in expression of adding exogenous GAGS. This is in line with earlier findings, that recovery of the barrier function after protamine treatment is completed after 24h. [2]  Interesting CSGALNACT1 and 2 do not increase after inflammation is induced, this is in line with findings by Rooney et al. [1], even though earlier research shows the digestion and/or replenishment of CS does affect barrier function directly.[2, 3] Rooney et al showed a slight tendency of increase of CSGALNACT1 and CSGALNACT2 at T24 after GAG therapy in damaged cells.[1] In our series no evident increase of CSGALNACT1 and 2 was seen. Our data for HAS2 shows that adding exogenous GAGs reduces the need for endogenous HA synthesis. These observations suggest that there is a protective effect of GAG therapy and that it modulates urothelial inflammatory responses. Future prospective could be to debate about the concentration of the given GAG therapy, with the negative feedback loop in mind.

GAG therapy influences urothelial GAG synthesis genes during normal conditions and damage. Adding exogenous GAGs (HA and/or CS) to healthy urothelial cells induces a negative feedback loop for the HA GAG synthesizing genes. This process is also seen in inflammatory conditions whereby GAG therapy attenuates activation of GAG synthesizing genes, thereby suggesting that GAG therapy not only works a liquid barrier patch, but also interacts with urothelial (barrier) repair mechanisms.

Rooney, P., et al., Effect of Glycosaminoglycan Replacement on Markers of Interstitial Cystitis In Vitro. Frontiers in pharmacology, 2020. 11: p. 575043-575043
Janssen, D.A., et al., The distribution and function of chondroitin sulfate and other sulfated glycosaminoglycans in the human bladder and their contribution to the protective bladder barrier. J Urol, 2013. 189(1): p. 336-42.
Rozenberg, B.B., et al., Improving the barrier function of damaged cultured urothelium using chondroitin sulfate. Neurourol Urodyn, 2020. 39(2): p. 558-564.

Continence 2S2 (2022) 100468
DOI: 10.1016/j.cont.2022.100468