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  • DDR expression is upregulated in the glomeruli and tubules o

    2020-05-22

    DDR1 expression is upregulated in the glomeruli and tubules of injured kidneys [7], [8], [19]; however, what role this receptor plays in kidney resident AG-120 is unclear. A plausible hypothesis is that DDR1 might contribute to kidney injury by directly exerting a pro-fibrotic action. In favor to this hypothesis, smooth muscle cells lacking DDR1 show decreased mRNA levels of non-fibrillar collagen, such as the basement membrane collagen IV [21], suggesting a pro-fibrotic effect of DDR1. However, mice lacking DDR1 also show increased fibrillar collagen deposition in the mammary gland [20] and DDR1-null smooth muscle show increased mRNA levels of fibrillar collagens I and III [21], suggesting that DDR1 is a negative regulator of fibrillar collagen synthesis. Thus, DDR1 can promote and/or inhibit collagen synthesis and this effect is dependent on the type of collagen. We provide evidence that in the glomerulus of the kidneys, DDR1 plays a deleterious effect by promoting the synthesis of both fibrillar and non-fibrillar collagens. We show that following 5/6 nephrectomy-induced injury DDR1KO mice have reduced glomerulosclerosis characterized by decreased collagen I and collagen IV deposition. Thus, our study suggests that, in contrast to breast cancer and/or smooth muscle cells, in the kidney DDR1 positively regulates extracellular matrix production thus playing a pro-fibrotic action. We also provide evidence that in order to promote collagen production, DDR1 requires collagen binding as mutation of R105 in the collagen binding site significantly decreases the ability of DDR1 to stimulate collagen production at baseline and/or following collagen stimulation. Thus, it is conceivable that in the course of kidney injury increased expression of collagen within the glomeruli results in increased DDR1 activation and further production of collagen. In addition, increased TGF-β production by injured resident cells and/or inflammatory cells could lead to increased DDR1 expression in a Smad4-depenent manner, as recently proposed for hepatocellular carcinoma cells [31]. Finally, endothelial stress, hypoxia and shear stress could also contribute to upregulation of DDR1 expression [32], thus creating a vicious cycle leading to chronic kidney disease. This statement seems to agree with the idea that in the context of hypertensive nephropathy, DDR1 functions as an amplifier of the initial lesion, which occurs independently of the initiating cause, and leads to the development of and progression to chronic kidney disease [32]. Because of DDR1 contribution to fibrotic diseases and cancer progression the interest in targeting DDR1 has significantly increased in the recent years. Approaches to block DDR1-mediated effects include: reduction of DDR1 expression by using antisense nucleotide [8], [33], inhibition of DDR1 binding to collagen with recombinant engineered bacterial collagen [34], inhibition of DDR1 oligomerization with selective monoclonal antibodies [35], and inhibition of DDR1 tyrosine kinase activity (see also [12]). As DDR1 ATP-competitive inhibitors with good selectivity have been developed [26], [36], [37], [38], [39] targeting the kinase activity of DDR1 has become an appealing strategy. The majority of tyrosine kinase inhibitors synthetized are ATP-competitive inhibitors which target either the kinase domains in the active form (type I inhibitors) or in the inactive form (type II inhibitors). While type I inhibitors tend to be promiscuous, because they usually target well-conserved active kinase binding sites, type II inhibitors tend to be more selective because they can interact with not-well-conserved exposed hydrophobic sites within the inactive kinase domain [38]. For our inhibition studies we used compound 1 discovered first as an inhibitor of tyrosine kinases of the Ephrin family [40]. Compound 1 is a type II kinase inhibitor as it targets the inactive conformation of the kinase [30]. In a screen against a panel of over 350 kinases, compound 1 was found to target a limited number of kinases with DDR1 and DDR2 among the top hits [30]. Inhibition of DDR1/2 was also confirmed in enzymatic assays with compound 1 inhibiting DDR1/2 in the nanomolar range, while requiring micromolar concentrations for other tyrosine kinases [30]. Thus compound 1 is highly potent against DDR1/2 and has good selectivity. Here, we confirm that compound 1 has high affinity for DDR1, (IC50 11.98nM) and we show that it blocks collagen-induced DDR1 autophosphorylation and collagen IV production in mesangial cells expressing human DDR1. In contrast, compound 1 had no significant effect on collagen production in cells lacking DDR1 although these cells express DDR2 (data not shown), suggesting that DDR1, but not DDR2, mediates collagen IV production in mesangial cells.