Abstract
Tubulo-interstitial nephritis antigen (TINag) is an extracellular matrix protein expressed in tubular basement membranes. Combined mutations in TINag and nephrocystin-1 genes lead to nephronophthisis with reduced cell survival. Because certain extracellular matrix proteins are known to modulate cell survival, studies were initiated in Lewis rats lacking TINag to assess if they are more susceptible to cisplatin-induced injury. Cisplatin induced a higher degree of tubular cell damage and apoptosis in regions where TINag is expressed in a parental Wistar strain. This was accompanied by an accentuated increase in serum creatinine and Kim-1 RNA and renal expression of Bax, p53, and its nuclear accumulation, mtDNA fragmentation, and a decrease of Bcl-2. Cisplatin induced fulminant apoptosis of HK-2 cells with increased caspase3/7 activity, mtDNA fragmentation, and a reduced cell survival. These effects were partially reversed in cells maintained on TINag substratum. Far Western/solid phase assays established TINag binding with integrin αvβ3 comparable with vitronectin. Transfection of cells with αv-siRNA accentuated cisplatin-induced apoptosis, aberrant translocation of cytochrome c and Bax, and reduced cell survival. The αv-siRNA decreased expression of integrin-recruited focal adhesion kinase (FAK) and p-FAK, while increasing the expression of p53 and p-p53. Similarly, p-AKT was reduced although ILK was unaffected. Inhibition of PI3K had similar adverse cellular effects. These effects were ameliorated in cells on TINag substratum. In vivo, a higher degree of decrease in the expression of p-FAK and pAKT was observed in Lewis rats following cisplatin treatment. These in vivo and in vitro studies demonstrate an essential role of TINag in cellular survival to maintain proper tubular homeostasis utilizing integrin αvβ3 and downstream effectors.
Highlights
Combined mutations in TINag and nephrocystin-1 genes lead to and proteoglycans, that are expressed normally in very minute nephronophthisis with reduced cell survival
In apposition with the extracellular matrix proteins are known to modulate cell sur- interstitium are the tubular basement membranes (TBMs) that vival, studies were initiated in Lewis rats lacking TINag to assess are made up of another set of integral ECM proteins, including if they are more susceptible to cisplatin-induced injury
The expression was seen in the tubular basement membranes (TBMs) of cortical proximal and distal tubules (Fig. 1A, arrowheads), and it was absent in the medullary tubules as well as in the glomeruli (Fig. 1A, arrows)
Summary
ECM, the basement membranes, have been long known to provide a scaffold for tissue organization by modulating cell/matrix signaling via integrin receptors, regulating the availability of growth factors and chemokines to maintain proper tissue homeostasis (10 –13). Among the various ECM basement membrane proteins, the collagen, laminin, and proteoglycans are known to support cellular survival, their growth, differentiation, and proliferation during embryonic and adult life. Mammalian TINag contains 11 exons and has a molecular mass of ϳ53 kDa. Mammalian TINag contains 11 exons and has a molecular mass of ϳ53 kDa It has a signal peptide, von Willebrand factor domain, follistatin module, procathepsin and ATP/GTP binding domains, and six potential N-glycosylation sites [15]. It does not have an RGD sequence, but it is believed to interact with laminin and type IV collagen [16]. A model of the Lewis strain of rats in which TINag seems to be undetectable in the tubular compartment, which perhaps is related to masking of its antigenic epitope or lack of protein expression, was used
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