Animación del modelo

The mechanisms underlying the initiation and progression o f TIP remain obscure [5], Since fibrotic renal diseases o f diverse etiology are commonly associated with vascular compromise, it was hypothesised that tissue hypoxia, either alone or in conjunction with other factors, may be a pro-fibrogenic stimulus [11]. Although all cellular components of the tubulo-interstitium (PTE, capillary endothelia and interstitial fibroblasts) are likely to be affected by decreased oxygen availability, the metabolic functions of PTE confer high oxygen demands, rendering these cells particularly vulnerable to fluctuations in oxygen levels in vivo and leading to the suggestion that tubular cells may be the primary target o f a hypoxic insult [13,14], Changes within proximal tubules occur early in the pathogenesis of TIE with the striking appearance o f a dense, thickened TBM surrounding hypertrophic and subsequently, atrophic, non­ functioning tubules [1]. This expanded TBM with altered matrix composition presumably has profound influence on PTE function and on paracrine interactions with other cell types; however the pathological stimulus(i) for these changes in the TBM is currently unclear. This project investigated the possibility that hypoxia may be a fibrogenic stimulus for PTE, eliciting changes in ECM metabolism {Fig. 6.7).

DAMAGE

1

i

PTE

FIBRO TIC

PH ENO TY PE

Fig. 6.1 Flow diagram demonstrating the potential effect o f ischaemia, arising fro m microvascular damage, on ECM metabolism in PTE.

The effects o f hypoxia on PTE were examined using an in vitro model with cells grown in monolayer. Although the limitations of this approach are recognised, this culture system allowed the examination of hypoxia per se without the multiple interacting factors present in vivo. Preliminary attempts were made to undertake experiments with PTE cultured in 3D collagen gels, since there is some evidence that PTE can form tubular structures when embedded in collagen-I, thus resembling their in vivo configuration. It was foreseen that such a model would allow examination of hypoxia-induced changes in PTE cultured alone and also in co-culture with other tubulo- interstitial cell-types, thus giving some insight into the effect of hypoxia on paracrine interactions between renal cells. Despite many attempts to perfect the 3D culture system however, the model proved difficult to manipulate, with retrieval o f cells almost impossible. It was therefore decided to use simple monolayer culture, which still allows co-culture with additional cell types grown on tissue cultue inserts, as a representative model of the in vivo environment.

The degree o f hypoxia chosen for experiments was based on the knowledge that under normal physiological conditions, cortical oxygenation levels are between 3-5% and that levels are likely to vary from this range, down to near anoxia in the event of microvascular damage [153]. PTE were subjected to 1%02 for 24hrs {Chapter 2),

conditions which are known to induce expression of EPO and various growth factors in other cell types, in vitro [123]. Since this degree of hypoxia had no effect on cell viability, but was sufficient to elicit changes in PTE protein synthesis and cellular respiration (as indicated by elevated GAPDH expression), this level of oxygenation was considered appropriate for the study of hypoxia induced ECM metabolism in PTE.

As discussed in Chapter 3, the accumulation of ECM material within the TBM in fibrosis results from increased synthesis and/or decreased degradation of matrix proteins [5]; hypoxia had marked effects on both aspects of matrix metabolism in PTE. Despite an overall suppression in total protein production (perhaps reflecting the decrease in cellular ATP available for protein synthesis), collagen production was stimulated approximately 5-fold by exposure to hypoxia for 24hrs, suggesting a specific effect of hypoxia on collagen proteins. Re-oxygenation, an in vitro analogy of reperfiision, further stimulated collagen production and continued to increase levels for up to 72hrs {Chapter 3), suggesting a long term effect of hypoxia on collagen protein despite the return to normal oxygen levels.

Since collagen IV is the major collagenous component of the TBM, it was of interest to determine whether hypoxia induced increased collagen production was due to an increase in production of collagen IV. Surprisingly, collagen IV mRNA levels decreased after hypoxia and remained suppressed on re-oxygenation, demonstrating for the first time, the (down) regulation of this collagen sub-type by oxygen depletion. In contrast, mRNA for the interstitial collagen, collagen I, was markedly increased by

hypoxia and remained elevated on return to normoxia, suggesting that increased collagen production in response to hypoxia was due to the induction of interstitial rather than BM collagen. A similar increase in expression of collagen I, accompanied by decreased collagen IV levels, has been reported in in vivo models of fibrosis [50]. Since matrix proteins act as regulators of cell growth, differentiation and function, qualitative and quantitative changes in TBM are likely to have profound effects on tubular cell behaviour.

ECM accumulation in TIP is partly attributed to decreased matrix turnover [5], therefore it was of interest to examine the effects of hypoxia on the MMP pathway. Hypoxia decreased both latent and active MMP-2 and MMP-9 enzymes and levels remained on re-oxygenation. While there is some in vitro evidence that MMP-9 is regulated by hypoxia in kératinocytes [166], this is the first demonstration of altered MMP-2 activity in any cell type. MMP-2 mRNA levels were not affected by hypoxia in PTE suggesting a translational or post-translational level of regulation. Examination of endogenous MMP inhibitor (TIMP-1 and -2) levels in response to hypoxia, showed increased TIMP-1 secretion but no change in mRNA expression, implying post- transcriptional regulation of the inhibitor in this period. In contrast, TIMP-2 mRNA levels were transiently suppressed during hypoxia demonstrating reciprocal effects of hypoxia on the two inhibitors. Both TIMP-1 and -2 mRNA levels were increased in the re-oxygenation phase.

The data in Chapter 3 demonstrate that in vitro, hypoxia acts as a pro- fibrogenic stimulus for PTE by increasing collagen production while simultaneously decreasing levels o f matrix degrading enzymes and increasing inhibitor levels. Similar effects o f hypoxia in vivo would potentially lead to an accumulation of matrix with clear repercussions on tubular cell function. In addition, thickening of the TBM leads to

physical separation o f tubules and capillaries, increasing the oxygen diffusion distance, and thereby subjecting the tubule to further ischaemia. Furthermore, increased metabolic activity o f PTE induced by increased filtered protein in the diseased kidney may exacerbate hypoxia and hence lead to further ECM deposition, maintaining a cycle of progressive, TBM thickening (Fig. 6.2).