Tubular epithelial cell activation and interstitial fibrosis. The role of glomerular ultrafiltration of growth factors in the nephrotic syndrome and diabetic nephropathy

Shi-Nong Wang and Raimund Hirschberg

Division of Nephrology & Hypertension, Harbor-UCLA Medical Center and UCLA, Torrance, California, USA

Correspondence and offprint requests to: Raimund Hirschberg, MD, Division of Nephrology & Hypertension, Harbor-UCLA Medical Center, Box 406, 1000 West Carson Street, Torrance, CA 90509, USA. E-mail: hirschberg{at}humc.edu.

Introduction

Chronic glomerular proteinuria or the nephrotic syndrome are associated with worsening tubulointerstitial injury and fibrosis in virtually all immunological or nonimmunological glomerular diseases [1]. Moreover, prospective, randomized clinical trials indicate that the degree of glomerular protein ultrafiltration is a major risk factor for onset and progression of both interstitial fibrosis and progressive renal failure [2].

Currently, therapeutic interventions are primarily aimed at reducing the rate of glomerular protein ultrafiltration such as with angiotensin I converting enzyme inhibitors. However, it is conceivable that additional treatment strategies may be developed that block pathophysiological, further downstream events, namely the interaction of ultrafiltered proteins with tubular cells and their activation.

Bioactive proteins in glomerular ultrafiltrate

In the nephrotic syndrome, the glomerular ultrafiltrate contains `bulk' plasma proteins such as serum albumin and globulins. Albumin is taken up by proximal tubular cells through megalin-assisted endocytosis. Albumin itself may not be harmful to tubular cells, but toxicity may derive from the metabolism of fatty acids that are bound to ultrafiltered albumin [3].

The glomerular permeability defect in the nephrotic syndrome also leads to glomerular ultrafiltration of bioactive growth factor proteins into tubular fluid. In adriamycin-induced nephropathy in rats, a model of selective proteinuria, insulin-like growth factor type I (IGF-I)-containing 50 kDa protein complexes are translocated into tubular fluid resulting in tubular fluid IGF-I levels of 1.35 nM [4]. In streptocotozin-induced diabetic nephropathy in rats, tubular fluid IGF-I levels are even greater, ~2.5 nM, due to glomerular ultrafiltration of IGF-I that is present in 50 kDa, as well as in 150 kDa, protein complexes [5]. Recently, this laboratory also demonstrated in rats with streptozotocin-induced diabetic nephropathy, that there is translocation of two other growth factor–cytokines into tubular fluid, namely hepatocyte growth factor (HGF) and transforming growth factor-ß (TGF-ß). The presence of HGF in diabetic, but not normal, rat glomerular ultrafiltrate collected by nephron micropuncture has been demonstrated by Western immunoblot analysis [6]. Moreover, proximal tubular fluid TGF-ß levels and bioactivity in diabetic rat glomerular ultrafiltrate has been measured in a highly sensitive and specific bioassay using mink lung epithelial cells that had been stably transfected with a TGF-ß-sensitive, truncated PAI-1 promoter fused to a luciferase reporter, and levels of active and total TGF-ß were measured as luciferase-induced luminescence in cell lysates. In normal rat glomerular ultrafiltrate, total TGF-ß levels are <10 pg/ml. In contrast, in rats with diabetic nephropathy, total TGF-ß levels in early proximal tubular fluid are, on average, 729 pg/ml, at least 14% of which is directly bioactive.

TGF-ß is present in serum in molecular mass forms of 100–220 kDa due to association with the latency-associated protein (LAP) as the LAP–TGF-ß complex (100 kDa). This TGF-ß precursor protein complex is the direct product of the TGF-ß gene. This complex is further bound to latent TGF-ß-binding protein (LTBP; 220 kDa). These complexes render serum TGF-ß inactive [7]. HGF is present in serum as the inactive 95 kDa monomeric precursor, but also as the 82 kDa mature heterodimeric protein. It is these high molecular mass forms of serum growth factors that undergo ultrafiltration into tubular fluid in the nephrotic syndrome or diabetic nephropathy.

Ultrafiltered growth factors present in tubular fluid are bioactive, at least in part. For example, incubation of cultured proximal tubular cells with glomerular ultrafiltrate obtained from rats with adriamycin nephropathy by nephron micropuncture, quickly phosphorylates IGF-I receptors [4]. The direct activity of aliquots of TGF-ß present in tubular fluid was demonstrated with the above mink lung epithelial cell bioassay. Although HGF-derived bioactivity has not been measured directly, Western blot studies indicate that all tubular fluid HGF is present in the mature, bioactive form. The mechanisms that cause activation of ultrafiltered growth factors have not been entirely sorted out, but appear to involve proteolysis of IGF-binding proteins [8], limited proteolysis of LTBP and LAP [7], and specific cleavage [9]. The relatively great urea concentration of tubular fluid, the acidic pH and certain enzymes that are luminously secreted by tubular cells such as urokinase, may play important roles in these processes [9,10].

Apical tubular membranes express specific growth factor receptors

For several decades apical tubular membranes have been studied extensively for their expression of various transport proteins. However, far less attention has been payed to the presence of specific growth factor/cytokine receptors and the membrane's function in signalling. To this end expression of signalling receptors in apical tubular membranes, such as AT-1 receptors in TALH and wide-spread apical expression of IGF-I receptors have been described by several laboratories. Recent immunohistological studies from this and other laboratories have demonstrated that apical membranes in proximal and distal tubules and in collecting ducts also express specific receptors, such as IGF-I receptors, HGF receptors (the p190met proto-oncogene) and TGF-ß type I and II receptors. The presence of specific, bioactive ligands in nephrosis-conditioned, but not in normal, tubular fluid (it is possible that very low levels of IGF-I, TGF-ß and HGF are also present in normal tubular fluid, but escape the sensitivity of currently available assays, nevertheless, data indicate that in states with glomerular proteinuria the levels of these growth factors in tubular fluid is far greater) and the expression of specific signalling receptors in apical membranes set the stage for activation of tubular cells in conditions with glomerular proteinuria.

Activation of tubular cells by ultrafiltered growth factors

The interaction of ultrafiltered IGF-I, HGF and TGF-ß with tubular epithelial cells through apical membranes was recently examined in cell-culture models of proximal tubular cells and medullary collecting duct cells. Cells were grown to confluence in 96-well plates or in Anopore support inserts allowing for discrete access to apical vs basolateral sites of the cells. Incubation of proximal tubular cells with rhHGF or rhTGF-ß raises the mRNA levels encoding the C-C-chemokine MCP-1, and the MCP-1 peptide levels increase ~2–3-fold. Moreover, about two-thirds of the MCP-1 are secreted through basolateral membranes. Incubation of cells with glomerular ultrafiltrate (1:5), collected from rats with diabetic nephropathy by micropuncture of early proximal tubules, also raises MCP-1 mRNA levels [11]. Importantly, co-incubation of cells with conditioned tubular fluid and neutralizing anti-HGF and anti-TGF-ß antibodies virtually quantitatively prevents the ultrafiltrate-induced rise in MCP-1 expression.

Apical stimulation of proximal tubular cells, as well as inner medullary collecting duct cells, with rhHGF or rhTGF-ß (but not rhIGF-I) also raises the expression and secretion of another C-C-chemokine, namely RANTES. In both proximal tubular cells and also to a lesser extent in inner medullary collecting duct cells, conditioned tubular fluid from rats with diabetic nephropathy also induces increased expression of the growth factor cytokine PDGF-B. This seems to be induced primarily by TGF-ß in diabetic glomerular ultrafiltrate as rhTGF-ß but not rhHGF induces PDGF-B expression in tubular cells.

These experimental findings provide strong evidence that in diabetic nephropathy, and probably also in other glomerular proteinurias, the ultrafiltration of bioactive growth factors such as IGF-I, TGF-ß and HGF, and possibly other as yet unidentified proteins activate tubular cells. `Activation' leads to increased expression and basolateral secretion of bioactive molecules such as chemokines (MCP-1, RANTES) and growth factor peptides (PDGF-B) into the peritubular interstitium.

Activated tubular cells initiate cell–cell interactions in the renal interstitium

Peritubular chemical gradients of C-C-chemokines attract and activate monocytes/macrophages (Figure 1Go). Indeed, even in diabetic nephropathy in rats, in early stages of the disease before any interstitial fibrosis can be histologically demonstrated there is a 2-fold increase in the number of ED-1 macrophages [11]. Immigrating macrophages express ample amounts of TGF-ß, which may be increased further by interaction with C-C-chemokines, specifically MCP-1 (unpublished observation). In fact, macrophages may be the most important source of interstitial TGF-ß, and its action on interstitial myofibroblasts appears to be a major mechanism causing accumulation of matrix proteins to form interstitial fibrosis. Indeed, recent studies from this laboratory indicate that TGF-ß (400 pM) increases fibronectin, Col{alpha}2I and Col{alpha}1III mRNA levels in cultured rat myofibroblasts. Activated tubular-cell-derived PDGF-B may further contribute to the production and deposition of interstitial extracellular matrix proteins by fibroblasts [12].



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Fig. 1. Schematic diagram illustrating how growth factors in nephrosis-conditioned glomerular ultrafiltrate induce or activate a cascade of cell–cell interactions. Ultrafiltered growth factors activate tubular cells via apical growth factor receptors. In response, activated tubular cells may themselves express increased amounts of extracellular matrix proteins. More importantly, activated tubular cells express increased amounts of chemokines such as the delayed-early genes MCP-1 and Rantes or the cytokine PDGF-B, which are secreted through basolateral membranes. Peritubular chemokine gradients attract immigrating macrophages. These express TGF-ß, which causes increased production of various extracellular matrix proteins by myofibroblasts. PDGF-B may also act in an autocrine loop with tubular cells as well as with macrophages and myofibroblasts and contributes to increases ECM-deposition.

IGF-I, insulin-like growth factor-I; HGF, hepatocyte growth factor; TGF-ß, transforming growth factor-ß; MCP-1, macrophage chemoattractant protein-1; Rantes, regulated upon activation, normal T-cell expressed and secreted; PDGF, platelet-derived growth factor; Mo, macrophage; ECM, extracellular matrix.

 
The direct effect of ultrafiltered IGF-I, HGF and TGF-ß on expression and basolateral secretion of extracellular matrix proteins by tubular cells may also contribute to interstitial fibrosis. Indirect evidence from in vitro studies suggests that ultrafiltered IGF-I induces increased production of type I and IV collagen in proximal tubular cells [4]. Activation of tubular cells by ultrafiltered HGF and TGF-ß may also directly increase the expression of various extracellular matrix proteins, but respective studies are currently pending. However, preliminary results suggest that this direct mechanism may play a less important role compared with the production of extracellular matrix proteins by myofibroblasts, which are stimulated indirectly by conditioned glomerular ultrafiltrate, via the activation of tubular cells.

In summary, in the nephrotic syndrome or in overt diabetic nephropathy there is glomerular ultrafiltration of high molecular (forms of) growth factor proteins, such as IGF-I, TGF-ß and HGF into the tubular fluid. In this environment these proteins become activated perhaps by limited proteolysis, proteolysis of their binding proteins or other mechanisms. Bioactive growth factors in tubular fluid activate tubular cells through signalling receptors in apical tubular membranes. Activated tubular cells secrete increased amounts of chemokines and cytokines into the peritubular interstitium. To a modest degree, activated tubular cells may themselves increase the formation of peritubular extracellular matrix.

Through the basolateral release of chemo- and cytokines, activated tubular cells may interact with macrophages and myofibroblasts in the interstitium causing increased production and deposition of extracellular matrix proteins. Hence, activation of tubular cells by ultrafiltered growth factors is a necessary, initial step resulting in further cell–cell interactions causing interstitial fibrosis. These experimental findings provide mechanistic insights as to the contribution of glomerular proteinuria to interstitial fibrosis and, hence, progressive, irreversible renal failure.

References

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