Obstructive nephropathy: insights from genetically engineered animals

Abstract

Congenital obstructive nephropathy is the primary cause for end-stage renal disease (ESRD) in children. An increasingly used animal model of obstructive nephropathy is unilateral ureteral obstruction (UUO). This model mimics, in an accelerated manner, the different stages of obstructive nephropathy leading to tubulointerstitial fibrosis: cellular infiltration, tubular proliferation and apoptosis, epithelial-mesenchymal transition (EMT), (myo)fibroblast accumulation, increased extracellular matrix (ECM) deposition, and tubular atrophy. During the last decade genetically modified animals are increasingly used to study the development of obstructive nephropathy. Although the use of these animals (mainly knockouts) has highlighted some pitfalls of this approach (compensation by closely related gene products, absence of temporal knockouts) it has brought important information about the role of specific gene-products in the pathogenesis of obstructive nephropathy. Besides confirming the important pathologic role for angiotensin II (Ang II) and transforming growth factor-beta (TGF-beta) in obstructive nephropathy, these animals have shown the complexity of the development of tubulointerstitial fibrosis involving a large number of closely functionally related molecules. More interestingly, the use of these animals has led to the discovery of unexpected and contradictory roles (both potentially pro- and antifibrotic) for Ang II, for ECM degrading enzymes matrix metalloproteinase 9 (MMP-9) and tissue plasminogen activators (PAs), for plasminogen activator inhibitor 1 (PAI-1), and for the adhesion molecule osteopontin (OPN) in obstructive nephropathy. Further use of these animals, especially in combination with pharmacologic tools, should help to better identify potential antifibrotic strategies in obstructive nephropathy.

Figure 1. Overview of the different stages of the development of obstructive nephropathy

Experimental ureteral obstruction (UUO) induces after a few hours cellular infiltration in the tubulointerstitium. These infiltrating cells (mainly macrophages) secrete growth factors and cytokines inducing a disequilibrium between apoptosis and proliferation of tubular cells, as well as inducing fibroblast activation and proliferation. Fibroblasts either infiltrate from the circulation into the interstitium, appear by EMT or appear by proliferation of the few resident fibroblasts. Activated fibroblasts secrete the ECM that is starting to accumulate into the interstitium as soon as myofibroblasts appear. As the obstruction continuous, ECM deposition becomes massive and uncontrolled apoptosis of tubular cells results in tubular atrophy. The involvement of different molecules in these stages of obstructive nephropathy, studied using genetically modified animals, is described in the text and in the subsequent figures.

Figure 2. Hypothetic representation of cellular infiltration in UUO based on data obtained with knockout mice

UUO induces macrophage infiltration in the tubulointerstitium. In UUO, the interaction between L-selectin and sulfatide seems to mediate the initial contact (rolling) between macrophages and the vascular endothelium. These rolling macrophages are exposed to adhesion molecules like chemokines and osteopontin expressed on endothelial cells. The chemokines bind to chemokine receptors (in UUO mainly the CCR1) on the macrophages. Osteopontin binds to the CD44 receptor. This results in firm adhesion and transendothelial migration of macrophages. The role of other important molecules (including ICAM-1, VCAM-1 and MCP-1) involved in cellular infiltration shown to be induced in UUO has not been studied yet and should give a more complete picture of cellular infiltration in UUO.

Figure 3. Proposed role of EMT in ureteral obstruction

UUO induces EMT which contributes significantly (36%) to the appearance of interstitial myofibroblasts. Myofibroblasts are the main source of profibrotic and inflammatory cytokines leading to matrix accumulation. The remainder of the fibroblasts comes from bone marrow (14–15%) and probably local fibroblast proliferation and activation [98]. EMT involves four key events leading to epithelial cell migration and invasion into the interstitium and transition into fibroblasts [49]. Interestingly one of these events, tubular basement membrane degradation, is blocked in tissue plasminogen activator (tPA) knockout mice (in contrast to what was expected for a tPA knockout) thereby blocking EMT and subsequent tubulointerstitial fibrosis.

Figure 4. The role and connections between the renin-angiotensin and kinin-kallikrein system and proteolytic systems in UUO

The profibrotic role of angiotensin II (AII) in UUO (see Figure 5) is counterbalanced by the production of bradykinin. It is proposed that during UUO, bradykinin is activating the G-protein coupled B2 receptor which activates the proteolytic plasminogen activator (PA, combined tissular and urokinase plasminogen activator activity) system (either directly or by inhibition of PAI-1) which in turn switches on metalloproteinases (MMPs) promoting matrix degradation (bold dashed line). Angiotensin converting enzyme (ACE)-inhibition thus blocks production of profibrotic AII and promotes accumulation of the antifibrotic peptide bradykinin. The role of the proteolytic enzymes in matrix degradation in UUO is depicted in the grey area in figure. Both pro and antifibrotic properties of this system have now been proposed: i) in UUO it was shown that plasmin can generate both profibrotic TGFβ and antifibrotic hepatocyte growth factor (HGF), ii) activation of MMPs degrades the extracellular matrix but stimulates EMT and iii) PAI-1 has been shown, besides its role to inhibit PA activity, to stimulate cellular infiltration in UUO. The dotted lines show other controversial effects PA activation TGFβ and inhibition of cellular infiltration by PAI-1 observed in other models of nephropathies (refs). Abbreviation: PAI-1, PA-inhibitor 1.

Figure 5. The central role for NF-κB in angiotensin II profibrotic effects in UUO

Angiotensin II (AII) accumulation during UUO stimulates two G-protein couples receptors, the AT₁ and AT₂. Although AT₂ knockout mice display less tubulointerstitial fibrosis stimulation of this receptor and the AT₁ receptor induces NF-κB family member activation which translocates to the nucleus and activates a vast panel of proinflammatory and profibrotic cytokines leading to sustained inflammation and matrix production. NF-κB activation is also leading to at least 2 positive autocrine regulation loops by the increased expression of TNFα and angiotensinogen. Abbreviations: IκB, inhibitor of κB; NF-κB, nuclear factor - κB; TNFα, tumor necrosis factor α; AT₁ and AT₂ receptor, angiotensin type 1 and 2 receptor.

Figure 6. Proposed schematic representation of the role of TGFβ in UUO

TGFβ has been shown to be an important regulator of many of the processus leading to tubulointerstitial fibrosis including EMT, cellular infiltration and apoptosis leading to tubular atrophy and interstitial accumulation of extracellular matrix proteins. Binding of TGFβ to the transmembrane serine/threonine kinase receptor type II (RII) leads to the recruitment-phosphorylation and activation of the type I receptor kinase (RI) which in turn, phosphorylates Smad2 and Smad 3 proteins thus able to bind Smad4 and translocate to the nucleus where they regulate the transcription of target genes by binding to their specific promoter-sequences. In addition to this classical TGFβ signaling pathway there is now increasing evidence that Smad-independent pathways like activation of RhoA and p38MAPKs are involved in EMT. Smad3 knockout mice are protected against UUO-induced tubulointerstitial fibrosis and showed the existence of a TGFβ autocrine loop during UUO. Furthermore, UUO on knockout mice showed that the production of active TGFβ is controlled by integrins, plasmin and decorin.