Relationship of clusterin with renal inflammation and fibrosis after the recovery phase of ischemia-reperfusion injury

Ischemic acute kidney injury (AKI) occurs when renal perfusion is decreased [1, 2], and it is the most common form of clinical AKI, accounting for up to 70 % of all AKI cases [3‐6]. With adequate therapy, quick recovery of kidney function to baseline may occur and is considered a hallmark of ischemic AKI [6, 7]. Many recent studies have documented long-term sequelae following AKI, including: increased risk of developing chronic kidney disease (CKD) [8‐12], worsening of preexisting CKD [8, 13], and progression to end-stage renal disease (ESRD) [8, 12, 14]. Increasing evidence in literature suggests that many pathological factors including microvascular rarefaction, hypoxia, inflammation and renal tubular injury play an important role in the progression of CKD [15‐20], but the precise pathophysiologic pathways mediating the progressive insults associated with AKI leading to CKD are still not fully understood, and thus there are currently no viable therapeutic options available to prevent CKD progression after renal injury.

Clusterin (CLU) is a 75–80 kDa disulfide-linked heterodimeric glycoprotein that is encoded by a single gene in both the human and mouse genomes [21, 22]. This protein not only is a major glycoprotein in physiological fluids, such as plasma, milk, urine, cerebrospinal fluid, and semen [21], but is also upregulated in renal tissues of both humans and experimental models by various forms of cellular stress, such as unilateral ureteral obstruction (UUO) [23], ischemia-reperfusion injury (IRI) [24‐26], as well as in rejected renal allografts and those native kidneys with intrinsic renal disease [27‐29]. However, the biological importance of CLU for the kidney is not fully understood. We and others have been investigating the biological impacts of CLU deficiency on adult kidneys using CLU knockout (KO) mice compared to CLU-expressing wild type (WT) control mice, and have demonstrated: (i) CLU KO in aging mice results in developing progressive glomerulopathy that is associated with glomerular antibody deposition [30]; (ii) CLU KO mice exhibit the higher levels of renal fibrosis in response to ureteral obstruction [31] or angiotensin II stimulation [32]; and (iii) our group has shown that CLU KO results in more severe renal IRI [26], in an experimental model of ischemic AKI, and also impairs renal repair after IRI [33]. However, the role of CLU in the progression to CKD from AKI has not been investigated yet. In the present study, the effects of CLU deficiency on the transition of ischemic AKI to CKD were investigated using CLU KO mice in a model of renal IRI compared to WT controls.

Recent literature suggests that the full recovery of renal function after AKI is suboptimal because the long-term effects of AKI are associated with development of CKD, worsening of preexisting CKD and progression to ESRD [8, 47, 48]. Thus, understanding of the pathways mediating the long-term pathologic effects of AKI may eventually lead to develop a strategy for reducing or preventing the incidence of CKD or ESRD. Our previous studies have demonstrated that CLU deficiency worsens IRI and delays injury repair in adult kidneys [22, 26, 33]; however, the role of CLU deficiency in the long-term effects of ischemic AKI has not been investigated. The present study demonstrates that although CLU expression does not prevent renal atrophy after IRI, CLU deficiency is associated with the loss of renal function, more damage in the renal tubules and an increase in both cellular infiltrates (particularly CD8⁺ T cells and Mac3⁺ macrophages) and tissue fibrosis. All these negative impacts of CLU deficiency on the kidney after IRI may be mainly mediated by a cascade network of down-regulation of EGF and up-regulation of CCL12, Col3a1, MMP9 and TIMP1.

During the early or initial phase of AKI, sub-lethal injury of both tubular epithelial and endothelial/vascular cells is seen along with acute inflammation, followed by cellular repair and organ integrity re-establishment until functional recovery [2, 15, 49]. However, even after kidney function returns to baseline, patients are still at increased risk for the development of CKD after 2.5–3.4 years [47, 50]. The AKI-to-CKD transition is confirmed in rodent models of ischemic AKI and 3–4 weeks after IRI, the kidneys exhibit remarkable accumulation of tissue fibrosis [51‐54]. Currently, the pathophysiology of AKI to CKD or AKI-initiated renal fibrosis is not completely clear, especially in humans, but experimental studies have identified several mechanisms, such as activation of memory or effector T cells and M2 macrophages [17, 54, 55], infiltration of bone marrow-derived myofibroblasts [51, 53], endothelial injury with vascular rarefaction and hypoxia [16, 17, 56, 57], and epigenetic changes and G2/M cell cycle arrest in epithelial cells [17, 58]. All of these components (pro-inflammatory immune cells, myofibroblasts, vascular rarefaction/hypoxia, and tubular epithelial cells) may play different roles during the transition from AKI to CKD in a cascade manner, but how they interact with each other remains for further investigation. Recently, Tanaka et al. [16] have proposed a central role of hypoxia in the AKI to CKD transition, and have hypothesized that after functional recovery from the early phase of AKI, the expression of vascular factors, such as vascular endothelial growth factor (VEGF) in tubular cells, is not yet restored, which results in capillary rarefaction leading to renal hypoxia, and consequently activation of inflammatory reactions, induces myofibroblast migration and/or differentiation and damages the regenerated tubules. All of these events together lead to tubulointerstitial fibrosis [16]. The primary role of hypoxia is supported by the fact that the density of blood vessels decreases by almost 45 % after 4 weeks of ischemic AKI [56], and treatment with VEGF preserves microvascular density and prevents renal fibrosis [59]. In this study, however, by PCR array analysis the expression of VEGF and EDN 1 (endothelin 1) was not different between CLU KO and WT kidneys (Additional file 2: Table S1), suggesting that the more severe fibrosis in CLU null kidneys may not be related to the hypoxia/vascular rarefaction, or the hypoxia is not a major factor for the CLU deficiency-related renal fibrosis after IRI.

It has been demonstrated that IRI initiates progressive renal weight or volume loss accompanying with tubular cell death (both apoptosis and necrosis) and interstitial infiltration [37‐39], and our group and others using WT mice compared with CLU negative controls (CLU KO mice) have demonstrated that CLU is an anti-apoptotic or a prosurvival molecule in the kidney against IRI [22, 26, 33, 40]. In the present study of the transition of IRI to renal fibrosis, the data show that CLU deficiency does not affect the kidney weight or atrophy (Fig. 1), but it is associated with a decrease in the number of intact tubules (Fig. 6), and at the same time with an increase in the cellular infiltrates in the interstitial spaces (CD8 T cells and macrophages) and α-SMA⁺ myofibroblasts (Figs. 5, 7 and 8), suggesting that the survival and/or proliferation of different kidney cells, particularly tubular epithelial cells versus (myo) fibroblasts, may be regulated by CLU differently. Also, we showed that up-regulation of MMP9, TIMP1 and perhaps the membrane-type MMP14 was found in CLU KO kidneys after IRI. In general, MMP9 directly degrades extracellular matrix and is produced by a range of immune cells (e.g. lymphocytes and macrophages), and other cells including fibroblasts and endothelial cells following the exposure to pro-inflammatory cytokines (e.g. TNF-α, IFN-γ and IL-1β) [60], which is in agreement with the increase in cellular infiltrates, particularly CD8⁺ T cells and macrophages, and perhaps IL-1α within the CLU KO kidneys. More interestingly, MMP9 produced by renal tubular cells, macrophages and myofibroblasts contributes to renal fibrogenesis via osteopontin cleavage, which in turn recruits macrophage and induces renal tubular epithelial-mesenchymal transition (EMT) [61, 62], suggesting that MMP9 but not TGF-β is a major fibrotic factor for fibrosis in CLU KO kidneys. The major biological function of TIMPs including TIMP1 is to inhibit MMPs by forming a 1:1 enzyme-inhibitor complex, but the role of TIMP1 in kidney fibrosis has not been well investigated as of today. TIMP1 binds to proMMP9 to forms a specific complex [63], that may play a role in proMMP9 activation. Also TIMP1 exhibit other cellular activities, such as stimulation of cell proliferation of keratinocytes [64] and other cells [65]. However, the underlying mechanism by which TIMP1 involves in renal fibrosis remains further investigation.

Based on the evidence in literature, it is postulated that CLU can restrain the transition of IRI to renal fibrosis in the kidney by several mechanisms. First, complement activation plays a role in the progression of fibrosis in the kidney, as previous studies have shown that inhibition of complement activation reduces chronic inflammatory injury to renal tubular cells [66, 67] and endothelial cells [68], which is associated with reduced tissue fibrosis [66, 68]. CLU is a major glycoprotein in all the physiological fluids including the plasma [21], and this secreted form of CLU inhibits lytic terminal complement cytotoxicity in many early studies [29, 69, 70]. Thus, CLU in the blood and/or locally secreted by renal epithelial cells in WT mice may inactivate the complement during the transition of IRI to tissue fibrosis, which at least partially reduces leukocyte inflammation (both CD8 T cells and macrophagies) and tubular atrophy. Secondly, intracellular CLU is a cytoprotective protein that inhibits cell apoptosis by interacting with BAX or GRP78 [71‐73], or promotes cell survival by activating Akt and NF-kB pathway [74, 75] and prosurvival autophagy [40, 76]. CLU expression has been confirmed in cultured glomerular mesangial and glomerular epithelial cells, and tubular epithelial cells [26, 40, 77‐79], and facilitates cell survival following the exposure to pro-inflammatory cytokines (IFN-γ and TNF-α) [26] and hypoxia [40]. Thus, any of these CLU-expressing kidney cells in WT kidneys may have better survival than those in CLU KO counterparts under the chronic inflammatory attack and/or hypoxia, resulting in the prevention of nephron damage as seen in Fig. 6. Finally, the lack of CLU expression in tubular epithelial cells causes cell cycle arrest at G2/M phase in vitro [33], and worsens fibrosis in the kidneys after IRI in this study. These data may be consistent with a previous study, showing that c-jun NH₂-terminal kinase (JNK) signaling mediates the cell cycle arrest at G2/M phase in proximal tubular cells, and treatment with a JNK inhibitor, or bypassing the G2/M arrest by administration of a p53 inhibitor rescues renal fibrosis after IRI [58]. Therefore, the potential of the down-regulation of EGF, G2/M phase interruption and more cell death probably result in the poorer maintenance of epithelial integrity of the nephron in the kidneys of CLU KO mice after IRI, which may contribute to more function loss (Fig. 4) as well as more space for infiltrates and myofibroblasts within the CLU null kidneys.

Myofibroblasts are commonly considered as the predominant effector cells in kidney fibrosis [80, 81], and a recent study using multiple genetically engineered mice has identified that a half of renal myofibroblasts are from local resident fibroblasts through proliferation, and the other half are derive through differentiation from bone marrow (35 %), the endothelial-to-mesenchymal transition (EndMT) (10 %) and EMT (5 %) [45], suggesting that renal fibrogenesis is involved in multiple cell types and multiple biological processes. The present study showed that more myofibroblasts were seen in CLU KO kidneys (Fig. 8), which is confirmed by PCR array, showing the higher transcript expression of Acta2 (α-SMA, 1.3753 fold increase with P = 0.0848), collagen (Col3a1), and fibrotic MMP9 [62] along with up-regulation of CCL12 (Table 3) and perhaps PDGF (a fibroblast growth factor). CCL12 stimulates the proliferation of infiltrating fibrocytes/bone marrow-derived myofibroblasts [82, 83], suggesting that a high level of CCL12 benefits the expansion of this myofibroblast population in CLU null kidneys. More interestingly, by immunohistochemical stain many tubules in these CLU KO kidneys are stained positively with α-SMA (Fig. 8), suggesting an important role of EMT in renal fibrosis in these kidneys. However, little is known about CLU expression and its function in renal fibroblast proliferation and survival, and myofibroblast differentiation from all different origins, which remain further investigation.

The transition of AKI to CKD has major clinical significance in patients, but its molecular pathways are not fully understood. CLU is a chaperone-like protein and has different activities in different cells or experimental systems [22]. Our previous studies have demonstrated that CLU plays an important role in the maintenance and restoration of renal tubular integrity during the early phase of IRI [26, 33]. The data from current study indicate that as compared to CLU-expressing controls CLU deficiency has negative impacts on the preservation of kidney function and its normal architecture during the transition of IRI to renal fibrosis or CKD development. Also, our PCR array reveals that MMP9 but not TGF-β may be a major fibrotic factor for CLU deficiency-induced renal fibrosis. However, further understanding of the molecular mechanisms underlying the negative impacts of CLU deficiency on the kidney is needed, which may lead to develop an effective strategy to reduce the incidence of CKD after AKI.