ACUTE KIDNEY INJURY AND CHRONIC KIDNEY DISEASE

Acute kidney injury (AKI) is a syndrome characterized by tubular injury and a sudden drop in glomerular filtration. Our current understanding of the pathophysiology of AKI is incomplete and this accounts for the lack of specific therapy. One key feature that has emerged in recent years is the close relationship between AKI and chronic kidney disease (CKD).1 Thus, CKD is the main risk factor for AKI and AKI contributes to progression of CKD. This suggests that AKI and CKD share pathogenic factors: from a pathogenic point of view CKD may be considered a low level, persistent AKI. Since pathogenic events are magnified in AKI and AKI has a shorter time course, AKI has advantages as a model for the identification and assessment of pathogenic factors. In this regard, proposed biomarkers of AKI are also altered in CKD, including Klotho.2,3

TRANSCRIPTOMICS

High throughput techniques such as transcriptomics and proteomics, may help identify novel potential pathogenic factors, therapeutic targets and biomarkers in a non-biased way.4-6 Transcriptomics is a high throughput technique that allows the identification of thousands of differentially expressed candidate genes. Such patterns of expression may themselves be used for diagnostic or prognostic purposes. Bioinformatics and biostatistics tools allow to manage thousands of genes simultaneous and help to prioritize molecules for further confirmatory studies. Novel therapeutic targets may be uncovered. We recently used a transcriptomic approach to identify new genes involved in AKI that could serve as biomarkers or therapeutic targets.7,8 This approach has successfully identified new players in diabetic nephropathy such as the lethal cytokine TRAIL; the MIF receptor CD74 and the intracellular lethal protein BASP1.9-12 

TWEAK AND Fn14

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, Apo3L, TNFSF12) is a member of the tumor necrosis factor superfamily (TNFSF).13-15 Other members of the family include TNF and Fas ligand, both of which play a key role in kidney injury.16-18 TNFSF ligands bind to one or more members of the TNF receptor superfamily (TNFRSF).19,20

The human TWEAK gene encodes a type II transmembrane glycoprotein. The TWEAK C-terminal extracellular domain contains the TNF homology domain that mediates self-trimerization and receptor-binding.13 The N-terminal intracellular domain contains several nuclear localization sequences (NLS)13,21-23 and a furin recognition site, suggesting that TWEAK can be cleaved.24 Most cells can express full-length membrane-anchored TWEAK (mTWEAK) and soluble TWEAK (sTWEAK).24,25 sTWEAK is formed by proteolysis of membrane TWEAK.13,25,26

Both sTWEAK and mTWEAK bind and activate fibroblast growth factor-inducible-14 (Fn14, TWEAK receptor, TNFRSF12A, CD266).24,27-29 Fn14 was initially described in fibroblasts as a growth factor-regulated early response gene.30 Fn14 is a type I transmembrane protein that when mature has 102-aa. Fn14 is the smallest member of TNFRSF. The intracellular Fn14 domain contains TNFR-associated factor (TRAF)-binding sites which activate signal cascade. Unlike TNF or Fas, Fn14 does not contain a death domain (DD).31 In addition, CD163 binds TWEAK and is thought to be a TWEAK scavenger receptor, since TWEAK-induced signaling through CD163 was not observed.32,33

TWEAK has multiple functions with potential physiopathological relevance for kidney injury that depend on the microenvironment, the cell type and the cell state of activation. TWEAK can regulate cell proliferation, cell death, cell migration, cell differentiation, tissue regeneration, neoangiogenesis and inflammation.34-43 TWEAK contributes to tissue injury in the central nervous system, liver, gut, the vasculature, skeletal muscle, heart and kidney.40,44-49

In the kidney TWEAK actions have been extensively studied in tubular epithelium. TWEAK induces proliferation in non-stressed renal tubular cells50 and apoptosis in tubular cells stressed by an inflammatory milieu.14,51 Furthermore TWEAK activates both canonical and non-canonical NFκB transcription factor signaling.14,52-54 Through these actions TWEAK promotes tubular injury in ischemic or toxic AKI54,55 and kidney hyperplasia following unilateral nephrectomy.56 Furthermore, TWEAK contributes to vascular injury and in CKD patients soluble TWEAK behaves as a biomarker of outcome, especially when interpreted in the context of systemic inflammation.57-61 A recent transcriptomics analysis of kidney tissue in AKI confirmed highly upregulated levels of Fn14 mRNA (Figure 1). Regulation of the TWEAK/Fn14 system often takes places through upregulation of receptor expression and, thus, of cell sensitivity to TWEAK actions.

CXCL16

Chemokines are small cytokines formerly known as intercrines62 that in the kidney tubulointerstitium may be expressed by both tubular cells and fibroblasts.63 Chemokines promote leukocyte trafficking, growth and activation in inflammatory sites.64 Chemokines promote kidney tubulointerstitial inflammation.65,66 Leukocytes recruited by chemokines have a key role in kidney tubulointerstitial tissue injury during AKI and CKD.67,68 Several chemokines were upregulated in the transcriptome of murine AKI.8 Some of them, such as MCP-1 and Rantes, had already been studied.54 These chemokines share with most chemokines their release as soluble mediators.65 CX3CL1 (fractalkine) and CXCL16 (SR-PSOX) were also found upregulated in the murine AKI the transcriptome.8 CX3CL1 and CXCL16 are the only two known membrane-anchored chemokines.69 CX3CL1 and CXCL16 are synthesized as transmembrane molecules and, as such, have specific functions that may go beyond their chemokines role. In addition, they can be cleaved from the cell surface to release a soluble chemoattractant that behaves as a classical chemokine.69 Fractalkine has been extensively studied in the context of kidney disease.70 However, much less was known about CXCL16 and kidney injury. Furthermore, CXCL16 expression correlated more closely than CX3CL1 with Fn14 expression.8 In addition, there is evidence that in humans urinary TWEAK and CXCL16 may be a potential diagnostic biomarkers of kidney diseases such as lupus nephritis.71,72 TWEAK is known to regulate the expression of several chemokines. These peculiarities made a complete understanding of the relationship between TWEAK and CXCL16 regulation in kidney cells of particular interest. 

CXCL16 was identified by different groups as a ligand for the CXC-chemokine receptor CXCR673 and as a scavenger receptor for phosphatidylserine and oxidized low density lipoprotein (oxLDL) and therefore was also termed SR-PSOX.74 Full length CXCL16 consists of an extracellular N-terminal chemokine domain, a glycosylated mucin-like stalk, a transmembrane-spanning region and a short cytoplasmic tail.73 Like CX3CL1, CXCL16 potentially functions as both a soluble chemokine and a membrane-bound adhesion molecule.75-78 CXCL16 regulated leukocyte chemotaxis, T cell recruitment and cell proliferation.79-87

In the kidney, CXCL16 is constitutively expressed in human mesangial cells, podocytes and tubular cells.79,86,87 There is evidence for differential regulation of CXCL16 expression in glomeruli or different tubular segments and in tubular injury of diverse etiology. CXCL16 expression is increased in various animal models of kidney injury and human nephropathies.72,79,86-89

Glomerular CXCL16 expression is increased in human membranous nephropathy.87 Glomerular and tubular CXCL16 was also increased in lupus mice and in anti-GBM nephritis.72,88,89 Functional studies suggest that CXCL16 promotes progression of damage in experimental glomerulonephritis.88 CXCL16 blockade significantly decreased monocyte/macrophage infiltration and glomerular and tubular injury.88,89 In this regard, besides effects on leukocytes, CXCL16 has direct actions on glomerular cells. Podocyte CXCL16 may regulate the uptake of oxLDL,87 while mesangial cell CXCL16 promotes mesangial cell migration and proliferation.79

In human allograft AKI, CXCL16 expression was increased focally in the apical side of tubules.86 By contrast, a low tubular CXCL16 expression was observed in interstitial rejection that was attributed to increases CXCL16 shedding. Thus, remnant CXCL16 was located to the basolateral membrane and surrounded by T cell infiltrates. In experimental toxic AKI, both prominent apical and basolateral CXCL16 expression were noted.8 Thus, other tubular cells, the interstitium and the tubular lumen were exposed to CXCL16 derived from tubular cells. Interestingly, both patterns did not overlap in many tubules.

No in vivo functional studies of CXCL16 targeting in tubulointerstitial kidney disease have been reported. In cell culture CXCL16 did not induce murine tubular epithelial cell proliferation or apoptosis, either alone or in combination with TWEAK.8 However, CXCL16 had a proinflammatory effect and increased TWEAK-induced gene expression of ICAM-1, MCP-1 and RANTES. In this regard, tubular cells expressed the CXCR6 receptor.8

In cultured glomerular cells CXCL16 is upregulated by TNF-α and IFN-γ.79,86,87 IFN-γ increased CXCL16 expression in cultured primary thick ascending limb cells and early distal tubular cells.86 TWEAK is a novel regulator of CXCL16 expression in tubular epithelial cells.8 TWEAK promoted CXCL16 expression through the canonical NFκB pathway in cultured tubular cells.8 Moreover, TWEAK increased renal CXCL16 expression and interstitial CD3 positive lymphocytes. Since neutralization of TWEAK decreased CXCL16 and CD3 lymphocyte infiltration in experimental AKI, TWEAK-induced CXCL16 expression may contribute to T cell recruitment and collaborate with TWEAK in promoting inflammation.

KLOTHO

Klotho is a protein with anti-aging properties which is highly expressed in tubular renal cells.90,91 Klotho is a single-pass transmembrane protein. The extracellular domain of Klotho may be proteolytically processed by ADAM10/17 and secreted. In addition, alternative splicing may give rise to a soluble secreted isoform.92 Trasmembrane Klotho binds to multiple fibroblast growth factor (FGF) receptors conferring them specific and high affinity for FGF23. FGF23 is a bone-derived hormone that regulates phosphate homeostasis and vitamin D metabolism. Thus, the main known function of Klotho is regulation of phosphate metabolism and evidence from mice in which phosphate was manipulated genetically or through diet suggests that aberrant phosphate homeostasis is a key contributor to the accelerated aging syndrome of Klotho -/- mice.93 Klotho also protects cells and tissues from oxidative stress and has anti-inflammatory properties through modulation of NFκB signaling.94

Klotho is downregulated during kidney diseases, such as long-term hypertension, diabetes mellitus, CKD,95 and in experimental AKI induced by ischemia-reperfusion or a folic acid overdose.7,96 In addition kidney Klotho was decreased in the course of systemic inflammation caused by inflammatory bowel disease and a neutralizing anti-TNF antibody attenuated bowel inflammation and reversed the repression of kidney Klotho expression.97 Consistent with these data, Klotho was downregulated in the transcriptome of murine AKI and Klotho expression was inversely correlated with Fn14 expression, suggesting that TWEAK, like TNF, may regulate Klotho expression. The reduction of kidney Klotho during nephrotoxic AKI persisted beyond recovery of renal function and was associated with decreased circulating Klotho. The persistent decrease in Klotho might be related to the increased mortality of AKI patients following recovery from AKI. Since Klotho may be nephroprotective,96,98-100 the persistent decrease in Klotho might also predispose to progression of CKD. However, these hypotheses await formal confirmation.

In nephrotoxic AKI, Klotho expression and renal function were preserved by TWEAK targeting thus identifying a potential regulator of Klotho expression in cultured cells.7 Indeed, in cultured tubular cells of proximal origin TWEAK and TNF promoted the NFκB-dependent downregulation of Klotho expression.7 TWEAK and TNF activate the canonical pathway for NFκB activation, but only TWEAK activates the non-canonical pathway.14,53 The reported downregulation of Klotho by TNF7,97 and the time course of Klotho mRNA downregulation, that is already observed at 3h, suggest activation of the canonical NFκB pathway. Indeed, RelA was necessary for TWEAK- and TNF-induced Klotho repression. For the first time it was observed TWEAK downregulates NFκB-mediated gene expression. Regulation of NFκB activation function is controlled through different mechanism, such as interaction of the p65/RelA subunit with histone deacetylase (HDAC) corepresor proteins.101-103 In this regard, HDAC inhibitors prevented repression of Klotho induced by TWEAK or TNF. In addition, recruitment of NFkB to chromatin is regulated in a promoter-specific manner. TWEAK induced histone H3 and H4 deacetylation at the murine Klotho promoter in renal tubular cells. 

INTERACTION BETWEEN INFLAMMATION AND AGEING: NFκB

From the above mentioned studies the NFκB emerges as a family of pleiotropic transcription factors with a key role at the interface between inflammation and ageing.52,104-107 This notion had been advanced before by proponents of the inflamma-aging concept.108 The term inflamm-aging has been used to describe the age-related increase in the systemic pro-inflammatory status of humans.109 

A wide range of stimuli relevant to tissue injury activate NFκB, including cytokines, growth factors, immune mediators, proteinuria and genotoxic or mechanical stretch.110,111 Activation of NFκB can proceed through classical/canonical, alternative/non-canonical NFκB and hybrid pathways.104,106,112 Classical NFκB activation is usually a rapid and transient response to a wide range of stimuli. Under basal conditions NFκB is inactive in the cytosol because it is bound to inhibitory IκB proteins. Activating stimuli activate the inhibitor of κB kinases (IKK), which phosphorylate IκBs, marking them for degradation by the proteasome. Degradation of IκB releases and activates NFκB dimers, such as those containing RelA. RelA containing dimers then migrate to the nucleus where they bind to κB DNA sequences in promoters and enhancers of target genes. In general canonical NFκB promote the transcription and expression of proinflammatory genes, as observed for CXCL16 in TWEAK-stimulated tubular cells. There are several negative feed-back mechanisms. Thus, suppressors of cytokine signaling (SOCS)-1 promotes the ubiquitination and proteasomal degradation of RelA-containing dimers, thus quenching the NFκB response.113 The SOCS1 overexpression decreases inflammation in experimental DN.114

As a result of NFκB integration of stimulus information it may both induce or repress individual gene transcription.115 However, the fact that NFκB can function as a repressor of gene expression is less well-known. Gene expression repression by NFκB may suppress the inflammatory response by recruiting inhibitory components of the NFκB system. Thus, antiinflammatory cytokines, such as IL-10 promote synthesis of nuclear located atypical IκB proteins B-cell lymphoma 3 (BCL-3), IκBζ and IκBNS, which bind to DNA-bound NFκB dimers and may repress transcription of inflammatory genes.113 In addition repression of gene expression by NFκB has been implicated in sepsis-induced downregulation of kidney aquaporin/V2 receptor and may have a role in resolution of inflammation.116,117 However, classical NFκB dimers containing RelA may also downregulate Klotho mRNA and Klotho-dependent anti-inflammatory and ageing pathways, as observed for TWEAK and TNF, and, thus, promote further injury in and outside the kidney.

CONCLUSIONS

In summary, transcriptomics of AKI tissue has identified TWEAK as a novel regulator of CXCL16 expression in renal tubular cells through activation of the RelA NFκB transcription factor. In addition, TWEAK, like TNFα, downregulated Klotho in renal tubular cells through a similar NFκB RelA-dependent mechanism. Since Klotho has anti-ageing and anti-inflammatory properties, these findings may have therapeutic implications in kidney injury and also for inflammation-associated premature aging. Thus targeting either TWEAK, through neutralizing anti-TWEAK antibodies currently undergoing clinical trials in lupus nephritis, or targeting NFκB, may potentially limit inflammation and the adverse consequences of inflammation on ageing.

Acknowledgments

This work has been supported by Sociedad Española de Nefrología. Additional funding for the group: FIS PS09/00447, CP07/0020, PI08/1083, PI081564, ISCIII-RETIC REDinREN/RD06/0016, Comunidad de Madrid S2010/BMD-2378, SAF 2007-60896, Ministerio de Ciencia y Tecnología PI10/00072. Salary: FIS to MCI and FIS Sara Borrel to ABS, MDSN, Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to AO.

Conflict of interest

 

The authors declare that there is no conflict of interest associated with this manuscript.

Key concepts

1. Tissue transcriptomics allows the non-biased analysis of gene expression and identification of potential novel therapeutic targets in tissue injury.

2. Acute kidney injury transcriptomics identified the simultaneous upregulation of inflammatory genes such as the TWEAK receptor FN14 and chemokines like CXCL16 and the downregulation of anti-inflammatory/anti-ageing genes such as Klotho.

3. TWEAK stimulation of tubular cells in culture reproduced the findings in AKI.

4. The transcription factor NFκB appears to be the key to both upregulation of proinflammatory genes and downregulation of Klotho in response to TWEAK.

5. Thus either targeting TWEAK, through neutralizing anti-TWEAK antibodies currently undergoing clinical trials in lupus nephritis, or targeting NFκB, may potentially limit inflammation and the adverse consequences of inflammation on ageing.

Figure 1. Gene expression for representative mediators of inflammation and ageing in experimental acute kidney injury (AKI): Transcriptomics results of kidney tissue