Pattern recognition receptors and the inflammasome in kidney disease. Jaklien C. Leemans, Lotte Kors, Hans-Joachim Anders and Sandrine Florquin

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1 Pattern recognition receptors and the inflammasome in kidney disease Jaklien C. Leemans, Lotte Kors, Hans-Joachim Anders and Sandrine Florquin Abstract Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NLRs) are families of pattern recognition receptors that, together with inflammasomes, sense and respond to highly conserved pathogen motifs and endogenous molecules released upon cell damage or stress. Evidence suggests that TLRs, NLRs and the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome have important roles in kidney diseases through regulation of inflammatory and tissue-repair responses to infection and injury. In this Review, we discuss the pathological mechanisms that are related to TLRs, NLRs and NLRP3 in various kidney diseases. In general, these receptors are protective in the host defence against urinary tract infection, but can sustain and self-perpetuate tissue damage in sterile inflammatory and immunemediated kidney diseases. TLRs, NLRs and NLRP3, therefore, have become promising drug targets to enable specific modulation of kidney inflammation and suppression of immunopathology in kidney disease. Leemans, J. C. et al. Nat. Rev. Nephrol. advance online publication 3 June 2014; doi: /nrneph REVIEWS Department of Pathology, Academic Medical Center, Meibergdreef 9, L2 111, 1105 AZ Amsterdam, Netherlands (J.C.L., L.K., S.F.). Nephrologisches Zentrum, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstraße 1, München, Germany (H. J.A.). Correspondence to: J.C.L. j.c.leemans@ amc.uva.nl Introduction Inflammation is a hallmark of almost all forms of renal injury and is an important factor in the development of many kidney diseases. This process has an important role in the induction, amplification and resolution of renal injury. The primary mechanism through which the kidney becomes inflamed involves pattern recognition receptors (PRRs). Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NLRs) are, respectively, transmembrane and intracellularly expressed PRRs that are essential to innate immune responses and tissue homeostasis. 1,2 Several members of the NLR family can oligomerize to form cytoplasmic multimeric protein inflammasome complexes that accomplish different, although often complementary, functions during immune responses. TLRs, NLRs and inflammasomes cooperate to signal infection, metabolic changes and tissue injury, and induce subsequent innate immune responses that are intended to regain tissue homeostasis. 1 3 Although inflammation mediated by TLRs, NLRs and inflammasomes can help to resolve and repair an initial insult, when the reaction is inappropriate or chronic it might increase tissue damage and lead to development of subsequent inflammatory disorders. Under normal and pathological conditions, PRRs are widely expressed in the kidney on renal resident immune cells and parenchymal cells (Table 1). After infection or nonpathogenic insult, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are released and sensed by these PRRexpressing cells (Figure 1, Table 2). The resulting innate inflammatory response coordinates the recruitment of Competing interests The authors declare no competing interests. leukocytes to the kidney, which in turn become activated by DAMPs and PAMPs and, together with resident renal cells, lead to an inflammatory response and shape adaptive immune responses (Figure 1). Moreover, ligation of PRRs in dendritic cells coordinates and regulates adaptive immune responses by stimulation of T cell and B cell responses, and can affect the quality of such responses. When renal inflammation is well controlled, the kidney is cleared from pathogens and/or cell debris, undergoes repair and returns to homeostasis. However, a maladaptive inflammatory response can lead to progressive kidney disease, which might increase the release of DAMPs and establish an increasingly aggravating cycle of renal disease (Figure 1). Substantial information indicates that TLRs, NLRs and the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome have important roles in the pathogenesis of multiple renal disorders (Figure 2, Table 2). In this Review, we provide an update on the expression and functional roles of TLRs and NLRs in renal pathology and discuss the opportunities and difficulties when targeting these receptors for the treatment of kidney diseases. Structure and biology of TLRs and NLRs The TLR family consists of functional identified members 10 in humans and 12 in mice that are localized at the plasma membrane or in endosomes. 4 A wide range of PAMPs and DAMPs, including lipopoly saccharide, peptidoglycan, heat shock proteins and nucleic acids, activate TLRs and induce downstream signalling. 5 7 TLRs have an extracellular recognition domain comprising leucine-rich repeats, a transmembrane region and an intracellular Toll/IL 1-receptor signalling domain (TIR). Upon ligand binding and subsequent formation NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 1

2 Key points Toll-like receptors (TLRs), nucleotide-binding oligomerization domain receptors (NLRs) and the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome regulate inflammatory and repair processes in the kidneys TLRs prevent invasion and growth of pathogens in the urinary tract Inappropriate activation of TLRs, NLRs and the NLRP3 inflammasome is a major cause of acute and chronic kidney disease Gene-association studies have revealed connections between TLR gene mutations and the development of several inflammatory kidney disorders TLRs, NLRs and the NLRP3 inflammasome represent attractive novel drug targets to prevent and intervene in kidney inflammation and suppress immunopathology in kidney disease of a sustainable homodimer or heterodimer, the TIR domain functions as a scaffold for the TIR domains of various adaptor molecules, such as MyD88 and TIRdomain-containing adapter-inducing interferon β (TRIF; also known as TICAM 1). 8 Binding and dimerization initi ate downstream signalling that leads to the secretion of proinflammatory cytokines and, in the case of endosomal TLRs, of type I interferons via various pathways. 7,9 Generally, all TLRs, except TLR3 and (partially) TLR4, signal via NF κb-dependent signalling pathways through the recruitment of MyD88. 7 In the NLR family, 22 members have been identified in humans and 34 in mice. 4 NLRs are expressed intracellularly and contain three different domains: a central nucleotide-binding and oligomerization domain, which enables ATP-driven activation of the signalling complex via oligomerization by the formation of high- molecularweight complexes; a C terminal leucine-rich repeat that acts as a sensing domain; and an N terminal effector domain that mediates downstream signalling. 10 The N terminal domains differ between NLR subgroups, but most commonly consist of a caspase-recruitment domain expressed by NOD1 and NOD2, and a pyrin domain displayed by NLRP NOD1, NOD2, NLRP3 and most TLRs have been extensively investigated in healthy kidneys and in various causes of renal injury (Tables 1 and 2). NOD1 and NOD2 are specialized NLRs that recog nize structures of bacterial peptidoglycan in the cytoplasm. Upon activation via caspase recruitment, domain inter actions cause NOD1 and NOD2 to recruit receptor- interacting serine/threonine-protein kinase 2 (RIP 2), which drives activation of proinflammatory-gene regulation through NF κb and MAPK. 11 No DAMPs have yet been established for NOD1 and NOD2. Upon activation, NLRP3 oligomerizes and recruits ASC, which in turn interacts with pro-caspase 1 to form a platform the inflammasome that stimulates the maturation and secretion of IL 1β, IL 18, IL 33 and a programmed form of cell death called pyroptosis. Priming of proinflammatory cytokines, for example by TLRs, is an important integrated step in the signalling of the NLRP3 inflammasome. 13 Activation of the NLRP3 inflammasome occurs in response to various PAMPs and DAMPs, such as bacterial toxins, different crystals, extracellular ATP and reactive oxygen species. 3,12 14 Given the molecular range of NLRP3 activators, direct recognition by NLRP3 seems unlikely in most cases. Rather, changes in intracellular ion concentrations, redox status and nutritional status seem to regulate NLRP3 inflammasome activation, which suggests the sensing of changes in cellular homeostasis. 12,13 Additionally, mitochondria have important roles in activation of the NLRP3 inflammasome. 13 Mitochondria not only form a platform for inflamma some assembly, but mitochondrial destabilization and dysfunction also mediate activation of the NLRP3 inflammasome by release of reactive oxygen species and mitochondrial DNA, or through direct binding of NLRP3 to the mitochondrial lipid cardiolipin A noncanonical NLRP3 inflammasome pathway has been described that engages caspase 11, which activates caspase 1 and leads to the release of IL 1β, IL 18 and IL 1α. 19 Resident renal dendritic cells, macrophages and infiltrating monocytes are capable of NLRP3 inflammasome signalling in the kidney, but whether renal nonimmune cells, such as intrinsic glomerular cells and tubular epithelial cells, have similar signalling capacity is unclear. Indeed, NLRP3 has several inflammasomeindependent effects in renal disease that are beyond cytokine maturation and pyroptosis (Table 2). 20 In general, the wide distribution of transmembrane TLRs and cytosolic NLRs in the kidney provides a sensing network for DAMPs and PAMPs that is critical to combat infection, and to stimulate inflammation that is needed to clear debris and initiate tissue repair. Excessive or uncontrolled PRR signalling and inflammasome activation, however, can be detrimental to the kidney and might lead to renal pathology (Figures 1 3). Urinary tract infections Urinary tract infections (UTIs) are common in humans, have notable medical and financial implications and impact on quality of life. The innate immune response during a UTI determines whether the host defence is effective and whether an individual will develop renal damage and scarring. 21 TLRs play an important part in the response to UTIs and tune the immune response to prevent pathogen invasion and growth. Whether NLRs have any role in the response to UTIs is unclear. The crucial role for TLR4 in the pathogenesis of UTIs caused by gram-negative bacteria was identified using mouse strains with loss-of-function mutations or deletions in the Tlr4 gene Innate immune response and bacterial clearance in the kidneys and bladder were impaired, but the mice generally remained asymptomatic and developed a state that resembled human asymptomatic bacteriuria. By contrast, mice deficient for Sigrr (also known as Tir8), a negative regulator of TIR signalling that is predominantly expressed in the kidney, showed improved bacterial clearance in the kidneys during gram-negative UTI due to an increased inflammatory response. 26 Various cell types drive these TLR-dependent processes in the kidney. Studies of bone-marrow- chimeric mice showed that Tlr4 in parenchymal and bone- marrowderived cells was crucial to stimulate inflammation and induce clearance of uropathogenic Escherichia coli in the bladder and kidneys. 27,28 Tlr4 was also involved in renal immunopathology by means of renal abscess formation ADVANCE ONLINE PUBLICATION

3 Table 1 Changes in renal and urinary tract PRR expression related to pathology PRR Physiological constitutional expression Increase expression after infection or injury TLR1 Tubular cells (mouse) 29 Kidney transplantation: acute rejected allograft (human)* 77 TLR2 Glomerular mesangial and endothelial cells, podocytes, epithelial cells of Bowman s capsule, tubular and collecting duct cells, peritubular endothelial cells and interstitial cells (mouse) 59,64 Peritubular endothelial cells, glomeruli and tubules (human) 104 Distal tubules (rat) 131 TLR3 Glomerular mesangial cells (mouse) 135 Glomeruli, distal tubules and vascular smooth-muscle cells (human) 156 TLR4 Glomerular mesangial cells, podocytes, collecting duct cells, peritubular endothelial cells (mouse) Proximal and distal tubules, bladder epithelial cells (mouse and human) 27,30,59,74,169 Distal tubules (rat) 88,131 TLR5 Total kidney and bladder (mouse) 45 ND TLR7 Total kidney (human) 77 Absent on intrinsic renal cells (mouse) 135 IRI: cytoplasm of tubules (mouse); 59,64 proximal tubules and mononuclear cells (rat) 182 Chronic cyclosporin toxic effects: proximal and distal tubules and mononuclear cells (rat) 131 Kidney transplantation: acute rejected allograft (human) 77 Lupus nephritis: glomerular endothelial cells and podocytes (mouse) 158 UUO: apical and cytoplasm of tubules (mouse); 102 tubular and tubulointerstitial cells (human) 102 Diabetic nephropathy: tubules and renal macrophages (mouse) 111,112 Kidney transplantation: acute rejected allograft (human) 77 Lupus nephritis and immune-complex glomerulonephritis : glomerular mesangial cells, infiltrating immune cells (mouse) 135,154 Hepatitis C associated glomerulonephritis: glomeruli (human) 156 IRI: tubules and infiltrating cells for >24 h after IRI and endothelial cells within 4 h after IRI (mouse); 56,59,62 proximal tubules and mononuclear cells (rat) 182 Sepsis: apical and in cytoplasm of proximal and distal tubules, peritubular and glomerular capillaries (rat) 88 Toxin-mediated AKI: proximal tubules (mouse) 96 Chronic cyclosporin toxic effects: proximal and distal tubules, mononuclear cells (rat) 131 Kidney transplantation: acute rejected allograft (human); 77 glomerular cells and infiltrating cells (rat) 183 Lupus nephritis: glomerular endothelial cells, podocytes (mouse) 158,169 UUO: total kidney (mouse) 100,103,104 Diabetic nephropathy: proximal and distal tubules, peritubular capillaries (human) 108 Lupus nephritis and immune complex glomerulonephritis: infiltrating macrophages, DCs (mouse) 135,142 Kidney transplantation: acute rejected allograft (human) 77 TLR8 Total kidney (human) 77 Kidney transplantation: acute rejected allograft (human) 77 TLR9 TLR11 Renal tubules, interstitial tissue (human) 168,185 Absent on intrinsic renal cells (mouse) 135 Kidney and bladder epithelial cells (mouse) 46 NOD1 Total kidney (mouse) 14 Tubular cells (mouse and human) 67 NOD2 NLRP3 Tubular cells, mesangial cells, podocytes (mouse) 14,67,117 Tubular cells, glomerular endothelial cells (human) 67,117 Glomerular podocytes and renal mononuclear cells (mouse) 118,124 Tubular cells (mouse and human) 69 Total kidney (rat) 119 Lupus nephritis: glomerular infiltrating monocytes (mouse); 141 proximal tubular cells, interstitial inflammatory cells (mouse and human); 141,184 glomeruli, podocytes (human) 168,185 ND ND Diabetic nephropathy: renal parenchymal cells, infiltrating immune cells (human and mouse) 117 IRI: total kidney (mouse) 68 Oxalate-induced AKI: intrarenal DCs (mouse) 123,124,126 Glomerulosclerosis in hyperhomocysteinaemia: podocytes (mouse) 118 Anti-GBM glomerulonephritis: tubulointerstitial (mouse) 186 Lupus nephritis: total kidney (mouse) 187 UUO: total kidney (mouse) 101,105 Diabetic/fructose/metabolic-syndrome-driven nephropathy: total kidney (mouse); 120 total kidney (rat) 116,119 *TLR expression is upregulated in the kidney after rejection of the allograft as, in those kidneys, the expression is increased compared with biopsies from transplant recipients without rejection. Aggravation of lupus nephritis and immune complex glomerulonephritis. Cell type not determined. Abbreviations: AKI, acute kidney injury; anti-gbm, antibodies against glomerular basement membrane; CKD, chronic kidney disease; DCs, dendritic cells; IRI, ischaemia reperfusion injury; ND, not determined; PRR, pattern recognition receptor; UUO, unilateral ureteral obstruction. Mouse primary renal tubular epithelial cells express several genes that encode TLRs, of which Tlr2 and Tlr4 mediate direct inflammatory responses to bacterial components. 29 Fischer et al. 22 delineated the downstream mechanism by which TLR4 controls UTIs in a mouse model. They found that epithelial TLR4 signalling involved glyco sphingo lipid receptors for P fimbriae and TRIF/TRIF-related adaptor molecule (TRAM; also known as TICAM 2). For type 1 fimbriae, TLR4 signalling involved mannosy lated glycoproteins that favored MyD88-dependent signalling. 22 Epithelial cells from the collecting ducts also participate in clearance of uropathogenic E. coli by activation of a proinflammatory immune response via TLR4-mediated MyD88-dependent and TLR4-independent signalling NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 3

4 Pathogen-mediated insult PAMPs DAMPs Non-pathogen-mediated insult DAMPs?? APC TEC EC Renal parenchymal cell-associated TLR/NLR/inflammasome Non-sterile TLR/NLR/inflammasome-mediated innate immune response Inflammatory mediators Leukocyte infiltration Sterile Increased DAMP release increases amplification of renal inflammation and injury DCs collaborate with adaptive immune system Controlled response Renal tissue repair Renal tissue homeostasis Maladaptive response Renal disease Ascending pyelonephritis, postpyelonephritic scarring, AKI, CKD, autoimmunity, diabetes-associated and metabolic-syndrome-associated nephropathy Figure 1 Roles of TLRs, NLRs and the NLRP3 inflammasome in renal tissue repair and remodelling and renal diseases. Responses to pathological changes in the kidneys can lead to controlled or maladaptive inflammatory responses. Direct mechanisms that can influence renal repair or disease mediated by TLRs, NLRs or the inflammasome are not clearly understood. Abbreviations: AKI, acute kidney disease; APC, antigen-presenting cell; CKD, chronic kidney disease; DAMP, damage-associated molecular pattern; DCs, dendritic cells; EC, endothelial cell; NLR, nucleotide-binding oligomerization domain receptor; NLRP3, NACHT, LRR and PYD domains-containing protein 3; PAMPs, pathogen-associated molecular patterns; TEC, tubular epithelial cell; TLR, toll-like receptor. pathways that require distinct MAPK-related signalling molecules. 30 Besides modulating inflammatory responses in UTIs, TLR4 and TLR2 may contribute to tubular dysfunction during bacterial infection by influencing renal epithelial transport. TLR4 and TLR2 agonists directly modify renal transport of bicarbonate in tubular epi thelial cells through distinct signalling pathways. 31,32 TLR4 also facilitates the lipid-raft-mediated transcellular translocation of uropathogenic E. coli strains across renal collecting duct cells in vitro. 33 Whether this function implies a role for TLR4 in disseminating cystitis to pyelonephritis in vivo remains unclear. These experimental research findings have been translated to human studies. TLR4 expression on neutrophils and monocytes was lower in, respectively, children with asymptomatic bacteriuria and adults with chronic UTIs than in age-matched controls. 34,35 This finding suggests that reduced TLR4-mediated innate immune responses increase the risk of UTI. Indeed, patients with asymptomatic bacteriuria who had reduced TLR4 expression due to mutations in the TLR4 promoter region had attenuated innate immune responses. 36 TLR4 polymorphisms are also related to the development of new and recurrent UTIs in children and adults, 35,37 but not to pyelonephritis. 38 The lack of relationship in the latter might be due to insufficient TLR-mediated inflammation reducing the clinical recognition of upper-tract UTIs. Although the role of TLR4 in UTIs has been studied extensively, less is known about the roles of other TLR family members. In a mouse model of gram-positive UTI, Tlr2 knockout mice showed similar bacterial outgrowth and inflammation in kidneys to wild-type mice, indicating that this TLR plays no part in the innate immune response. 39 Fischer et al. 22 found that Tlr2 knockout mice responded with a normal granulocyte influx in urine in reaction to P fimbriated E. coli, although the effects on bacterial outgrowth in the urinary tract were not assessed. In vitro mouse studies have shown that TLR2 in proximal tubule cells mediates early inflammation caused by lepto spiral outer membrane proteins. 40 By contrast, several studies in humans have shown that TLR2 polymorphisms are related to increased risk of UTI or asymptomatic bacteriuria, 41,42 but are not associated with a history of recurrent cystitis or pyelonephritis. 38 Fewer children with acute pyelonephritis and acute lobar nephronia, however, had a TLR2 CC genotype (1350T>C) than controls, 43 whereas the frequencies of single-nucleotide polymorphisms (SNPs) in TLR1, TLR4, TLR5 and TLR6 did not differ. 43,44 In adult women, a TLR5 polymorphism that was predicted to prematurely truncate TLR5 was associated with increased risk of recurrent cystitis but not with pyelonephritis; a TLR1 SNP correlated with protection against pyelonephritis. 38 Partly in line with this human study, Tlr5 knockout mice were more susceptible than non deficient mice to upper-tract and lower-tract UTIs caused by E. coli, possibly due to decreased inflammation in the bladder at an earlier time. 45 Besides having SNPs in genes encoding TLRs, Zhang and co-workers speculated that humans are susceptible 4 ADVANCE ONLINE PUBLICATION

5 Table 2 TLRs and NLRs involved in renal infection and injury Renal disorder Study type Possible ligands Renal effect Reference(s) TLR1 UTI Clinical ND Protective if mutated 36 TLR2 UTI UTI, ABU or pyelonephritis AKI Infection in mouse with Enterococcus faecalis 22 and Escherichia coli 39 Gram-positive E. faecalis, P fimbriated uropathogenic E. coli No role 22,39 Clinical ND Harmful if mutated (UTI, ABU) 41,42 IRI, mouse Sepsis-induced (CLP), mouse Biglycan, HMGB1, Gp96 and histones ND Kidney transplant Transplantation, mouse HMGB1, biglycan, HSP70, HSP60, Gp94 and fibrinogen Protective if mutated (pyelonephritis and acute lobar nephronia) Harmful 53,57,59,60,90 Harmful 130 Glomerulonephritis Lupus nepritis, mouse ND Harmful 158,161,167 CKD TLR3 Diabetic nephropathy, mouse (STZ-induced) HMGB1 Harmful 111,112 Adenine overload, mouse 2,8-Dihydroxyadenine Harmful 127 UUO, mouse HMGB1, biglycan and Gp96 Initiates inflammation, no role in fibrosis 102,104 Acute rejection Clinical ND Harmful if mutated 75,82 Glomerulonephritis Lupus nephritis, mouse dsrna, poly(i:c) RNA Harmful TLR4 UTI Infection, in mouse with uropathogenic E. coli Gram-negative bacteria, uropathogenic E. coli fimbriae and LPS Protective 22 25,27,28,30 UTI or ABU Clinical ND Protective (UTI, ABU) 34,35 Harmful if mutated (UTI) 35,37 34,35,37 AKI IRI, mouse and human HMGB1, histones Harmful 54,56,57,61,71 Sepsis-induced (CLP), mouse Systemic LPS-induced, mouse Toxin-mediated (cisplatin), mouse Kidney transplant Transplantation, mouse HMGB1, biglycan, HSP70, HSP60, Gp94, fibrinogen Glomerulonephritis CKD TLR5 UTI ND Harmful 90 LPS Harmful 91 ND Harmful 96 Harmful 130 Clinical ND Protective if mutated Lupus nephritis/anti-gbm/ pristane-gn, mouse Diabetic nephropathy, mouse (STZ-induced) and human LPS Harmful , 166,169 HMGB1, S100A8 Harmful Adenine overload, mouse 2,8-Dihydroxyadenine Harmful 127 UUO, mouse ND Promotes renal fibrosis 100,103 Infection, mouse with uropathogenic E. coli Uropathogenic E. coli, flaggelin Protective 45 Clinical ND Harmful if mutated 38 AKI IRI, mouse ND Protective 65 TLR 7 Glomerulonephritis Lupus nephritis, mouse snrnp Harmful 137, 144, 145 NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 5

6 Table 2 (cont.) TLRs and NLRs involved in renal infection and injury Renal disorder Study type Possible ligands Renal effect Reference(s) TLR9 CKD UUO, mouse ND No role 104 AKI Glomerulonephritis TLR11 UTI NOD1/2 Sepsis-induced (CLP), mouse Lupus nephritis/ apoferritin-gn/igan/ Pristane-GN, mouse Infection, mouse with uropathogenic E. coli ND Harmful 92 dsdna, chromatin Lupus nephritis: harmful, 133 protective 136 Apoferritin-GN/IgAN/ Pristane-GN: harmful Uropathogenic E. coli Protective 46 AKI IRI, mouse ND Harmful 67 NOD2 Progressive renal injury NLRP3 Systemic LPS/PGNinduced, mouse Diabetic nephropathy, mouse (HFD/STZ-induced) and human LPS/peptidoglycan Harmful 95 ND Harmful 117 AKI IRI, mouse Biglycan, hyaluronic acid Harmful 68 Glomerulonephritis Oxalate, mouse Calcium oxalate Harmful 123 Heterologous anti-gbm, mouse ND No role 186 CKD UUO, mouse ND Protective in early phase, harmful in late phase Fructose- and MetSyn-driven nephropathy Oxalate, mouse Calcium oxalate Harmful 124 Adenine overload, mouse 2,8-Dihydroxyadenine Harmful 127 Diet, mouse and rat (fed with fructose or western-type diet) 133, 136, 141, 145, 159, 160, 162, ,105 ND Harmful 119,120 Abbreviations: ABU, asymptomatic bacteriuria; AKI, acute kidney injury; anti-gbm, antibodies against glomerular basement membrane; CKD, chronic kidney disease; CLP, caecal ligation puncture; ds, double-stranded; HFD, high fat diet; IgAN, IgA nephropathy; IRI, ischaemia reperfusion injury; LPS, lipopolysaccharide; MetSyn, metabolic syndrome; ND, not determined; poly (I:C), polyinosinic:polycytidylic acid; snrnp, small nuclear ribonucleoproteins; STZ, streptozotocin; UTI, urinary tract infection; UUO, unilateral urethral obstruction. to UTIs because they do not have the full-length TLR11 protein. 46 In mice, Tlr11 mrna is highly expressed in urothelium and tubular epithelial cells, and its protein specifically recognizes uropathogenic E. coli. 46 The rates of bladder infections with uropathogenic E. coli were equal in Tlr11 knockout and wild-type mice, whereas the rate of kidney infections was notably higher in knockout mice, and substantial leukocyte infiltration was noted. 46 Thus, in humans, genetic variability in TLRs might lead to differences in susceptibility to UTIs and where the infection is localized. In general, experimental and human studies demonstrate that TLRs provide protection against UTIs, but the roles of individual TLR family members can differ between humans and mice. Studies of SNPs in patients provide an excellent opportunity to investigate the role of TLRs in the pathogenesis of human UTIs, but the analysis of these polymorphisms is probably only marginally useful for clinical practice as it can only distinguish between patients who are more or less susceptible to acquiring a UTI rather than an absolute risk. Acute kidney injury Acute kidney injury (AKI) often occurs owing to tubular apoptosis or necrosis, and is generally induced by ischaemia reperfusion injury, 47 sepsis 48 or toxins. 49 TLRs and NLRs are constitutively expressed in many parts of the kidney (Table 1). They initiate renal injury and mediate disproportional inflammatory responses, which can be detrimental and might worsen kidney injury (Table 2). 14,50 52 Renal ischaemia reperfusion injury Renal ischaemia is a major cause of AKI and can lead to delayed allograft function or acute rejection. Potential DAMPs induced by experimental ischaemia reperfusion injury include, amongst others, HMGB1, histones, heat shock proteins, hyaluronan and biglycan (Table 2). In hypoxic tubular epithelial cells, stimulation of the ERK pathway by TLR2 is regulated by heat shock protein Gp96 (also known as endoplasmin). 53 Blockade of HMGB1 before and after renal ischaemia reperfusion injury improves tubular damage, whereas administration of 6 ADVANCE ONLINE PUBLICATION

7 Pyelonephritis Sepsisinduced AKI Toxin-mediated AKI Renal ischaemia reperfusion injury Renal-cell-associated/leukocyte-associated TLR/NLR/inflammasome APC TEC EC Renal transplantation Immune complex glomerulonephritis Diet-driven and metabolic-syndromedriven nephropathy Diabetic nephropathy CKD Crystal-related nephropathies Figure 2 Pathophysiological role of TLRs, NLRs and the NLRP3 inflammasome in various renal disorders. Abbreviations: AKI, acute kidney disease; APC, antigenpresenting cell; CKD, chronic kidney disease; EC, endothelial cell; NLR, nucleotidebinding oligomerization domain receptor; NLRP3, NACHT, LRR and PYD domains-containing protein 3; TEC, tubular epithelial cell; TLR, toll-like receptor. recombinant HMGB1 exacerbates injury. 5,55 Importantly, neither approach altered the risk of ischaemia reperfusion injury in Tlr4 knockout mice, which supports the HMGB1 TLR4 pathway as having a critical role in this process Similarly, direct injection of histones induced structural features of AKI dependent on TLR2 and TLR4 signalling, and injection of antihistone IgG diminished renal inflammation, dysfunction and tubular necrosis after ischaemia reperfusion injury. 57 Kidney injury could also be associated with increased intestinal permeability and translocation of intestinal PAMPs that, in turn, increase susceptibility of the kidneys to injury via TLR activation, as described in a model of liver cirrhosis. 58 No evidence was found, however, of bac ter ial translocation in a model of bilateral renal ischaemia reperfusion injury, and intestinal permeability was not affected (I. Stroo, personal communication). Wolfs and colleagues 59 demonstrated that Tlr2 and Tlr4 mrna is constitutively expressed in tubules and that expression is increased after renal ischaemia reperfusion injury in mice (Table 1). Moreover, murine tubular epithelial cells require functional TLR2 or TLR4 to coordinate an immune response to ischaemic injury. 60,61 Chimeric mice deficient for renal Tlr2 or Tlr4 display less tubular damage and renal dysfunction than wild-type mice because of impaired leukocyte influx in the former. 56,60 The therapeutic potential of TLR2 antisense therapy for renal ischaemia reperfusion injury was evaluated because these nucleotides are mainly taken up by tubular epithelial cells. 60 Treatment protected mice against renal dysfunction and renal damage after ischaemia reperfusion injury, presumably by blockade of neutrophil influx. 60 TLR4 expression on endothelial cells is also increased early after renal reperfusion, where it might regulate expression of adhesion molecules. 62 Comparison of single Tlr2 or Tlr4 knockout mice and mice with double Tlr2 and Tlr4 knockout showed no difference in protection. 63 Likewise, no significant differences were found in renal dysfunction and inflammation after ischaemia reperfusion injury between mice with or without the downstream signalling molecules Myd88 and Trif. 61,64 This lack of difference might reflect adaptive mechanisms in knockout animals or an ability of TLRs to signal via uncharacterized pathways. In contrast to the overall proinflammatory properties of TLR2 and TLR4, activation of TLR5 by a pharmacological agonist before and after renal ischaemia reperfusion injury confers a notable degree of protection against neutrophil infiltration, renal dysfunction and tissue injury, although the exact mechanism remains unclear. 65 Deletion of Tlr9 has no effect on the development of renal ischaemia reperfusion injury. 66 Nod1 and Nod2 double-knockout or Nod2 singleknockout mice had increased functional protection against renal ischaemia reperfusion injury compared with wild-type and Nod1 single-knockout mice. 67 This functional protection correlated with decreased apoptosis of tubular epithelial cells and inflammation. 67 Iyer et al. 68 compared wild-type and Nlrp3 knockout mice and found that NLRP3 triggers an acute and detrimental inflammatory response to acute tubular necrotic cell death after renal ischaemia reperfusion injury. The absence of Nlrp3 was associated with marked conservation of renal function and protected mice against death after lethal renal ischaemia. 68 Studies by Shigeoka and colleagues 69 and Kim and colleagues 70 confirmed these findings. In contrast to Nlrp3 knockout mice, those deficient for Asc, (also known as Pycard) were not protected against harmful effects 1 day after renal ischaemia reperfusion injury. 67,68 On day 5, however, while analysing long-term survival, Asc knockout mice were found to have partial protection against renal injury. 68 Conflicting evidence has been reported for the effects of caspase 1 in protection against renal ischaemia reperfusion injury. 69,71 Together, these data suggest that NLRP3-mediated protection is at least partly independent of the NLRP3 inflammasome. In vitro data indicate that NLRP3 is activated in macrophages by ATP released from mitochondria of necrotic cells and is primed by biglycan and hyaluronic acid, which are both upregulated in kidneys after renal ischaemia reperfusion injury. 68 Renal transplantation TLR stimulation can, in theory, directly and/or indirectly affect the alloimmune response. 72 In line with the data obtained in animals models of renal ischaemia reperfusion injury, 55,59 when compared with living-donor kidneys, tubular HMGB1, TLR4 and MyD88 expression was upregulated in deceased-donor kidneys, 73 and fewer recipients of kidneys carrying a loss-of-function TLR4 mutation had delayed graft function than those without such mutations. 74 Polymorphisms in TLR3 have also been associated with the occurrence of delayed renal graft function. 75 The direct effect of DAMP-transmitted TLR signals on recipient immune cells can amplify the response. In a mouse model of renal transplantation, Myd88 knockout NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 7

8 REVIEWS Therapy Controlled TLR/NLR/inflammasome-mediated renal inflammatory response Maladaptive TLR/NLR/inflammasome-mediated renal inflammatory response Normal kidney Diseased kidney Host defence and tissue repair Pathogen clearance Tissue debris clearance Tissue repair programme Pathogen dissemination and collateral tissue damage Tubular necrosis Renal fibrosis Glomerulosclerosis Figure 3 TLRs, NLRs and the NLRP3 inflammasome drive host defence and tissue repair mechanisms by the clearance of pathogens and cell debris and by regulation of repair pathways. When inflammatory pathways are maladaptive, ascending or blood-borne pathogens can disseminate to the kidney or surrounding renal tissue might be damaged. The balance between renal host defence, tissue repair and prevention of collateral kidney damage must, therefore, be maintained when blocking inflammation mediated by TLRs, NLRs and the inflammasome to prevent or treat kidney diseases. Abbreviations: NLR, nucleotide-binding oligomerization domain receptor; NLRP3, NACHT, LRR and PYD domains-containing protein 3; TLR, toll-like receptor. mice developed donor antigen-specific tolerance, which protected them from acute and chronic rejection via the induction of regulatory T cells.76 In a series of 36 human renal biopsy samples from patients with acute rejection, levels of mrna were raised for all TLR genes except TLR6 and TLR10, and levels of TOLLIP (a negative regulator of Toll-like receptor signalling) mrna were reduced compared with those in biopsy samples from patients without acute rejection. This finding suggests a proinflammatory state (Table 1).77 Several studies have described significantly reduced acute rejection episodes and better survival in patients with TLR4 loss-of-function polymorphisms78,79 or in patients receiving grafts from donors with TLR4 lossof-function polymorphisms,80 but others have shown no relationship.81 Polymorphisms in TLR3 in recipients have also been associated with severe acute rejection,75,82 but no notable effects of TLR2, TLR3, TLR4, TLR5 and TLR9 polymorphism in recipients on graft survival or renal function have been observed.75 Polymorphisms in the TLR4 gene in the recipient have been associated with increased risks of cytomegalo virus, 83 bacterial and opportunistic infections. 78 By contrast, Krüger and colleagues 72 observed no significant effect of TLR2, TLR3, TLR4, TLR5 and TLR9 polymorphism on the incidence of any infection.75 Together, the data suggest double-edged consequences of TLR polymorphism: benefits for renal allografts but harmful effects in terms of infections. Conflicting results for SNPs might reflect differences between ethnicities and/or insufficiently powered studies. Large prospective studies are required to define the clinical consequences of genetic vari ations and to explore the potential benefits of genotyping markers relevant to renal transplantation. The contribution of NLRs in the outcome of renal transplantation remains to be studied. Sepsis-induced AKI Septic AKI is common in critical care units and associated with a high risk of mortality. The role of TLRs in sepsis-induced renal failure is related to recognition of exogenous PAMPs and local DAMPs that are produced during (ischaemic) tissue injury. Hence, critically ill patients have high plasma levels of self-dna,84 mitochondrial DNA85 and HMGB1.86 In septic animals, TLR4 expression is increased in renal tubules.87,88 In a model of polymicrobial sepsis induced by caecal puncture, wildtype but not MyD88 knockout mice developed AKI,89 whereas Tlr2 or Tlr4 single-knockout mice were partially protected.90 Renal protection in MyD88 knockout mice might be explained by reduced levels of tumour necrosis factor (TNF) in serum; increased levels of TNF activate 8 ADVANCE ONLINE PUBLICATION

9 renal TNFR1 (tumour necrosis factor receptor 1), which can lead to acute renal failure. 91 Considering that all TLRs, except TLR3, signal through MyD88, other TLRs would also be expected to be involved in sepsis-induced AKI. The role of local versus extrarenal TLR4 signalling in acute renal failure was investigated by Cunningham et al., 91 who performed renal cross-transplantation between wild-type and Tlr4 knockout mice injected with lipopolysaccharide. Two main conclusions were drawn from these experiments. First, systemic TLR4 has an important role in lipopolysaccharide models of sepsis-induced AKI because it led to the release of TNF into the bloodstream. Second, renal TLR4 is an important modulator of the renal response to sepsis, because it acts independently of systemic TLR4 to recognize lipopolysaccharide and leads to local TNF synthesis, followed by renal TNFR1-mediated acute renal failure. 91 TLR activation in sepsis-induced AKI is not only attributed to the recognition of extracellular PAMPs as chloroquine, an inhibitor of endosomal TLRs, is protective in sepsis-induced AKI. 92 The same protective phenotype was observed in Tlr9 knockout mice. 92 Participation of NLRs in renal injury in response to sepsis is not well described, although a few studies have reported the involvement of NOD2, NLRP3 and inflammasome components in sepsis. 89,90 Stroo and co-workers showed that, despite comparable systemic and metabolic responses, Nod1 and Nod2 double-knockout mice were partly protected against renal dysfunction and damage to tubular epithelial cells. 95 Toxin-mediated AKI The kidney is susceptible to toxin injury by haemodynamic alterations or direct kidney injury. One of the major side effects of the chemotherapy agent cisplatin is AKI. In bone-marrow chimeric mice, Zhang et al. 96 showed that renal parenchymal TLR4, rather than myeloid TLR4, mediated the nephrotoxic effects of cisplatin via activation of p38 MAPK pathway. By contrast, in vitro studies have shown that tubular adult renal stem or progenitor cells are protective against cisplatin toxic effects through the engagement of TLR2, which prevented apoptosis and increased proliferation of renal proximal tubular epithelial cells. 97,98 The latter findings support the concept that TLRs have important roles in tissue homeostasis and repair. Nevertheless, more research is needed to assess whether this phenomenon also takes place in vivo. Kim et al. 70 established that NLRP3 was not involved in cisplatin-induced AKI, despite having previously demonstrated that caspase 1 is a mediator of cisplatininduced AKI. 71,99 The contributions of other TLR and NLR family members in toxin-induced AKI remain to be elucidated. Chronic kidney disease Irrespective of the nature of the primary renal insult, renal fibrosis is the result of nephron loss and sustained tissue remodelling and, therefore, is associated with endstage renal disease. So far, data on AKI strongly support the role of TLRs and NLRs in signalling renal damage. This concept could also apply to tissue remodelling in CKD. The unilateral ureteral obstruction model is often used to decipher the mechanisms of renal fibrosis. Expression of NLRP3, several TLRs and DAMPs, such as Gp96, biglycan and HMGB1, increases in the kidney upon unilateral ureteral obstruction, whereas expression of SIGRR is downregulated (Table1). 104 Accordingly, notably increased TLR2 expression was found in kidneys of patients with obstructive hydronephrosis and severe forms of IgA nephro pathy. 102 In Tlr4 knockout mice, TLR4 attenuates tubular damage but mediates renal fibrosis upon unilateral ureteral obstruction 100,103 independently from the local inflammatory response. 100 TLR4 probably increases renal fibrinogenesis by altering the susceptibility of tubular epithelial cells towards TGF β signalling. 100 By contrast, the absence of TLR2, SIGRR, TLR9 or MYD88 does not affect the development of tubular atrophy and renal fibrosis. 102,104 Nlrp3 knockout mice developed enhanced early tubular damage and interstitial oedema after unilateral ureteral obstruction, compared with wild-type mice, whereas the rates of renal fibrosis and inflammation were similar. 101 Increased interstitial oedema in Nlrp3 knockout mice might be explained by increased tubular and vascular permeability owing to reduced expression of inter cellular junction components. The early protective role of NLRP3 in unilateral ureteral obstruction, however, is not extended or reversed in the late phase of disease. Indeed, Vilaysane and colleagues described less tubular injury, renal inflammation and fibrosis 14 days after unilateral ureteral obstruction in the absence of NLRP This phenotype was associated with the activation of the inflammasome-dependent pathway of NLRP Additionally, NLRP3 was shown to augment TGF β signalling in tubular epithelial cells indepen dently of the NLRP3 inflammasome. 106 Bonemarrow chi meras revealed that NLRP3 mediates the injurious and inflammatory processes in bone-marrowderived cells and parenchymal compartments in unilateral ureteral obstruction. Biglycan acts as a DAMP for some TLRs and NLRP3 in unilateral ureteral obstruction, by driving caspase 1-mediated maturation and secretion of IL 1β. 107 Finally, NLRP3 expression was increased in renal biopsy samples from patients with various renal diseases when compared with normal renal tissue, which eventually reflected increased inflammation in diseased kidneys. 105 Together, these results imply that activation of TLRs and NLRs in acute and chronic renal injury can lead to different outcomes. Diabetes and metabolic syndrome In patients with type 2 diabetes mellitus or metabolic syndrome, the development of CKD is an important complication. Renal disease secondary to these metabolic disorders has been associated with dysregulation of the innate immune response via TLRs and NLRs. TLRs in diabetic nephropathy In patients with type 2 diabetes mellitus and nephro pathy, TLR4 and HMGB1, but not TLR2, are upregulated in the NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 9

10 kidneys (Table 1), which correlates strongly with macrophage influx. This TLR response was specific to diabetic nephropathy and not secondary to proteinuria. 108 In unilaterally nephrectomized mice with streptozocininduced diabetes, TLR4 was upregulated in the tubuli and promoted tubulointerstitial inflammation and diabetic nephropathy. 109 Mice deficient for TLR4 had less renal inflammation, albuminuria and renal dysfunction independent of blood glucose levels. In mice only treated with streptozocin, TLR4 does not seem to be involved in early and mild changes of diabetic nephro pathy. 110 Only the combination of streptozocin-induced diabetes and hyperlipidaemia (achieved with a 45% fat diet) was associated with less-severe albuminuria and nephropathy in TLR4 knockout mice than in wild-type mice. 110 This protective effect was associated with reduced phosphorylation of interferon regulatory factor 3 (IRF3). Other than HMGB1, potential ligands that might be involved in worsening TLR-mediated diabetic nephro pathy are S100 A8 S100 A9 complexes, as S100 A8 is upregulated in glomerular macrophages in mice with diabetes and hyperlipidaemia and in patients with diabetes. 110 In contrast to the stable TLR2 expression in humans with type 2 diabetes mellitus, 108 expression is increased in tubular epithelial cells 111 and peritoneal and renal macrophages 112 in mice with streptozocin-induced diabetes. Importantly, TLR2 induced a proinflammatory response and incipient nephropathy in a mouse model of type 1 diabetes. 112 Renal hypertrophy (as assessed by the kidney weight to body weight ratio) and albuminuria were attenuated in streptozocin-treated Tlr2 knockout mice compared with controls; this attenuation was associated with inhibition of MyD88-dependent signalling, NF kb activity and proinflammatory mediators. 112 In vitro findings suggest that glucose is the trigger that leads to TLR4 and TLR2 upregulation on tubular epithelial cells, increased activation of these cells and secretion of HMGB1. 108,111 In addition, mouse mesangial cells and cultured podocytes increased expression of TLR4 and cytokines after exposure to, respectively, glucose and glucose combined with free fatty acids. 113,114 Knockdown of TLR4 by small-interfering RNA eliminated cytokine production. No potential TLR ligand was identified. A therapeutic approach in which signalling pathways in macrophages mediated by TLR4, TLR2 and TLR6 were inhibited by the immunomodulator GIT 27 led to suppressed diabetic nephropathy through metabolic and anti glo merulosclerotic mechanisms in mice with type 2 diabetes (db/db model). 114 In another study, inhibition of TLR4 with the synthetic antagonist CRX 526 was renoprotective in Nos3 knockout mice with diabetes and advanced nephropathy, and was accompanied by decreased parenchymal inflammation and decreased fibrosis. 109 NLRs in diabetic nephropathy In patients with type 2 diabetes-associated nephro pathy, NLRP3 expression is significantly increased in renal tubular epithelial cells, which correlates positively with urinary IL 1β and IL 18 levels. 115 Study of a human renal proximal tubular cell line showed a time-dependent and dose-dependent effect of glucose in NLRP3 inflammasome activation. Activation of the NLRP3 inflammasome by high levels of glucose and subsequent interstitial inflammation in patients with diabetic nephropathy might be mediated through ATP P2X4 signalling, which might represent a potential anti-inflammatory therapeutic target for delaying diabetic nephropathy. 115 Wang and colleagues 116 investigated a link between NLRP3 and diabetic nephropathy in a rat model where nephropathy with hyperuricaemia and dyslipidaemia was induced by streptozocin. They observed activation of the renal NLRP3 inflammasome and raised expression of IL 1β and IL 18 with a subsequent increase of renal injury. In streptozocin-treated rats, treatment with agents to reduce urate concentrations (allopurinol and quer cetin) was associated with reduced renal NLRP3 inflammasome activation, hyperuricaemia, renal dysfunction, dyslipid aemia and renal inflammation. 116 In a recent study, NOD2 was shown to be upregulated in both the human diabetic kidney and in kidneys from streptozocin-treated diabetic mice on a high-fat diet. 117 Moreover, in these mice, Nod2 promoted renal injury by exacerbating inflammation and podocyte insulin resistance. 117 Together, these data suggest that besides TLRs, NLRs can sense potential metabolic harm in diabetic states, which might translate into inflammation and subsequent diabetic nephropathy. TLRs and NLRs in metabolic syndrome In contrast to the well-studied role of TLRs in diabetic nephropathy, little is known about their functional roles in diet-driven renal disease. Only the combination of diabetes induced with streptozocin and a high-fat diet was associated with reduced severity of albuminuria and nephropathy in TLR4 knockout mice compared with wild-type mice. 110 More research has been done on NLRP3 in diet-driven kidney disease. In experimental hyperhomocysteinaemia induced by feeding mice with a folate-free diet, Zhang et al. 118 noted early activation of the NLRP3 inflammasome in glomeruli (that is, in podocytes). Inhibition of local ASC or caspase 1 was associated with reduced albuminuria, podocyte injury and glomerulo sclerosis at later stages of nephropathy. 118 Hu and co-workers reported that rats with fructosedriven nephropathy develop renal inflammation, lipid accumulation and NLRP3 inflammasome activation. 119 Given that fructose induces hyperuricaemia and dyslipidaemia and that uric acid and lipids are activators of the NLRP3 inflammasome, Hu et al. 119 used allopurinol, quercetin and rutin to indirectly study the role of the renal NLRP3 inflammasome. They found that fructoseinduced hyperuricaemia and dyslipidaemia were restored by treatment with these agents to lower urate concentrations. These findings are consistent with reduced renal expression of NLRP3, ASC, caspase 1, IL 1β, IL 18, IL 6 and TNF and decreased interstitial leukocyte influx. 119 To study the role of NLRP3 in fructose-driven nephropathy in a more direct way, Nlrp3 knockout mice were given fructose in water (15%). 120 NLRP3 was implicated in the induction of hyperuricaemia and renal macrophage 10 ADVANCE ONLINE PUBLICATION

11 influx independently from IL β, but did not influence albuminuria, renal steatosis or insulin resistance. 120 In the same study, the role of NLRP3 in renal damage associated with metabolic syndrome was investigated by feeding Nlrp3 knockout and wild-type mice a Western diet rich in cholesterol. 116 Renal Nlrp3 mrna levels were higher in wild-type mice with metabolic syndrome than in control mice without metabolic syndrome. Animals deficient for Nlrp3 were protected against hyperuric aemia, micro albuminuria and nephropathy (renal macro phage influx, fibrosis, steatosis and vacuolization of tubules), also independently of IL 1β. 120 Further results imply that NLRP3 drives diet-associated nephropathy by means of macro phage infiltration and cholesterol accumu lation, the latter owing to upregulated expression of SREBP 2 (sterol regulatory element- binding protein 2) and the low-density lipoprotein receptor. Possible NLRP3 activators in metabolic syndrome, such as uric acid and cholesterol crystals were not detected in kidneys, which could mean that other, as yet unidentified, DAMPs are responsible for NLRP3-dependent and diet-dependent nephropathy. In conclusion, TLR4 and NLRP3 seem to mediate and sustain renal inflammation and abnormal lipid metabolism in response to excessive nutrient intake, which can result in nephropathy. Crystal-related nephropathies Secretion of IL 1β driven by the NLRP3 inflammasome is recognized to be the essential pathophysiological element of crystal-induced and particle-induced sterile inflammation 121 and, therefore, is likely to also apply to crystal-related nephropathies. 14,20,122 The uptake of calcium oxalate crystals by dendritic cells of the inter stitial compartment has been demonstrated in experimental studies. 123 This process is associated with potassium efflux from the cell and activation of the NLRP3 inflammasome to mediate release of IL 1β, 123 which ligates with IL 1 receptors on all sorts of renal cells. The result is induction of renal inflammation that accounts largely for crystal-induced AKI. 123 Nlrp3 knockout mice are protected from CKD caused by calcium oxalate crystal deposition, 124 which is a condition that mimics kidney disease in primary hyperoxaluria and other hereditary crystallopathies. Whether this process is dependent on or independent of IL 1β is unclear. 125 As different types of crystals activate the NLRP3 inflammasome in an identical manner, this mechanism could contribute to all crystallopathies that are associated with diffuse crystallization and/or crystal translocation into the interstitial compartment, such as adenine or urate nephropathy and cystinosis. 126 Adenine overload induces 2,8-dihydroxyadenine crystal precipitation inside the kidney and is associated with tubular atrophy and renal fibrosis. Adenine-induced renal inflammation and CKD were largely abrogated in mice deficient for TLR2, TLR4, NLRP3 and CASP1, 127 which supports the role of these PRRs in chronic crystal nephropathies. Uromodulin (also known as Tamm Horsfall urinary glycoprotein) is a sticky multimeric protein that is exclusively secreted by the thick ascending limb of the distal tubule. It forms particles that can activate myeloid cells in a NLRP3-dependent manner. 128 Renal crystals are generally covered by uromodulin, and injury to distal tubules facilitates uromodulin leakage into the interstitium, where it activates interstitial dendritic cells via TLR4 and NLRP So far, uromodulin is the only known DAMP that is exclusively expressed in the kidney. Thus, NLRP3 signalling clearly contributes to crystal-related AKI and CKD, but whether the latter involves inflammasomedependent or inflammasome-independent NLRP3 signalling remains unclear. Chronic allograft dysfunction Chronic allograft dysfunction is the major cause of organ loss in transplant recipients and is characterized by interstitial fibrosis, tubular atrophy and arterio pathy. In a murine model of renal transplantation, Wang and colleagues 130 studied the effect of transplanting wildtype grafts into wild-type, Tlr2 single-knockout, Tlr4 single-knockout, Tlr2 and Tlr4 double-knockout, Myd88 knockout or Trif knockout mice. In all recipients, expression of HMGB1, biglycan and HSP70 was observed within 7 days of transplantation and persisted for up to 6 weeks. Moreover, the magnitude of DAMP expression in allografts was higher than in isografts, and correlated with increased inflammation and decreased renal allograft function. 130 The rate of chronic allograft injury was significantly lower in all knockout recipients than in wild-type recipients. These data document a role for TLRs in alloimmunity against renal allografts, but do not necessarily indicate a role for TLR signalling in the donor kidney. Another major cause of chronic renal allograft dysfunction is toxic effects from calcineurin inhibitors. In a model of chronic cyclosporin-induced toxic effects in rats, Lim et al. 131 observed upregulation of TLR2 and TLR4 in renal tubules and of the putative ligand HSP70, but their potential roles in the development of cyclosporin-related toxic effects was not evaluated. Evidence for TLR and DAMP signalling to mediate chronic transplant injury in humans is only associative. A significant increase in TLR4 and MyD88 transcripts was observed in monocytes from patients with chronic allograft rejection compared with that in tolerant patients. TLR4 transcripts were also significantly higher in renal biopsy samples from patients with chronic rejection than in samples from recipients with histologically normal grafts. 132 Immune complex glomerulonephritis Robust data indicate that expression of nucleic-acidspecific TLRs has a functional role in lupus nephritis In patients with lupus, endogenous nucleic acids are important autoantigens and autoadjuvants that ligate TLR7 or TLR9 inside dendritic cells, macro phages and B cells. 134,138,139 Immune complexes that contain immuno stimulatory nucleic acids can be internalized into endosomal compartments via Fc domain Fc receptor interactions in mononuclear phagocytes and via B cell-receptor interactions in B cells. 134 Extracellular NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 11