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Lipid accumulation is associated with various forms of acute renal injury; however, the causative factors and pathways underpinning this lipid accumulation have not been thoroughly investigated. In this study, we performed lipidomic profiling of renal tissue following ischaemia–reperfusion injury (IRI). We identified a significant accumulation of cholesterol and specific phospholipids and sphingolipids in kidneys 24 h after IRI. In light of these findings, we hypothesised that pathways involved in lipid metabolism may also be altered. Through the analysis of published microarray data, generated from sham and ischaemic kidneys, we identified nephron‐specific metabolic pathways affected by IRI and validated these findings in ischaemic renal tissue. In silico analysis revealed the downregulation of several energy and lipid metabolism pathways, including mitochondrial fatty acid beta‐oxidation (FAO), peroxisomal lipid metabolism, fatty acid (FA) metabolism, and glycolysis. The pentose phosphate pathway (PPP), which is fuelled by glycolysis, was the only metabolic pathway that was upregulated 24 h following IRI. In this study, we describe the effect of renal IRI on metabolic pathways and how this contributes to lipid accumulation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Item does not contain fulltext Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury. To further test this, wild-type and S100A9 knockout mice (deficient for S100A8/A9 complex) were subjected to renal I/R. The expression of S100A8/A9 was significantly increased 1 day after I/R and was co-localized with Ly6G (mouse neutrophil marker)-positive cells. These knockout mice displayed similar renal dysfunction and damage and neutrophil influx compared with wild-type mice at this early time point. Interestingly, S100A9 knockout mice displayed altered tissue repair 5 and 10 days post I/R, as reflected by increased renal damage, sustained inflammation, induction of fibrosis, and increased expression of collagens. This coincided with enhanced expression of alternatively activated macrophage (M2) markers, while the expression of classically activated macrophage (M1) markers was comparable. Similarly, S100A9 deficiency affected M2, but not M1 macrophage polarization in vitro. During the repair phase following acute kidney injury, S100A9 deficiency affects M2 macrophages in mice leading to renal fibrosis and damage. Thus, S100A8/A9 plays a crucial part in controlling macrophage-mediated renal repair following I/R.

Renal ischemia reperfusion (IR)-injury induces activation of innate immune response which sustains renal injury and contributes to the development of delayed graft function (DGF). Triggering receptor expressed on myeloid cells-1 (TREM-1) is a pro-inflammatory evolutionary conserved pattern recognition receptor expressed on a variety of innate immune cells. TREM-1 expression increases following acute and chronic renal injury. However, the function of TREM-1 in renal IR is still unclear. Here, we investigated expression and function of TREM-1 in a murine model of renal IR using different TREM-1 inhibitors: LP17, LR12 and TREM-1 fusion protein. In a human study, we analyzed the association of non-synonymous single nucleotide variants in the TREM1 gene in a cohort comprising 1263 matching donors and recipients with post-transplant outcomes, including DGF. Our findings demonstrated that, following murine IR, renal TREM-1 expression increased due to the influx of Trem1 mRNA expressing cells detected by in situ hybridization. However, TREM-1 interventions by means of LP17, LR12 and TREM-1 fusion protein did not ameliorate IR-induced injury. In the human renal transplant cohort, donor and recipient TREM1 gene variant p.Thr25Ser was not associated with DGF, nor with biopsy-proven rejection or death-censored graft failure. We conclude that TREM-1 does not play a major role during experimental renal IR and after kidney transplantation.

Background: Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (E. coli) are one of the most prominent infections that have serious impact on kidney functioning and the development of chronic kidney disease. NOD-like receptor (NLR)X1 is an innate immune receptor that is important for immune metabolism and regulation, with as yet an unknown role in UTI and the pathophysiology of pyelonephritis. Methods: Wild-type (WT) and NLRX1 Knock-out (KO) female mice were subjected to UTI by intravesically inoculation of uropathogenic E. coli and sacrificed at 24h and 48h after infection after which bacterial burden and the inflammatory response in the bladder and kidney were studied. Ex vivo we studied the role of NLRX1 during the LPS induced pro-inflammatory cytokine response and phagocytosis of E. coli by granulocytes and monocytes. Results: Here, we report that during early experimental UTI NLRX1 absence reduces bacterial clearance in the bladder and dampens the inflammatory cytokine response, whereas in the kidney NLRX1 does not affect bacterial burden or cytokine response. In addition, we found that NLRX1 is not essential for the pro-inflammatory cytokine secretion by granulocytes and monocytes in response to LPS nor for bacterial phagocytosis. Conclusion: Together, we report that NLRX1 is important in enhancing the early host defense against uropathogenic E. coli in the bladder but does not affect the development of pyelonephritis.

NLRX1 is a mitochondrial innate immune receptor involved in viral immunity. Stokman et al. found that loss of NLRX1 increased cellular mitochondrial activity, production of reactive oxygen species, and apoptosis during oxidative stress in kidney injury., Mitochondrial dysfunction is the most prominent source of oxidative stress in acute and chronic kidney disease. NLRX1 is a receptor of the innate immune system that is ubiquitously expressed and localized in mitochondria. We investigated whether NLRX1 may act at the interface of metabolism and innate immunity in a model of oxidative stress. Using a chimeric mouse model for renal ischemia-reperfusion injury, we found that NLRX1 protects against mortality, mitochondrial damage, and epithelial cell apoptosis in an oxidative stress–dependent fashion. We found that NLRX1 regulates oxidative phosphorylation and cell integrity, whereas loss of NLRX1 results in increased oxygen consumption, oxidative stress, and subsequently apoptosis in epithelial cells during ischemia-reperfusion injury. In line, we found that NLRX1 expression in human kidneys decreased during acute renal ischemic injury and acute cellular rejection. Although first implicated in immune regulation, we propose that NLRX1 function extends to the control of mitochondrial activity and prevention of oxidative stress and apoptosis in tissue injury.

Item does not contain fulltext An accumulating body of evidence shows that gut microbiota fulfill an important role in health and disease by modulating local and systemic immunity. The importance of the microbiome in the development of kidney disease, however, is largely unknown. To study this concept, we depleted gut microbiota with broad-spectrum antibiotics and performed renal ischemia-reperfusion (I/R) injury in mice. Depletion of the microbiota significantly attenuated renal damage, dysfunction, and remote organ injury and maintained tubular integrity after renal I/R injury. Gut flora-depleted mice expressed lower levels of F4/80 and chemokine receptors CX3CR1 and CCR2 in the F4/80+ renal resident macrophage population and bone marrow (BM) monocytes than did control mice. Additionally, compared with control BM monocytes, BM monocytes from gut flora-depleted mice had decreased migratory capacity toward CX3CL1 and CCL2 ligands. To study whether these effects were driven by depletion of the microbiota, we performed fecal transplants in antibiotic-treated mice and found that transplant of fecal material from an untreated mouse abolished the protective effect of microbiota depletion upon renal I/R injury. In conclusion, we show that depletion of gut microbiota profoundly protects against renal I/R injury by reducing maturation status of F4/80+ renal resident macrophages and BM monocytes. Therefore, dampening the inflammatory response by targeting microbiota-derived mediators might be a promising therapy against I/R injury.

Contains fulltext : 109256.pdf (Publisher’s version ) (Open Access) The receptor for advanced glycation end products (RAGE) mediates a variety of inflammatory responses in renal diseases, but its role in renal ischemia/reperfusion (I/R) injury is unknown. We showed that during renal I/R, RAGE ligands HMGB1 and S100B are expressed. However, RAGE deficiency does not affect renal injury and function upon I/R-induced injury.

Background The chemokine stromal cell-derived factor-1 (SDF-1) is thought to be involved in mediating tissue repair by promoting migration of bone marrow stem or progenitor cells to the site of injury. Increased levels of renal SDF-1 are found after kidney injury. However, recently, we showed that SDF-1 does not play an important role in the migration of haematopoietic stem cells to the post-ischaemic kidney. The function of increased post-ischaemic renal SDF-1 expression in modulating renal ischaemia/reperfusion injury remains, therefore, unknown. Methods We studied the role of SDF-1 in renal ischaemia/reperfusion injury by locally decreasing SDF-1 expression and subsequent SDF-1 signalling in the corticomedullary region of the kidney using antisense oligonucleotide treatment in mice. Results Renal SDF-1 protein increased significantly in the early phase of ischaemia/reperfusion injury. Antisense treatment resulted in a reduction of corticomedullary SDF-1 expression which was accompanied by severely increased tubular injury and decreased renal function. We did not observe any difference in mobilization or retention of CXCR4-positive haematopoietic stem or progenitor cells after induction of renal ischaemia. Rather, antisense-treated animals showed markedly increased apoptosis of the tubular epithelium accompanied by an increased renal inflammatory response. Conclusions. These data indicate a new role for SDF-1 in renal pathogenesis by mediating tubular epithelial protection against ischaemic injury and suggest that SDF-1 by itself is not crucial for the influx of haematopoietic stem or progenitor cells towards the ischaemic injured kidney.

Dying cells are capable of activating the innate immune system and inducing a sterile inflammatory response. Here, we show that necrotic cells are sensed by the Nlrp3 inflammasome resulting in the subsequent release of the proinflammatory cytokine IL-1β. Necrotic cells produced by pressure disruption, hypoxic injury, or complement-mediated damage were capable of activating the Nlrp3 inflammasome. Nlrp3 inflammasome activation was triggered in part through ATP produced by mitochondria released from damaged cells. Neutrophilic influx into the peritoneum in response to necrotic cells in vivo was also markedly diminished in the absence of Nlrp3. Nlrp3-deficiency moreover protected animals against mortality, renal dysfunction, and neutrophil influx in an in vivo renal ischemic acute tubular necrosis model. These findings suggest that the inhibition of Nlrp3 inflammasome activity can diminish the acute inflammation and damage associated with tissue injury.

Background. Haematopoietic stem cells (HSC) have been shown to migrate to the ischemic kidney. The factors that regulate the trafficking of HSC to the ischemic damaged kidney are not fully understood. The stromal cell-derived factor-1 (SDF-1)/CXCR4-axis has been identified as the central signalling axis regulating trafficking of HSC to the bone marrow. Therefore, we hypothesized that SDF-1/CXCR4 interactions are implicated in the migration of HSC to the injured kidney. Methods. HSC were isolated from mouse bone marrow and labelled with a cell tracker. Acceptor mice were subjected to unilateral ischemia and received HSC intravenously directly after reperfusion. In addition, in separate groups of acceptor mice, endogenous SDF-1 or HSC-associated CXCR4 was blocked or kidneys were injected with SDF-1. Results. Exogenous HSC could be detected in the tubules and interstitium of the kidney 24 h after ischemic injury. Importantly, the amount of HSC in the ischemic kidney was markedly higher compared to the contralateral kidney. Neutralizing endogenous SDF-1 or HSC-associated CXCR4 did not prevent the migration of HSC. No increase in the number of labelled HSC could be observed after local administration of SDF-1, as was also determined in bilateral kidney ischemia. Conclusion. In conclusion, systemically administered HSC preferentially migrate to the ischemic injured kidney. This migration could not be prevented by blocking the SDF-1/CXCR4-axis or increased after local administration of SDF-1.

Acute kidney injury is often the result of ischemia reperfusion injury, which leads to activation of coagulation and inflammation, resulting in necrosis of renal tubular epithelial cells. Platelets play a central role in coagulation and inflammatory processes, and it has been shown that platelet activation exacerbates acute kidney injury. However, the mechanism of platelet activation during ischemia reperfusion injury and how platelet activation leads to tissue injury are largely unknown. Here we found that renal ischemia reperfusion injury in mice leads to increased platelet activation in immediate proximity of necrotic cell casts. Furthermore, platelet inhibition by clopidogrel decreased cell necrosis and inflammation, indicating a link between platelet activation and renal tissue damage. Necrotic tubular epithelial cells were found to release extracellular DNA, which, in turn, activated platelets, leading to platelet-granulocyte interaction and formation of neutrophil extracellular traps ex vivo . Renal ischemia reperfusion injury resulted in increased DNA-platelet and DNA-platelet-granulocyte colocalization in tissue and elevated levels of circulating extracellular DNA and platelet factor 4 in mice. After renal ischemia reperfusion injury, neutrophil extracellular traps were formed within renal tissue, which decreased when mice were treated with the platelet inhibitor clopidogrel. Thus, during renal ischemia reperfusion injury, necrotic cell–derived DNA leads to platelet activation, platelet-granulocyte interaction, and subsequent neutrophil extracellular trap formation, leading to renal inflammation and further increase in tissue injury.

BACKGROUND: The plasticity of bone marrow-derived stem cells, also comprising haematopoietic stem cells, has been shown to extend to renal epithelial lineages. Yet, the low rate of their contribution to the injured kidney has led to questions regarding their significance in tissue repair after acute injury. We describe here the effect of stem cell mobilization therapy on the progression of renal fibrosis in a mouse model of chronic obstructive nephropathy. METHODS: Mice were subjected to unilateral ureter obstruction (UUO) and treated with stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) or saline. Circulating cells were analysed by flow cytometry; labelled bone marrow c-KIT(HIGH) cells were injected into animals subjected to UUO. Granulocytes, macrophages, cellular proliferation or apoptosis and myofibroblasts were detected by immunostaining. Collagen deposition was determined by measuring renal hydroxyproline contents. Cytokine levels were measured by ELISA. RESULTS: SCF/G-CSF treatment of mice induced significant haematopoietic stem and progenitor cell mobilization from the bone marrow. Although these cells are able to migrate to the obstructed kidney, they did not influence renal damage, fibrosis and inflammatory cell influx. CONCLUSIONS: Although SCF/G-CSF treatment significantly enhanced the availability of haematopoietic stem cells to the obstructed kidney, the progression of renal fibrosis could not be delayed or halted. Our results indicate that effective stem cell mobilization does not alter renal fibrosis

SummaryAcute renal failure, a serious condition characterised by a drastic decline in renal function, often follows ischaemia/reperfusion (I/R) episodes. I/R is characterised by necrosis, inflammation and activation of coagulation, in concert causing renal tissue damage. In this context, activated protein C (APC) might be of importance in the pathogenesis of renal I/R. APC is a serine protease which has anticoagulant but also several anti-inflammatory and cytoprotective effects such as protection of endothelial barrier function. It was our objective to study the role of cytoprotective and anticoagulant functions of APC during renal I/R. C57BL/6j mice subjected to renal I/R were treated with intraperitoneally injected exogenous human APC, or two mutant forms of APC(200 µg/kg) which specifically lack anticoagulant or signalling properties. In a different experiment mice received specific monoclonal antibodies (20 mg/kg) that block the cytoprotective and/or anticoagulant properties of endogenous APC. Treatment with APC reduced tubular injury and enhanced renal function without altering the inflammatory response and did reduce renal fibrin deposition. Administration of APC mutant lacking anticoagulant properties reduced renal damage and enhanced renal function. Blocking the anticoagulant and cytoprotective functions of endogenous APC resulted in elevated tubular damage and reduced tubular cell proliferation, however, without influencing renal function or the inflammatory response. Furthermore, blocking both the anticoagulant and cytoprotective effects of APC resulted in dramatic renal interstitial haemorrhage, indicative of impaired vascular integrity. Blocking only the anticoagulant function of APC did not result in interstitial bleeding. In conclusion, the renoprotective effect of APC during I/R is independent of its anticoagulant properties.

TLRs are conserved pattern recognition receptors that detect motifs of pathogens and host material released during injury. For unknown reasons, renal TLR2 mRNA is mainly expressed by tubular cells and is enhanced upon renal ischemia/reperfusion (I/R) injury. We evaluated the role of TLR2 in I/R injury using TLR2(-/-) and TLR2(+/+) mice, TLR2 antisense oligonucleoddes, and chimeric mice deficient in leukocyte or renal TLR2. Tubular-cells needed TLR2 to produce significant cytokine and chemokine amounts upon ischemia in vitro. TLR2 played a proinflammatory and detrimental role in vivo after I/R injury, as reflected by a reduction in the amount of local cytokines and chemokines, leukocytes, and the level of renal injury and dysfucntion in TLR2(-/-) mice compared with controls. Analysis of chimeric mice suggested that TLR2 expressed on renal parenchyma plays a crucial role in the induction of inflammation and injury. TLR2-antisense treatment protected mice from renal dysfunction, neutrophil influx, and tubular apoptosis after I/R injury compared with nonsense treatment. In summary, we identified renal-associated TLR2 as an important initiator of inflammatory responses leading to renal injury and dysfunction in I/R injury. These data imply that TLR2 blockade could provide a basis for therapeutic strategies to treat or prevent renal ischemic injury

Systemic inflammatory response syndrome (SIRS) typically causes multiple-organ dysfunction/failure, including acute kidney injury (AKI). CD44 comprises a family of 85–200 kDa transmembrane glycoproteins that are widely expressed by multiple cell types and involved in a variety of inflammatory diseases. The multiple functions of CD44 have been attributed to the existence of numerous CD44 isoforms generated by alternative mRNA splicing as well as by extensive post-translational modifications. Renal CD44 expression is minimal in healthy adult kidneys, whereas in inflammatory renal disorders CD44 expression is markedly induced particularly in injured tubular epithelial cells (TEC) both in human diseases and in animal models. The function of CD44 on TEC remains unclear; previously we showed that the shortest isoform CD44 standard (CD44s) and the long CD44 variant 3-10 (CD44v3) exert opposite effects in fibrotic settings. To assess the contribution of tubular expression of CD44s and CD44v3 in SIRS-associated AKI, we used WT and unique transgenic mice expressing CD44s or CD44v3 specifically on proximal TEC. Mice were subjected to intraperitoneal injection of LPS and were sacrificed 4 and 24 hours later. The presence of CD44-isoforms in TEC did not alter the onset of kidney dysfunction or lymphocyte influx but affected the induction of KIM-1 and pro-/anti-inflammatory cytokines after LPS injection. Transgenic kidneys expressing CD44s/CD44v3 displayed more KIM-1 expression, less TNF-α and IL-1β at 4 hours compared to WT kidneys. At 24 hours, CD44v3-expressing kidneys showed elevated IL-10 and TLR4 negative regulator mRNA levels. Proximal TEC-CD44 influenced the renal inflammatory milieu and TEC-CD44v3 associated with expression of antiinflammatory molecules.

Temporal expression of chemokines is a crucial factor in the regulation of renal ischemia/reperfusion (I/R) injury and repair. Beside their role in the migration and activation of inflammatory cells to sites of injury, chemokines are also involved in other processes such as angiogenesis, development and migration of stem cells. In the present study we investigated the role of the chemokine MCP-1 (monocyte chemoattractant protein-1 or CCL2), the main chemoattractant for monocytes, during renal I/R injury. MCP-1 expression peaks several days after inducing renal I/R injury coinciding with macrophage accumulation. However, MCP-1 deficient mice had a significant decreased survival and increased renal damage within the first two days, i.e. the acute inflammatory response, after renal I/R injury with no evidence of altered macrophage accumulation. Kidneys and primary tubular epithelial cells from MCP-1 deficient mice showed increased apoptosis after ischemia. Taken together, MCP-1 protects the kidney during the acute inflammatory response following renal I/R injury.

Pyelonephritis, a common complication of urinary tract infections, is frequently associated with kidney scarring and may lead to end-stage renal disease. During bacterial infections inflammatory and coagulation pathways and their mutual interaction are playing pivotal roles in the host response. Given that thrombomodulin (TM) is crucially involved in the interplay between coagulation and inflammation, we aimed to investigate the roles of its EGF and lectin-like domains in inflammation during acute pyelonephritis. Indeed, the EGF-like and the lectin-like domains of TM, are especially known to orchestrate inflammation and coagulation in different ways. Acute pyelonephritis was induced by intravesical inoculation of 1 × 10(8) CFU of uropathogenic Escherichia coli in two strains of TM transgenic mice. TM(pro/pro) mice carry a mutation in the EGF-like domain making them unable to activate protein C, an anticoagulant and anti-inflammatory protein. TM(LeD/LeD) mice lack the lectin-like domain of TM, which is critical for its anti-inflammatory and cytoprotective properties. Mice were sacrificed 24 and 48 h after inoculation. Bacterial loads, the immune response and the activation of coagulation were evaluated in the kidney and the bladder. TM(LeD/LeD) mice showed elevated bacterial load in bladder and kidneys compared to WT mice, whereas TM(pro/pro) had similar bacterial load as WT mice. TM(LeD/LeD) mice displayed a reduced local production of pro-inflammatory cytokines and neutrophil renal infiltration. Activation of coagulation was comparable in TM(LeD/LeD) and WT mice. From these data, we conclude that the lectin-like domain of thrombomodulin is critically involved in host defence against E. coli induced acute pyelonephritis.

Chronic kidney diseases (CKDs) are characterized by tubular atrophy and interstitial fibrosis. We previously showed that in obstructive nephropathy de novo CD44 renal expression contributes to renal fibrosis but attenuates tubular damage/apoptosis. As CD44-standard (CD44s) has been linked to TGF-beta 1-mediated actions and CD44-variant-3 (CD44v3) favors HGF-c-Met binding, we compared the functional properties of these CD44 isoforms in the progression of obstructive nephropathy, using specific CD44-variant knockout/knockin mice. The presence of CD44v3 diminished tubular damage during obstructive nephropathy, decreased apoptosis, and increased proliferation of tubular epithelial cells, and prevented renal fibrosis development. In contrast, expression of CD44s led to increased tubular damage and tubular epithelial cell apoptosis, and more renal fibrosis. A relative increase in renal p-catenin expression, HGF production, and HGF/c-Met signaling, together with a relative inhibition of TGF-beta 1 downstream signaling and TGF-beta type I receptor expression, was found in CD44v3 mice compared with CD44s littermates. In line with this, Wnt3a/HGF treatment of tubular cells resulted in higher beta-catenin/p-AKT levels in CD44v3 tubular epithelial cells, whereas TGF-beta 1 induced a mild collagen I upregulation in CD44v3(+) mouse embryonic fibroblasts as compared with CD44s(+) cells. Thus, CD44s and CD44v3 exert opposite roles in the progression of obstructive nephropathy, with CD44v3-v10 being the protective isoform that delays evolution of the renal pathology.

Ischemia/reperfusion injury is a major cause of acute kidney injury. Improving renal repair would represent a therapeutic strategy to prevent renal dysfunction. The innate immune receptor Nlrp3 is involved in tissue injury, inflammation, and fibrosis; however, its role in repair after ischemia/reperfusion is unknown. We address the role of Nlrp3 in the repair phase of renal ischemia/reperfusion and investigate the relative contribution of leukocyte- versus renal-associated Nlrp3 by studying bone marrow chimeric mice. We found that Nlrp3 expression was most profound during the repair phase. Although Nlrp3 expression was primarily expressed by leukocytes, both leukocyte- and renal-associated Nlrp3 was detrimental to renal function after ischemia/reperfusion. The Nlrp3-dependent cytokine IL-1β remained unchanged in kidneys of all mice. Leukocyte-associated Nlrp3 negatively affected tubular apoptosis in mice that lacked Nlrp3 expression on leukocytes, which correlated with reduced macrophage influx. Nlrp3-deficient ( Nlrp3KO ) mice with wild-type bone marrow showed an improved repair response, as seen by a profound increase in proliferating tubular epithelium, which coincided with increased hepatocyte growth factor expression. In addition, Nlrp3KO tubular epithelial cells had an increased repair response in vitro , as seen by an increased ability of an epithelial monolayer to restore its structural integrity. In conclusion, Nlrp3 shows a tissue-specific role in which leukocyte-associated Nlrp3 is associated with tubular apoptosis, whereas renal-associated Nlrp3 impaired wound healing.

Metabolic syndrome (MetSyn) is a major health concern and associates with the development of kidney disease. The mechanisms linking MetSyn to renal disease have not been fully elucidated but are known to involve hyperuricemia, inflammation, and fibrosis. Since the innate immune receptor Nlrp3 is an important mediator of obesity and inflammation, we sought to determine whether Nlrp3 is involved in the development of MetSyn-associated nephropathy by giving wild-type or Nlrp3-knockout mice a Western-style compared to a normal diet or water without or with fructose. A plausible driver of pathology, the Nlrp3-dependent cytokine IL-1β was not increased in the kidney. Interestingly, Nlrp3-dependent renal cholesterol accumulation, another well-known driver of renal pathology, was enhanced during MetSyn. We also determined the role of Nlrp3 and fructose-fortified water on the development of MetSyn and kidney function since fructose is an important driver of obesity and kidney disease. Surprisingly, fructose did not induce MetSyn but, irrespective of this, did induce Nlrp3-dependent renal inflammation. The presence of Nlrp3 was crucial for the development of Western-style diet–induced renal pathology as reflected by the prevention of renal inflammation, fibrosis, steatosis, microalbuminuria, and hyperuricemia in the Nlrp3-knockout mice. Thus, Nlrp3 may mediate renal pathology in the context of diet-induced MetSyn.

Podocytes of the kidney adhere tightly to the underlying glomerular basement membrane (GBM) in order to maintain a functional filtration barrier. The clinical importance of podocyte binding to the GBM via an integrin-laminin-actin axis has been illustrated in models with altered function of alpha 3 beta 1 integrin, integrin-linked kinase, laminin-521, and alpha-actinin 4. Here we expanded on the podocyte-GBM binding model by showing that the main poclocyte adhesion receptor, integrin alpha 3 beta 1, interacts with the tetraspanin CD151 in situ in humans. Deletion of Cd151 in mouse glomerular epithelial cells led to reduced adhesive strength to laminin by redistributing alpha 3 beta 1 at the cell-matrix interface. Moreover, in vivo podocyte-specific deletion of Cd151 led to glomerular nephropathy. Although global Cd151-null B6 mice were not susceptible to renal disease, as has been shown previously, increasing blood and transcapillary filtration pressure induced nephropathy in these mice. Importantly, blocking the angiotensin-converting enzyme in renal disease-susceptible global Cd151-null FVB mice prolonged their median life span. Together, these results establish CD151 as a crucial modifier of integrin-mediated adhesion of podocytes to the GBM and show that blood pressure is an important factor in the initiation and progression of Cd151 knockout-induced nephropathy

Our immune system has to constantly strike a balance between activation and inhibition of an inflammatory response to combat invading pathogens and avoid inflammation-induced collateral tissue damage. Toll interleukin-1 receptor 8 (IL-1R-8)/single Ig domain IL-1R-related molecule (TIR8/SIGIRR) is an inhibitor of Toll-like receptor (TLR)/IL-1R signaling, which is predominantly expressed in the kidney. The biological role of renal TIR8 during infection is, however, unknown. We therefore evaluated renal TIR8 expression during Escherichia coli pyelonephritis and explored its role in host defense using TIR8 −/− versus TIR8 +/+ mice. We found that TIR8 protein is abundantly present in the majority of cortical tubular epithelial cells. Pyelonephritis resulted in a significant downregulation of TIR8 mRNA in kidneys of TIR8 +/+ mice. TIR8 inhibited an effective host response against E. coli , as indicated by diminished renal bacterial outgrowth and dysfunction in TIR8 −/− mice. This correlated with increased amounts of circulating and intrarenal neutrophils at the early phase of infection. TIR8 −/− tubular epithelial cells had increased cytokine/chemokine production when stimulated with lipopolysaccharide (LPS) or heat-killed E. coli , suggesting that TIR8 played an anti-inflammatory role during pathogen stimulation by inhibiting LPS signaling. These data suggest that TIR8 is an important negative regulator of an LPS-mediated inflammatory response in tubular epithelial cells and dampens an effective antibacterial host response during pyelonephritis caused by uropathogenic E. coli .

Background Renal ischemia leads to apoptosis of tubular epithelial cells and results in decreased renal function. Tissue repair involves re-epithelialization of the tubular basement membrane. Survival of the tubular epithelium following ischemia is therefore important in the successful regeneration of renal tissue. The cytokine stem cell factor (SCF) has been shown to protect the tubular epithelium against apoptosis. Methodology/Principal Findings In a mouse model for renal ischemia/reperfusion injury, we studied how expression of c-KIT on tubular epithelium and its ligand SCF protect cells against apoptosis. Administration of SCF specific antisense oligonucleotides significantly decreased specific staining of SCF following ischemia. Reduced SCF expression resulted in impaired renal function, increased tubular damage and increased tubular epithelial apoptosis, independent of inflammation. In an in vitro hypoxia model, stimulation of tubular epithelial cells with SCF activated survival signaling and decreased apoptosis. Conclusions/Significance Our data indicate an important role for c-KIT and SCF in mediating tubular epithelial cell survival via an autocrine pathway.

BACKGROUND: Inflammation is commonly followed by the release of endogenous proteins called danger associated molecular patterns (DAMPs) that are able to warn the host for eminent danger. S100A8/A9 subunits are DAMPs that belong to the S100 family of calcium binding proteins. S100A8/A9 complexes induce an inflammatory response and their expression correlates with disease severity in several inflammatory disorders. S100A8/A9 promote endotoxin- and Escherichia (E.) coli-induced sepsis showing its contribution in systemic infection. The role of S100A8/A9 during a local infection of the urinary tract system caused by E. coli remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the contribution of S100A8/A9 in acute urinary tract infection (UTI) by instilling 2 different doses of uropathogenic E. coli transurethrally in wild type (WT) and S100A9 knockout (KO) mice. Subsequently, we determined bacterial outgrowth, neutrophilic infiltrate and inflammatory mediators in bladder and kidney 24 and 48 hours later. UTI resulted in a substantial increase of S100A8/A9 protein in bladder and kidney tissue of WT mice. S100A9 KO mice displayed similar bacterial load in bladder or kidney homogenate compared to WT mice using 2 different doses at 2 different time points. S100A9 deficiency had little effect on the inflammatory responses to E. Coli-induced UTI infection, as assessed by myeloperoxidase activity in bladder and kidneys, histopathologic analysis, and renal and bladder cytokine concentrations. CONCLUSIONS: We show that despite high S100A8/A9 expression in bladder and kidney tissue upon UTI, S100A8/A9 does not contribute to an effective host response against E. Coli in the urinary tract system.

Chemokines are important players in the migration of leukocytes to sites of injury and are also involved in angiogenesis, development and wound healing. In this study, we performed microarray analyses to identify chemokines that play a role during the inflammatory and repair phase after renal ischemia/reperfusion (I/R) injury and investigated the temporal relationship between chemokine expression, leukocyte accumulation and renal damage/repair. C57BI/6 mice were subjected to unilateral ischemia for 45 min and sacrificed 3 h, 1 day and 7 days after reperfusion. From ischemic and contralateral kidney, RNA was isolated and hybridized to a microarray. Microarray results were validated with quantitative real-time reverse transcription-PCR (QRT-PCR) on RNA from an independent experiment. (Immuno)histochemical analyses were performed to determine renal damage/repair and influx of leukocytes. Twenty out of 114 genes were up-regulated at one or more reperfusion periods. All these genes were up-regulated 7 days after I/R. Up-regulated genes included CC chemokines MCP-1 and TARC, CXC chemokines KC and MIP-2 alpha, chemokine receptors Ccr1 and Cx3cr1 and related genes like matrix metalloproteinases. Microarray data of 1 and 7 days were confirmed for 17 up-regulated genes by ORT-PCR. (Immuno)histochemical analysis showed that the inflammatory and repair phase after renal I/R injury take place after, respectively, 1 and 7 days. Interestingly, chemokine expression was highest during the repair phase. In addition, expression profiles showed a biphasic expression of all up-regulated CXC chemokines coinciding with the early inflammatory and late repair phase. In conclusion, we propose that temporal expression of chemokines is a crucial factor in the regulation of renal I/R injury and repair

Acute pyelonephritis, frequently caused by Escherichia coli, is a substantial health problem. Plasminogen activator inhibitor type-1 (PAI-1) not only inhibits plasminogen activation but is also involved in cell migration. To determine if it has a role in host defense, we induced pyelonephritis in PAI-1 gene knockout and wild-type mice by intravesical inoculation with uropathogenic E. coli 1677. Bacterial growth was determined on blood agar plates in portions of the kidneys homogenized in sterile saline. Kidney levels of PAI-1 were increased in infected compared to control mice, suggesting a physiological role for PAI-1 during pyelonephritis. The knockout mice had significantly more bacterial outgrowth in kidney homogenates compared to the wild-type mice. Strikingly, higher colony-forming units were accompanied by increased levels of the cytokines TNF-alpha, IL-1beta, and IL-6 in the kidneys of knockout mice, but levels of the chemokines KC and MIP-2 were not different. Remarkably, plasma levels of KC were higher, but renal neutrophil influx was significantly lower, in the knockout than in the wild-type mice. Our study shows that PAI-1 is critically involved in host defense against E. coli-induced acute pyelonephritis, in part, by modulating neutrophil influx.

The role that the enterococcal surface protein Esp plays in the capacity of Enterococcus faecium to adhere to uroepithelial cells and the role that it plays in urinary tract infection and peritonitis was investigated in vitro and in vivo, respectively, using Esp-expressing E. faecium (E1162) and its isogenic Esp-deficient mutant (E1162 Delta esp). Esp expression enhanced in vitro binding to bladder and kidney epithelial cells. In mice, higher numbers of E1162 were cultured from kidneys and bladders after the induction of urinary tract infection, compared with E1162 Delta esp numbers. This was accompanied by a higher frequency of bacteremia, higher cytokine levels in kidney tissue, and renal insufficiency. Esp had no effect on the course of E. faecium peritonitis.

Background Acute pyelonephritis is one of the most common bacterial infections. Tissue-type plasminogen activator (tPA) is a potent fibrinolytic agent, but can play a role in inflammatory processes as well. Methods We induced pyelonephritis in tPA(-/-) and C57BL/6 wild-type (WT) mice by intravesical inoculation with 10(10) CFU uropathogenic Escherichia coli 1677. The mice were killed after 24 and 48 h, after which bacterial outgrowth and cytokine levels in kidney homogenates were determined. Influx of neutrophils was quantified by myeloperoxidase-ELISA. Neutrophil phagocytosis and oxidative burst were measured. Results The tPA(-/-) kidneys contained significantly higher numbers of E. coli CFU, accompanied by higher levels of interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). The number of infiltrating neutrophils was similar in tPA(-/-) and WT mice at both time points, suggesting that tPA(-/-) neutrophils have a lower ability to eliminate E. coli. Phagocytosis of E. coli organisms was not diminished in tPA(-/-) neutrophils. Interestingly, tPA(-/-) neutrophils showed a significantly lower ability to generate an oxidative burst reaction upon stimulation with E. coli than WT neutrophils. Incubation with recombinant tPA reversed this effect completely. Conclusions These results show that deletion of the tPA-gene in mice leads to lower bactericidal potential of tPA(-/-) neutrophils, which results in significantly more bacterial outgrowth during experimental pyelonephritis.

Tissue fibrosis and chronic inflammation are common causes of progressive organ damage, including progressive renal disease, leading to loss of physiological functions. Recently, it was shown that Toll-like receptor 2 (TLR2) is expressed in the kidney and activated by endogenous danger signals. The expression and function of TLR2 during renal fibrosis and chronic inflammation has however not yet been elucidated. Therefore, we studied TLR2 expression in human and murine progressive renal diseases and explored its role by inducing obstructive nephropathy in TLR2(-/-) or TLR2(+/+) mice. We found that TLR2 is markedly upregulated on tubular and tubulointerstitial cells in patients with chronic renal injury. In mice with obstructive nephropathy, renal injury was associated with a marked upregulation and change in distribution of TLR2 and upregulation of murine TLR2 danger ligands Gp96, biglycan, and HMGB1. Notably, TLR2 enhanced inflammation as reflected by a significantly reduced influx of neutrophils and production of chemokines and TGF-beta in kidneys of TLR2(-/-) mice compared with TLR2(+/+) animals. Although, the obstructed kidneys of TLR2(-/-) mice had less interstitial myofibroblasts in the later phase of obstructive nephropathy, tubular injury and renal matrix accumulation was similar in both mouse strains. Together, these data demonstrate that TLR2 can initiate renal inflammation during progressive renal injury and that the absence of TLR2 does not affect the development of chronic renal injury and fibrosis.

Toll-like receptors (TLRs) can detect endogenous danger molecules released upon tissue injury resulting in the induction of a proinflammatory response. One of the TLR family members, TLR4, is constitutively expressed at RNA level on renal epithelium and this expression is enhanced upon renal ischemia/reperfusion (I/R) injury. The functional relevance of this organ-specific upregulation remains however unknown. We therefore investigated the specific role of TLR4 and the relative contribution of its two downstream signaling cascades, the MyD88-dependent and TRIF-dependent cascades in renal damage by using TLR4-/-, MyD88-/- and TRIF-mutant mice that were subjected to renal ischemia/reperfusion injury. Our results show that TLR4 initiates an exaggerated proinflammatory response upon I/R injury, as reflected by lower levels of chemokines and infiltrating granulocytes, less renal damage and a more preserved renal function in TLR4-/- 2 mice as compared to wild type mice. In vitro studies demonstrate that renal tubular epithelial cells can coordinate an immune response to ischemic injury in a TLR4-dependent manner. In vivo we found that epithelial- and leukocyte-associated functional TLR4 contribute in a similar proportion to renal dysfunction and injury as assessed by bone marrow chimeric mice. Surprisingly, no significant differences were found in renal function and inflammation in MyD88-/- and TRIF-mutant mice compared with their wild types, suggesting that selective targeting of TLR4 directly may be more effective for the development of therapeutic tools to prevent I/R injury than targeting the intracellular pathways used by TLR4. In conclusion, we identified TLR4 as a cellular sentinel for acute renal damage that subsequently controls the induction of an innate immune response

Objective EMR2 and CD97 are closely related members of the epidermal growth factor (EGF)–TM7 family of adhesion class 7-span transmembrane (TM7) receptors. Chondroitin sulfates (CS) have recently been identified as ligands for EMR2 and CD97. CS have been implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the expression of EMR2 and the distribution of EMR2 and CD97 ligands within RA synovial tissue (ST). Methods ST samples were obtained by arthroscopy from 19 patients with RA, 13 patients with inflammatory osteoarthritis (OA), and 13 patients with reactive arthritis (ReA). Immunohistochemistry was performed with a monoclonal antibody against EMR2, and stained STs were analyzed by digital image analysis. Coexpression of EMR2 with cell lineage– and activation-specific markers was determined by double immunofluorescence microscopy. To evaluate the expression of EMR2 and CD97 ligands in RA synovium, binding assays were performed using EMR2- and CD97-specific multivalent fluorescent probes. Results EMR2 expression in the synovial sublining was found to be significantly higher in RA patients compared with OA and ReA control patients. Most EMR2+ cells were macrophages and dendritic cells expressing costimulatory molecules and tumor necrosis factor α. Dermatan sulfate was shown to be the ligand of the largest isoforms of EMR2 and CD97 in rheumatoid synovium. In addition, the smaller isoforms of CD97, but not those of EMR2, bound CD55 on fibroblast-like synoviocytes. Conclusion The EGF-TM7 receptors EMR2 and CD97 are abundantly expressed on myeloid cells in ST of RA patients where their cognate ligands dermatan sulfate and CD55 are detected. These results suggest that these interactions may facilitate the retention of activated macrophages in the synovium.

Progressive renal disease is characterized by tubulo-interstitial injury with ongoing inflammation and fibrosis. The Nlrp3 inflammasome contributes to these pathophysiological processes through its canonical effects in cytokine maturation. Nlrp3 may additionally exert inflammasome-independent effects following tissue injury. Hence, in this study we investigated potential non-canonical effects of Nlrp3 following progressive renal injury by subjecting WT and Nlrp3-deficient (−/−) mice to unilateral ureter obstruction (UUO). Our results revealed a progressive increase of renal Nlrp3 mRNA in WT mice following UUO. The absence of Nlrp3 resulted in enhanced tubular injury and dilatation and an elevated expression of injury biomarker NGAL after UUO. Moreover, interstitial edema was significantly elevated in Nlrp3−/− mice. This could be explained by increased intratubular pressure and an enhanced tubular and vascular permeability. In accordance, renal vascular leakage was elevated in Nlrp3−/− mice that associated with reduced mRNA expression of intercellular junction components. The decreased epithelial barrier function in Nlrp3−/− mice was not associated with increased apoptosis and/or proliferation of renal epithelial cells. Nlrp3 deficiency did not affect renal fibrosis or inflammation. Together, our data reveal a novel non-canonical effect of Nlrp3 in preserving renal integrity and protection against early tubular injury and interstitial edema following progressive renal injury.