Atorvastatin Attenuates Diet-Induced Non-Alcoholic Steatohepatitis in APOE*3-Leiden Mice by Reducing Hepatic Inflammation.

Patients with metabolic syndrome are often prescribed statins to prevent the development of cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma cholesterol lowering (-30%) and anti-atherogenic effects (severe lesion size -48%), atorvastatin significantly reduced hepatic steatosis (-22%), the number of aggregated inflammatory cells in the liver (-80%) and hepatic fibrosis (-92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated mice showed less immunohistochemically stained areas of inflammation markers. Atorvastatin prevented accumulation of free cholesterol in the form of cholesterol crystals (-78%). Cholesterol crystals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its activation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1ß (-61%) and IL-18 (-26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crystal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in dyslipidemic patients.

Semaglutide Has Beneficial Effects on Non-Alcoholic Steatohepatitis in Ldlr-/-.Leiden Mice.

Semaglutide, a glucagon-like peptide-1 receptor agonist, is an antidiabetic medication that has recently been approved for the treatment of obesity as well. Semaglutide is postulated to be a promising candidate for the treatment of non-alcoholic steatohepatitis (NASH). Here, Ldlr-/-.Leiden mice received a fast-food diet (FFD) for 25 weeks, followed by another 12 weeks on FFD with daily subcutaneous injections of semaglutide or vehicle (control). Plasma parameters were evaluated, livers and hearts were examined, and hepatic transcriptome analysis was performed. In the liver, semaglutide significantly reduced macrovesicular steatosis (-74%, p < 0.001) and inflammation (-73%, p < 0.001) and completely abolished microvesicular steatosis (-100%, p < 0.001). Histological and biochemical assessment of hepatic fibrosis showed no significant effects of semaglutide. However, digital pathology revealed significant improvements in the degree of collagen fiber reticulation (-12%, p < 0.001). Semaglutide did not affect atherosclerosis relative to controls. Additionally, we compared the transcriptome profile of FFD-fed Ldlr-/-.Leiden mice with a human gene set that differentiates human NASH patients with severe fibrosis from those with mild fibrosis. In FFD-fed Ldlr-/-.Leiden control mice, this gene set was upregulated as well, while semaglutide predominantly reversed this gene expression. Using a translational model with advanced NASH, we demonstrated that semaglutide is a promising candidate with particular potential for the treatment of hepatic steatosis and inflammation, while for the reversal of advanced fibrosis, combinations with other NASH agents may be necessary.

A Novel, Orally Bioavailable, Small-Molecule Inhibitor of PCSK9 With Significant Cholesterol-Lowering Properties In Vivo.

Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibits the clearance of low-density lipoprotein (LDL) cholesterol (LDL-C) from plasma by directly binding with the LDL receptor (LDLR) and sending the receptor for lysosomal degradation. As the interaction promotes elevated plasma LDL-C levels, and therefore a predisposition to cardiovascular disease, PCSK9 has attracted intense interest as a therapeutic target. Despite this interest, an orally bioavailable small-molecule inhibitor of PCSK9 with extensive lipid-lowering activity is yet to enter the clinic. We report herein the discovery of NYX-PCSK9i, an orally bioavailable small-molecule inhibitor of PCSK9 with significant cholesterol-lowering activity in hyperlipidemic APOE∗3-Leiden.CETP mice. NYX-PCSK9i emerged from a medicinal chemistry campaign demonstrating potent disruption of the PCSK9-LDLR interaction in vitro and functional protection of the LDLR of human lymphocytes from PCSK9-directed degradation ex vivo. APOE∗3-Leiden.CETP mice orally treated with NYX-PCSK9i demonstrated a dose-dependent decrease in plasma total cholesterol of up to 57%, while its combination with atorvastatin additively suppressed plasma total cholesterol levels. Importantly, the majority of cholesterol lowering by NYX-PCSK9i was in non-HDL fractions. A concomitant increase in total plasma PCSK9 levels and significant increase in hepatic LDLR protein expression strongly indicated on-target function by NYX-PCSK9i. Determinations of hepatic lipid and fecal cholesterol content demonstrated depletion of liver cholesteryl esters and promotion of fecal cholesterol elimination with NYX-PCSK9i treatment. All measured in vivo biomarkers of health indicate that NYX-PCSK9i has a good safety profile. NYX-PCSK9i is a potential new therapy for hypercholesterolemia with the capacity to further enhance the lipid-lowering activities of statins.

Dual targeting of hepatic fibrosis and atherogenesis by icosabutate, an engineered eicosapentaenoic acid derivative.

BACKGROUND & AIMS: While fibrosis stage predicts liver-associated mortality, cardiovascular disease (CVD) is still the major overall cause of mortality in patients with NASH. Novel NASH drugs should thus ideally reduce both liver fibrosis and CVD. Icosabutate is a semi-synthetic, liver-targeted eicosapentaenoic acid (EPA) derivative in clinical development for NASH. The primary aims of the current studies were to establish both the anti-fibrotic and anti-atherogenic efficacy of icosabutate in conjunction with changes in lipotoxic and atherogenic lipids in liver and plasma respectively. METHODS: The effects of icosabutate on fibrosis progression and lipotoxicity were investigated in amylin liver NASH (AMLN) diet (high fat, cholesterol and fructose) fed ob/ob mice with biopsy-confirmed steatohepatitis and fibrosis and compared with the activity of obeticholic acid. APOE*3Leiden.CETP mice, a translational model for hyperlipidaemia and atherosclerosis, were used to evaluate the mechanisms underlying the lipid-lowering effect of icosabutate and its effect on atherosclerosis. RESULTS: In AMLN ob/ob mice, icosabutate significantly reduced hepatic fibrosis and myofibroblast content in association with downregulation of the arachidonic acid cascade and a reduction in both hepatic oxidised phospholipids and apoptosis. In APOE*3Leiden.CETP mice, icosabutate reduced plasma cholesterol and TAG levels via increased hepatic uptake, upregulated hepatic lipid metabolism and downregulated inflammation pathways, and effectively decreased atherosclerosis development. CONCLUSIONS: Icosabutate, a structurally engineered EPA derivative, effectively attenuates both hepatic fibrosis and atherogenesis and offers an attractive therapeutic approach to both liver- and CV-related morbidity and mortality in NASH patients.

Deletion of NLRX1 increases fatty acid metabolism and prevents diet-induced hepatic steatosis and metabolic syndrome.

NOD-like receptor (NLR)X1 (NLRX1) is an ubiquitously expressed inflammasome-independent NLR that is uniquely localized in mitochondria with as yet unknown effects on metabolic diseases. Here, we report that NLRX1 is essential in regulating cellular metabolism in non-immune parenchymal hepatocytes by decreasing mitochondrial fatty acid-dependent oxidative phosphorylation (OXPHOS) and promoting glycolysis. NLRX1 loss in mice has a profound impact on the prevention of diet-induced metabolic syndrome parameters, non-alcoholic fatty liver disease (NAFLD) progression, and renal dysfunction. Despite enhanced caloric intake, NLRX1 deletion in mice fed a western diet (WD) results in protection from liver steatosis, hepatic fibrosis, obesity, insulin resistance, glycosuria and kidney dysfunction parameters independent from inflammation. While mitochondrial content was equal, NLRX1 loss in hepatocytes leads to increased fatty acid oxidation and decreased steatosis. In contrast, glycolysis was decreased in NLRX1-deficient cells versus controls. Thus, although first implicated in immune regulation, we show that NLRX1 function extends to the control of hepatocyte energy metabolism via the restriction of mitochondrial fatty acid-dependent OXPHOS and enhancement of glycolysis. As such NLRX1 may be an attractive novel therapeutic target for NAFLD and metabolic syndrome.

Depletion of Gut Microbiota Protects against Renal Ischemia-Reperfusion Injury.

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.

Intragraft Blood Dendritic Cell Antigen-1-Positive Myeloid Dendritic Cells Increase during BK Polyomavirus-Associated Nephropathy.

Although both polyomavirus infection and T cell-mediated rejection (TCMR) are characterized by tubulointerstitial inflammation in the renal allograft, these conditions are treated with opposing therapeutic regimens. To gain more insight into the differences between antiviral and alloimmune responses, we performed a case-control study, in which we immunophenotyped the inflammatory infiltrates in renal biopsy specimens with BK polyomavirus-associated nephropathy (BKPyVAN) and specimens with TCMR. Compared with TCMR, BKPyVAN was diagnosed later after transplantation; therefore, BKPyVAN specimens showed more chronic damage than TCMR specimens showed. However, TCMR and BKPyVAN specimens had comparable levels of tubulointerstitial inflammation. Adjustment for confounders in various multivariable models revealed more blood dendritic cell antigen-1(+) (BDCA-1(+)) myeloid dendritic cells (mDCs) present during BKPyVAN (odds ratio, 2.31; 95% confidence interval, 1.03 to 5.16; P=0.04) than during TCMR. Double immunostaining for SV40 and BDCA-1 showed that, during BKPyVAN, BDCA-1(+) mDCs localized in proximity to the polyomavirus-infected tubular epithelial cells. We ensured that time of biopsy after transplantation was not a confounding factor by including additional specimens with late TCMR and protocol biopsy specimens matched for biopsy time. These additional specimens showed amounts of BDCA-1(+) mDCs comparable with amounts in the early TCMR specimens. These results suggest that BDCA-1(+) mDCs, known to be involved in the antiviral immune response during various viral infections, might have a pivotal role during BKPyVAN infection in the grafted kidney.

Epac-Rap signaling reduces oxidative stress in the tubular epithelium.

Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-2'-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-2'-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate-induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-2'-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-2'-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress.

Bile acids trigger cholemic nephropathy in common bile-duct-ligated mice.

UNLABELLED: Tubular epithelial injury represents an underestimated but important cause of renal dysfunction in patients with cholestasis and advanced liver disease, but the underlying mechanisms are unclear. To address the hypothesis that accumulation and excessive alternative urinary elimination of potentially toxic bile acids (BAs) may contribute to kidney injury in cholestasis, we established a mouse model for detailed in vivo time course as well as treatment studies. Three-day common bile duct ligation (CBDL) induced renal tubular epithelial injury predominantly at the level of aquaporin 2-positive collecting ducts with tubular epithelial and basement membrane defects. This was followed by progressive interstitial nephritis and tubulointerstitial renal fibrosis in 3-, 6-, and 8-week CBDL mice. Farnesoid X receptor knockout mice (with a hydrophilic BA pool) were completely protected from CBDL-induced renal fibrosis. Prefeeding of hydrophilic norursodeoxycholic acid inhibited renal tubular epithelial injury in CBDL mice. In addition, we provide evidence for renal tubular injury in cholestatic patients with cholemic nephropathy. CONCLUSION: We characterized a novel in vivo model for cholemic nephropathy, which offers new perspectives to study the complex pathophysiology of this condition. Our findings suggest that urinary-excreted toxic BAs represent a pivotal trigger for renal tubular epithelial injury leading to cholemic nephropathy in CBDL mice.

Erratum: Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

[This corrects the article DOI: 10.1016/j.omtn.2023.04.004.].

NLRX1 Prevents M2 Macrophage Polarization and Excessive Renal Fibrosis in Chronic Obstructive Nephropathy.

BACKGROUND: Chronic kidney disease often leads to kidney dysfunction due to renal fibrosis, regardless of the initial cause of kidney damage. Macrophages are crucial players in the progression of renal fibrosis as they stimulate inflammation, activate fibroblasts, and contribute to extracellular matrix deposition, influenced by their metabolic state. Nucleotide-binding domain and LRR-containing protein X (NLRX1) is an innate immune receptor independent of inflammasomes and is found in mitochondria, and it plays a role in immune responses and cell metabolism. The specific impact of NLRX1 on macrophages and its involvement in renal fibrosis is not fully understood. METHODS: To explore the specific role of NLRX1 in macrophages, bone-marrow-derived macrophages (BMDMs) extracted from wild-type (WT) and NLRX1 knockout (KO) mice were stimulated with pro-inflammatory and pro-fibrotic factors to induce M1 and M2 polarization in vitro. The expression levels of macrophage polarization markers (Nos2, Mgl1, Arg1, and Mrc1), as well as the secretion of transforming growth factor ß (TGFß), were measured using RT-PCR and ELISA. Seahorse-based bioenergetics analysis was used to assess mitochondrial respiration in naïve and polarized BMDMs obtained from WT and NLRX1 KO mice. In vivo, WT and NLRX1 KO mice were subjected to unilateral ureter obstruction (UUO) surgery to induce renal fibrosis. Kidney injury, macrophage phenotypic profile, and fibrosis markers were assessed using RT-PCR. Histological staining (PASD and Sirius red) was used to quantify kidney injury and fibrosis. RESULTS: Compared to the WT group, an increased gene expression of M2 markers-including Mgl1 and Mrc1-and enhanced TGFß secretion were found in naïve BMDMs extracted from NLRX1 KO mice, indicating functional polarization towards the pro-fibrotic M2 subtype. NLRX1 KO naïve macrophages also showed a significantly enhanced oxygen consumption rate compared to WT cells and increased basal respiration and maximal respiration capacities that equal the level of M2-polarized macrophages. In vivo, we found that NLRX1 KO mice presented enhanced M2 polarization markers together with enhanced tubular injury and fibrosis demonstrated by augmented TGFß levels, fibronectin, and collagen accumulation. CONCLUSIONS: Our findings highlight the unique role of NLRX1 in regulating the metabolism and function of macrophages, ultimately protecting against excessive renal injury and fibrosis in UUO.

Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

A gene-silencing platform (miQURE) has been developed and successfully used to deliver therapeutic microRNA (miRNA) to the brain, reducing levels of neurodegenerative disease-causing proteins/RNAs via RNA interference and improving the disease phenotype in animal models. This study evaluates the use of miQURE technology to deliver therapeutic miRNA for liver-specific indications. Angiopoietin-like 3 (ANGPTL3) was selected as the target mRNA because it is produced in the liver and because loss-of-function ANGPTL3 mutations and/or pharmacological inhibition of ANGPTL3 protein lowers lipid levels and reduces cardiovascular risk. Overall, 14 candidate miRNA constructs were tested in vitro, the most potent of which (miAngE) was further evaluated in mice. rAAV5-miAngE led to dose-dependent (≤-77%) decreases in Angptl3 mRNA in WT mice with ≤-90% reductions in plasma ANGPTL3 protein. In dyslipidemic APOE∗3-Leiden.CETP mice, AAV5-miAngE significantly reduced cholesterol and triglyceride levels vs. vehicle and scrambled (miSCR) controls when administrated alone, with greater reductions when co-administered with lipid-lowering therapy (atorvastatin). A significant decrease in total atherosclerotic lesion area (-58% vs. miSCR) was observed in AAV5-miAngE-treated dyslipidemic mice, which corresponded with the maintenance of a non-diseased plaque phenotype and reduced lesion severity. These results support the development of this technology for liver-directed indications.

Focal adhesion kinase signaling mediates acute renal injury induced by ischemia/reperfusion.

Renal ischemia/reperfusion (I/R) injury is associated with cell matrix and focal adhesion remodeling. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that localizes at focal adhesions and regulates their turnover. Here, we investigated the role of FAK in renal I/R injury, using a novel conditional proximal tubule-specific fak-deletion mouse model. Tamoxifen treatment of FAK(loxP/loxP)//γGT-Cre-ER(T2) mice caused renal-specific fak recombination (FAK(ΔloxP/ΔloxP)) and reduction of FAK expression in proximal tubules. In FAK(ΔloxP/ΔloxP) mice compared with FAK(loxP/loxP) controls, unilateral renal ischemia followed by reperfusion resulted in less tubular damage with reduced tubular cell proliferation and lower expression of kidney injury molecule-1, which was independent from the postischemic inflammatory response. Oxidative stress is involved in the pathophysiology of I/R injury. Primary cultured mouse renal cells were used to study the role of FAK deficiency for oxidative stress in vitro. The conditional fak deletion did not affect cell survival after hydrogen peroxide-induced cellular stress, whereas it impaired the recovery of focal adhesions that were disrupted by hydrogen peroxide. This was associated with reduced c-Jun N-terminal kinase-dependent phosphorylation of paxillin at serine 178 in FAK-deficient cells, which is required for focal adhesion turnover. Our findings support a role for FAK as a novel factor in the initiation of c-Jun N-terminal kinase-mediated cellular stress response during renal I/R injury and suggest FAK as a target in renal injury protection.

Epac-Rap signaling reduces cellular stress and ischemia-induced kidney failure.

Renal ischemia-reperfusion injury is associated with the loss of tubular epithelial cell-cell and cell-matrix interactions which contribute to renal failure. The Epac-Rap signaling pathway is a potent regulator of cell-cell and cell-matrix adhesion. The cyclic AMP analogue 8-pCPT-2'-O-Me-cAMP has been shown to selectively activate Epac, whereas the addition of an acetoxymethyl (AM) ester to 8-pCPT-2'-O-Me-cAMP enhanced in vitro cellular uptake. Here we demonstrate that pharmacological activation of Epac-Rap signaling using acetoxymethyl-8-pCPT-2'-O-Me-cAMP preserves cell adhesions during hypoxia in vitro, maintaining the barrier function of the epithelial monolayer. Intrarenal administration in vivo of 8-pCPT-2'-O-Me-cAMP also reduced renal failure in a mouse model for ischemia-reperfusion injury. This was accompanied by decreased expression of the tubular cell stress marker clusterin-α, and lateral expression of ß-catenin after ischemia indicative of sustained tubular barrier function. Our study emphasizes the undervalued importance of maintaining tubular epithelial cell adhesion in renal ischemia and demonstrates the potential of pharmacological modulation of cell adhesion as a new therapeutic strategy to reduce the extent of injury in kidney disease and transplantation.

Chemokine expression in renal ischemia/reperfusion injury is most profound during the reparative phase.

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. C57Bl/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-2alpha, 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 QRT-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.

SDF-1 provides morphological and functional protection against renal ischaemia/reperfusion 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.

Haematopoietic stem cell migration to the ischemic damaged kidney is not altered by manipulating the SDF-1/CXCR4-axis.

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.

Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney.

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 oligonucleotides, 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 dysfunction 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.

Enhanced mobilization of bone marrow cells does not ameliorate renal fibrosis.

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.

Aryl hydrocarbon receptor expression by macrophages and lymphocytes within infiltrates in BK polyomavirus associated nephropathy.

BACKGROUND: BK virus nephropathy (BKPyVN) is a major complication after renal transplantation. Little is known about the intra renal immune response during BKPyVN. The role of macrophages remains elusive. The activation of aryl hydrocarbon receptor (AHR) - a transcription factor involved in drug metabolism - plays a key role in inflammation and viral tolerance through modulation of macrophages polarization. Since AHR has not been studied in kidney transplantation, our aim was to compare the AHR expression within renal grafts in BKPyVN with T-cell mediated rejection (TCMR) as a control. METHODS: We evaluated AHR expression in kidney grafts from BKPyVN (n=8) with TCMR as control (n=6) among cases with available frozen material for AHR gene intragraft transcription measurement and stainings for AHR, CD68 and CD45. RESULTS: AHR transcription was higher in BKPyVN grafts versus TCMR (p=0.03). While CD68+ or CD45+ cell expression did not differ within infiltrates (median score=3 in both groups; p=1.0 and 0.69, respectively), a higher proportion of nuclear AHR expression was found in BKPyVN for CD68+ and CD45+ cells when compared with TCMR (score median 2 vs 0; p=0.007 and 1 vs 0; p=0.013, respectively). CONCLUSIONS: We describe for the first time a higher expression of AHR in inflammatory cell infiltrates from BKPyVN versus TCMR renal biopsies. Further studies are required to explore AHR as a potential target in the modulation of inflammatory response in BKPyVN with known modulating ligands.