Lipoxin Mimetics and the Resolution of Inflammation.

Inflammation and its timely resolution are critical to ensure effective host defense and appropriate tissue repair after injury and or infection. Chronic, unresolved inflammation typifies many prevalent pathologies. The key mediators that initiate and drive the inflammatory response are well defined and targeted by conventional anti-inflammatory therapeutics. More recently, there is a growing appreciation that specific mediators, including arachidonate-derived lipoxins, are generated in self-limiting inflammatory responses to promote the resolution of inflammation and endogenous repair mechanisms without compromising host defense. We discuss the proresolving biological actions of lipoxins and recent efforts to harness their therapeutic potential through the development of novel, potent lipoxin mimetics generated via efficient, modular stereoselective synthetic pathways. We consider the evidence that lipoxin mimetics may have applications in limiting inflammation and reversing fibrosis and the underlying mechanisms.

Genetic variants affecting mitochondrial function provide further insights for kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a complex disorder that has become a high prevalence global health problem, with diabetes being its predominant pathophysiologic driver. Autosomal genetic variation only explains some of the predisposition to kidney disease. Variations in the mitochondrial genome (mtDNA) and nuclear-encoded mitochondrial genes (NEMG) are implicated in susceptibility to kidney disease and CKD progression, but they have not been thoroughly explored. Our aim was to investigate the association of variation in both mtDNA and NEMG with CKD (and related traits), with a particular focus on diabetes. METHODS: We used the UK Biobank (UKB) and UK-ROI, an independent collection of individuals with type 1 diabetes mellitus (T1DM) patients. RESULTS: Fourteen mitochondrial variants were associated with estimated glomerular filtration rate (eGFR) in UKB. Mitochondrial variants and haplogroups U, H and J were associated with eGFR and serum variables. Mitochondrial haplogroup H was associated with all the serum variables regardless of the presence of diabetes. Mitochondrial haplogroup X was associated with end-stage kidney disease (ESKD) in UKB. We confirmed the influence of several known NEMG on kidney disease and function and found novel associations for SLC39A13, CFL1, ACP2 or ATP5G1 with serum variables and kidney damage, and for SLC4A1, NUP210 and MYH14 with ESKD. The G allele of TBC1D32-rs113987180 was associated with higher risk of ESKD in patients with diabetes (OR:9.879; CI95%:4.440-21.980; P = 2.0E-08). In UK-ROI, AGXT2-rs71615838 and SURF1-rs183853102 were associated with diabetic nephropathies, and TFB1M-rs869120 with eGFR. CONCLUSIONS: We identified novel variants both in mtDNA and NEMG which may explain some of the missing heritability for CKD and kidney phenotypes. We confirmed the role of MT-ND5 and mitochondrial haplogroup H on renal disease (serum variables), and identified the MT-ND5-rs41535848G variant, along with mitochondrial haplogroup X, associated with higher risk of ESKD. Despite most of the associations were independent of diabetes, we also showed potential roles for NEMG in T1DM.

Albuminuria-Related Genetic Biomarkers: Replication and Predictive Evaluation in Individuals with and without Diabetes from the UK Biobank.

Increased albuminuria indicates underlying glomerular pathology and is associated with worse renal disease outcomes, especially in diabetic kidney disease. Many single nucleotide polymorphisms (SNPs), associated with albuminuria, could be potentially useful to construct polygenic risk scores (PRSs) for kidney disease. We investigated the diagnostic accuracy of SNPs, previously associated with albuminuria-related traits, on albuminuria and renal injury in the UK Biobank population, with a particular interest in diabetes. Multivariable logistic regression was used to evaluate the influence of 91 SNPs on urine albumin-to-creatinine ratio (UACR)-related traits and kidney damage (any pathology indicating renal injury), stratifying by diabetes. Weighted PRSs for microalbuminuria and UACR from previous studies were used to calculate the area under the receiver operating characteristic curve (AUROC). CUBN-rs1801239 and DDR1-rs116772905 were associated with all the UACR-derived phenotypes, in both the overall and non-diabetic cohorts, but not with kidney damage. Several SNPs demonstrated different effects in individuals with diabetes compared to those without. SNPs did not improve the AUROC over currently used clinical variables. Many SNPs are associated with UACR or renal injury, suggesting a role in kidney dysfunction, dependent on the presence of diabetes in some cases. However, individual SNPs or PRSs did not improve the diagnostic accuracy for albuminuria or renal injury compared to standard clinical variables.

Genome-wide meta-analysis and omics integration identifies novel genes associated with diabetic kidney disease.

AIMS/HYPOTHESIS: Diabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets. METHODS: We performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets. RESULTS: The meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR<60 ml/min per 1.73 m2) and DKD (microalbuminuria or worse) phenotype (p=9.8×10-9; although not withstanding correction for multiple testing, p>9.3×10-9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN-RESP18, GPR158, INIP-SNX30, LSM14A and MFF; p<2.7×10-6). Integration of GWAS with human glomerular and tubular expression data demonstrated higher tubular AKIRIN2 gene expression in individuals with vs without DKD (p=1.1×10-6). The lead SNPs within six loci significantly altered DNA methylation of a nearby CpG site in kidneys (p<1.5×10-11). Expression of lead genes in kidney tubules or glomeruli correlated with relevant pathological phenotypes (e.g. TENM2 expression correlated positively with eGFR [p=1.6×10-8] and negatively with tubulointerstitial fibrosis [p=2.0×10-9], tubular DCLK1 expression correlated positively with fibrosis [p=7.4×10-16], and SNX30 expression correlated positively with eGFR [p=5.8×10-14] and negatively with fibrosis [p<2.0×10-16]). CONCLUSIONS/INTERPRETATION: Altogether, the results point to novel genes contributing to the pathogenesis of DKD. DATA AVAILABILITY: The GWAS meta-analysis results can be accessed via the type 1 and type 2 diabetes (T1D and T2D, respectively) and Common Metabolic Diseases (CMD) Knowledge Portals, and downloaded on their respective download pages ( https://t1d.hugeamp.org/downloads.html ; https://t2d.hugeamp.org/downloads.html ; https://hugeamp.org/downloads.html ).

Dietary restriction and medical therapy drives PPARα-regulated improvements in early diabetic kidney disease in male rats.

The attenuation of diabetic kidney disease (DKD) by metabolic surgery is enhanced by pharmacotherapy promoting renal fatty acid oxidation (FAO). Using the Zucker Diabetic Fatty and Zucker Diabetic Sprague Dawley rat models of DKD, we conducted studies to determine if these effects could be replicated with a non-invasive bariatric mimetic intervention. Metabolic control and renal injury were compared in rats undergoing a dietary restriction plus medical therapy protocol (DMT; fenofibrate, liraglutide, metformin, ramipril, and rosuvastatin) and ad libitum-fed controls. The global renal cortical transcriptome and urinary 1H-NMR metabolomic profiles were also compared. Kidney cell type-specific and medication-specific transcriptomic responses were explored through in silico deconvolution. Transcriptomic and metabolomic correlates of improvements in kidney structure were defined using a molecular morphometric approach. The DMT protocol led to ∼20% weight loss, normalized metabolic parameters and was associated with reductions in indices of glomerular and proximal tubular injury. The transcriptomic response to DMT was dominated by changes in fenofibrate- and peroxisome proliferator-activated receptor-α (PPARα)-governed peroxisomal and mitochondrial FAO transcripts localizing to the proximal tubule. DMT induced urinary excretion of PPARα-regulated metabolites involved in nicotinamide metabolism and reversed DKD-associated changes in the urinary excretion of tricarboxylic acid (TCA) cycle intermediates. FAO transcripts and urinary nicotinamide and TCA cycle metabolites were moderately to strongly correlated with improvements in glomerular and proximal tubular injury. Weight loss plus pharmacological PPARα agonism is a promising means of attenuating DKD.

Therapeutic potential of the FPR2/ALX agonist AT-01-KG in the resolution of articular inflammation.

The resolution of inflammation is a dynamic process, characterized by the biosynthesis of pro-resolving mediators, including the lipid Lipoxin A4 (LXA4). LXA4 acts on the N-formyl peptide receptor 2 (FPR2/ALX) to mediate anti-inflammatory and pro-resolving effects. In order to exploit the therapeutic potential of endogenous LXA4 in the context of inflammation we have recently developed synthetic LXA4 mimetics (sLXms) including a dimethyl-imidazole-containing FPR2/ALX agonist designated AT-01-KG. Here, we have investigated the effect of treatment with AT-01-KG in established models of articular inflammation. In a model of gout, mice were injected with MSU crystals and treated with AT-01-KG at the peak of inflammatory response. The treatment decreased the number of neutrophils in the knee exudate, an effect which was accompanied by low levels of myeloperoxidase, CXCL1 and IL-1ß in periarticular tissue. AT-01-KG treatment led to reduced tissue damage and hypernociception. The effects of AT-01-KG on neutrophil accumulation were not observed in MSU treated FPR2/3-/-mice. Importantly, AT-01-KG induced resolution of articular inflammation by increasing neutrophil apoptosis and subsequent efficient efferocytosis. In a model of antigen-induced arthritis, AT-01-KG treatment also attenuated inflammatory responses. These data suggest that AT-01-KG may be a potential new therapy for neutrophilic inflammation of the joints.

Promoting resolution in kidney disease: are we nearly there yet?

PURPOSE OF REVIEW: Nephrology lacks effective therapeutics for many of the presentations and diseases seen in clinical practice. In recent decades, we have come to understand the central place of inflammation in initiating and propagating kidney disease, and, research in more recent years has established that the resolution of inflammation is a highly regulated and active process. With this, has evolved an appreciation that this aspect of the host inflammatory response is defective in kidney disease and led to consideration of a therapeutic paradigm aiming to harness the activity of the molecular drivers of the resolution phase of inflammation. Fatty-acid-derived Specialized pro-resolving mediators (SPMs), partly responsible for resolution of inflammation have gained traction as potential therapeutics. RECENT FINDINGS: We describe our current understanding of SPMs for this purpose in acute and chronic kidney disease. These studies cement the place of inflammation and its defective resolution in the pathogenesis of kidney disease, and highlight new avenues for therapy. SUMMARY: Targeting resolution of inflammation is a viable approach to treating kidney disease. We optimistically look forward to translating these experimental advances into tractable therapeutics to treat kidney disease.

Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen.

BACKGROUND: Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. METHODS: To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. RESULTS: Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). CONCLUSIONS: The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

Liraglutide Attenuates Preestablished Atherosclerosis in Apolipoprotein E-Deficient Mice via Regulation of Immune Cell Phenotypes and Proinflammatory Mediators.

We have shown that the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide (Lir) inhibits development of early atherosclerosis in vivo by modulating immune cell function. We hypothesized that Lir could attenuate pre-established disease by modulating monocyte or macrophage phenotype to induce atheroprotective responses. Human atherosclerotic plaques obtained postendarterectomy and human peripheral blood macrophages were treated ex vivo with Lir. In parallel, apolipoprotein E-deficient (ApoE-/-) mice received a high-fat, high-cholesterol diet to induce atherosclerosis for 8 weeks, after which ApoE-/- mice received 300 µg/kg of Lir daily or vehicle control for a further 4 weeks to investigate the attenuation of atherosclerosis. Lir inhibited proinflammatory monocyte chemoattractant protein-1 secretion from human endarterectomy samples and monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin (IL)-1ß secretion from human macrophages after ex vivo treatment. An increase in CD206 mRNA and IL-10 secretion was also detected, which implies resolution of inflammation. Importantly, Lir significantly attenuated pre-established atherosclerosis in ApoE-/- mice in the whole aorta and aortic root. Proteomic analysis of ApoE-/- bone marrow cells showed that Lir upregulated the proinflammatory cathepsin protein family, which was abolished in differentiated macrophages. In addition, flow cytometry analysis of bone marrow cells induced a shift toward reduced proinflammatory and increased anti-inflammatory macrophages. We concluded that Lir attenuates pre-established atherosclerosis in vivo by altering proinflammatory mediators. This is the first study to describe a mechanism through which Lir attenuates atherosclerosis by increasing bone marrow proinflammatory protein expression, which is lost in differentiated bone marrow-derived macrophages. This study contributes to our understanding of the anti-inflammatory and cardioprotective role of GLP-1RAs. SIGNIFICANCE STATEMENT: It is critical to understand the mechanisms through which liraglutide (Lir) mediates a cardioprotective effect as many type 2 diabetic medications increase the risk of myocardial infarction and stroke. We have identified that Lir reduces proinflammatory immune cell populations and mediators from plaque-burdened murine aortas in vivo and augments proresolving bone marrow-derived macrophages in attenuation of atherosclerotic disease, which provides further insight into the atheroprotective effect of Lir.

Specialized pro-resolving mediators in diabetes: novel therapeutic strategies.

Diabetes mellitus (DM) is an important metabolic disorder characterized by persistent hyperglycemia resulting from inadequate production and secretion of insulin, impaired insulin action, or a combination of both. Genetic disorders and insulin receptor disorders, environmental factors, lifestyle choices and toxins are key factors that contribute to DM. While it is often referred to as a metabolic disorder, modern lifestyle choices and nutrient excess induce a state of systemic chronic inflammation that results in the increased production and secretion of inflammatory cytokines that contribute to DM. It is chronic hyperglycemia and the low-grade chronic-inflammation that underlies the development of microvascular and macrovascular complications leading to damage in a number of tissues and organs, including eyes, vasculature, heart, nerves, and kidneys. Improvements in the management of risk factors have been beneficial, including focus on intensified glycemic control, but most current approaches only slow disease progression. Even with recent studies employing SGLT2 inhibitors demonstrating protection against cardiovascular and kidney diseases, kidney function continues to decline in people with established diabetic kidney disease (DKD). Despite the many advances and a greatly improved understanding of the pathobiology of diabetes and its complications, there remains a major unmet need for more effective therapeutics to prevent and reverse the chronic complications of diabetes. More recently, there has been growing interest in the use of specialised pro-resolving mediators (SPMs) as an exciting therapeutic strategy to target diabetes and the chronic complications of diabetes.

Lipoxins Regulate the Early Growth Response-1 Network and Reverse Diabetic Kidney Disease.

Background The failure of spontaneous resolution underlies chronic inflammatory conditions, including microvascular complications of diabetes such as diabetic kidney disease. The identification of endogenously generated molecules that promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. Lipoxins (LXs) are lipid mediators that promote the resolution of inflammation.Methods We investigated the potential of LXA4 and a synthetic LX analog (Benzo-LXA4) as therapeutics in a murine model of diabetic kidney disease, ApoE-/- mice treated with streptozotocin.Results Intraperitoneal injection of LXs attenuated the development of diabetes-induced albuminuria, mesangial expansion, and collagen deposition. Notably, LXs administered 10 weeks after disease onset also attenuated established kidney disease, with evidence of preserved kidney function. Kidney transcriptome profiling defined a diabetic signature (725 genes; false discovery rate P≤0.05). Comparison of this murine gene signature with that of human diabetic kidney disease identified shared renal proinflammatory/profibrotic signals (TNF-α, IL-1ß, NF-κB). In diabetic mice, we identified 20 and 51 transcripts regulated by LXA4 and Benzo-LXA4, respectively, and pathway analysis identified established (TGF-ß1, PDGF, TNF-α, NF-κB) and novel (early growth response-1 [EGR-1]) networks activated in diabetes and regulated by LXs. In cultured human renal epithelial cells, treatment with LXs attenuated TNF-α-driven Egr-1 activation, and Egr-1 depletion prevented cellular responses to TGF-ß1 and TNF-αConclusions These data demonstrate that LXs can reverse established diabetic complications and support a therapeutic paradigm to promote the resolution of inflammation.

Wireless Electrochemiluminescence at Nafion-Carbon Microparticle Composite Films.

Thin films of a composite of nafion and carbon microparticles have been deposited on nonconducting substrates and their conductivity as well as their ability to generate electrochemiluminescence investigated. The films exhibit very low conductivity (<6 × 103 S m-1) for low particle loadings, but once the percolation threshold is reached (volume percentage of 71 ± 8% carbon particles), the conductivity increases dramatically and a maximum conductivity of 2.0 ± 0.1 × 107 S m-1 is achieved. The electrochemical properties of the composites, including heterogeneous electron transfer rates, were probed using cyclic voltammetry. Significantly, bipolar, or wireless, electrochemiluminescence can be generated with films that contain >65% (by volume) carbon particles using [Ru(bpy)3]2+ as the luminophore and tripropylamine as the coreactant, at an electric field of 14 V cm-1. Under these conditions, the complete film is sufficiently conducting to become polarized in the external electric field and the electrochemiluminescence intensity correlates strongly with the film conductivity. These results demonstrate the usefulness of particle arrays for the wireless generation of electrochemiluminescence at relatively low electric field strengths.

Profibrotic IHG-1 complexes with renal disease associated HSPA5 and TRAP1 in mitochondria.

The highly conserved mitochondrial protein induced in high glucose-1 (IHG-1) functions to maintain mitochondrial quality and is associated with the development of fibrosis in diabetic nephropathy. Towards identifying novel approaches to treating diabetic kidney disease, IHG-1-protein-protein interactions were investigated using epitope-tagged immunoprecipitation analyses followed by mass spectrometry. Here we show that IHG-1 is solely expressed in mitochondria and localised to the inner mitochondrial membrane, the region where mitochondrial reactive oxygen species are generated. Chaperones HSPA5 and TRAP1 and cold shock protein YBX1 were identified as IHG-1 binding partners. All three proteins are important in the cellular response to oxidative stress and play important roles in mitochondrial transcription and DNA repair. Both redox imbalance and IHG-1 stimulate TGF-ß signalling. IHG-1, HSPA5 and YBX1 all show increased expression in diabetic nephropathy, chronic kidney disease and in the Unilateral Ureteral Obstruction model of kidney fibrosis. Increased IHG-1 expression in UUO correlated with loss of TRAP1 expression. IHG-1 may target TRAP1 for degradation. When IHG-1 is no longer localised to mitochondria, it retains the ability to interact with the cold shock protein YBX1, facilitating anti-fibrotic actions in the nucleus. Targeting these proteins may offer alternative treatments for fibrotic kidney disease.

Mesenchymal Stem Cells Deliver Exogenous MicroRNA-let7c via Exosomes to Attenuate Renal Fibrosis.

The advancement of microRNA (miRNA) therapies has been hampered by difficulties in delivering miRNA to the injured kidney in a robust and sustainable manner. Using bioluminescence imaging in mice with unilateral ureteral obstruction (UUO), we report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c (miR-let7c-MSCs), selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared with nontargeting control (NTC)-MSCs. miR-let7c-MSC therapy attenuated kidney injury and significantly downregulated collagen IVα1, metalloproteinase-9, transforming growth factor (TGF)-ß1, and TGF-ß type 1 receptor (TGF-ßR1) in UUO kidneys, compared with controls. In vitro analysis confirmed that the transfer of miR-let7c from miR-let7c-MSCs occurred via secreted exosomal uptake, visualized in NRK52E cells using cyc3-labeled pre-miRNA-transfected MSCs with/without the exosomal inhibitor, GW4869. The upregulated expression of fibrotic genes in NRK52E cells induced by TGF-ß1 was repressed following the addition of isolated exosomes or indirect coculture of miR-let7c-MSCs, compared with NTC-MSCs. Furthermore, the cotransfection of NRK52E cells using the 3'UTR of TGF-ßR1 confirmed that miR-let7c attenuates TGF-ß1-driven TGF-ßR1 gene expression. Taken together, the effective antifibrotic function of engineered MSCs is able to selectively transfer miR-let7c to damaged kidney cells and will pave the way for the use of MSCs for therapeutic delivery of miRNA targeted at kidney disease.

Detection and Live-Cell Imaging of a Micro-RNA Associated with the Cancer Neuroblastoma.

The ability of a molecular beacon to detect miR-132, a microRNA associated with the childhood cancer neuroblastoma, is reported in solution and within live cells. The stem-loop structure comprises a sequence complementary to miR-132, modified with a 6-FAM dye and dabcyl quencher on either end. In the absence of the target, self-binding occurs bringing the luminophore and quencher into close proximity, significantly decreasing the emission intensity. In the presence of miR-132, the signal is greatly enhanced, with a linear increase in intensity for mole ratios of beacon-to-target between 0.25 and 2.00. The structure differentiates between target and mismatched nucleic acid sequences, e.g., in the presence of a single-base mismatch, no increase in emission intensity beyond the background is observed. The stem-loop can be introduced into neuroblastoma cancer cells by electroporation, allowing miR-132 to be imaged within live cells. miR-132 appears to be localized within the nucleus of the cells, where its concentration is of the order of 1 µM. Significantly, transfection of the cells with a miR-132 mimic causes the emission intensity to more than double, demonstrating the sensitivity of the approach to changes in miR-132 concentration in live cells. This behavior opens up significant theranostic applications, such as the possibility of rapidly identifying retinoic acid resistant patients as well as providing a means to monitor therapeutic efficacy.

Paricalcitol protects against TGF-ß1-induced fibrotic responses in hypoxia and stabilises HIF-α in renal epithelia.

Epithelial injury and tubulointerstitial fibrosis (TIF) within a hypoxic microenvironment are associated with progressive loss of renal function in chronic kidney disease [CKD]. Transforming growth factor beta-1 (TGF-ß1) is an important mediator of renal fibrosis. Growing evidence suggests that Vitamin D [1,25-(OH)2D] and its analogues may have a renoprotective effect in CKD. Here we examined the protective effect of the vitamin D analogue paricalcitol [PC; 19-nor-1α,3ß,25-trihydroxy-9,10-secoergosta-5(Z),7(E) 22(E)-triene] on the responses of human renal epithelial cells to TGF-ß1. PC attenuated TGF-ß1-induced Smad 2 phosphorylation and upregulation of the Notch ligand Jagged-1, α-smooth muscle actin and thrombospondin-1 and prevented the TGF-ß1-mediated loss of E-Cadherin. To mimic the hypoxic milieu of CKD we cultured renal epithelial cells in hypoxia [1% O2] and observed similar attenuation by PC of TGF-ß1-induced fibrotic responses. Furthermore, in cells cultured in normoxia [21% O2], PC induced an accumulation of hypoxia-inducible transcription factors (HIF) 1α and HIF-2α in a time and concentration [1 µM-2 µM] dependent manner. Here, PC-induced HIF stabilisation was dependent on activation of the PI-3Kinase pathway. This is the first study to demonstrate regulation of the HIF pathway by PC which may have importance in the mechanism underlying renoprotection by PC.

Study of microRNA in diabetic nephropathy: isolation, quantification and biological function.

In recent years, several studies have reported dysregulation of microRNA expression in disease with a growing interest focussed on targeting microRNAs as a novel therapy for human disease. This is especially true in diabetic nephropathy where the expression of several microRNAs is dysregulated, contributing to the increased expression and accumulation of extracellular matrix proteins and increased pro-fibrotic signalling, ultimately resulting in renal fibrosis. The development of various techniques and microRNA reagents has enabled work to progress very rapidly in this area. In the present article, the authors describe the methods they have used that have enabled them to contribute to our current understanding of the role of microRNAs in diabetic nephropathy.

New susceptibility loci associated with kidney disease in type 1 diabetes.

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ~2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2 × 10(-8)) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0 × 10(-9)). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-ß1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1 × 10(-7)), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.

Targeting cellular drivers and counter-regulators of hyperglycaemia- and transforming growth factor-ß1-associated profibrotic responses in diabetic kidney disease.

Diabetic kidney disease occurs in >30% of patients with type 2 diabetes mellitus and is characterized at source by a maladaptive response in the renal parenchyma to exposure to a glucotoxic-lipotoxic diabetic milieu that courses coincident with hypertension. The consequence of these maladaptive responses is progressive renal injury, which is exacerbated by the development of a chronic inflammatory infiltrate associated with the development of tubulointerstitial fibrosis. The evolution of tubulointerstitial fibrosis is correlated with the loss of functional renal mass and descent towards renal failure. Transforming growth factor-ß1 (TGF-ß1) is a recognized mediator of the profibrotic response of mesangial cells and renal tubular epithelial cells to hyperglycaemia. While euglycaemia remains the goal in the treatment of type 2 diabetes mellitus, the prevention, arrest and reversal of microvascular complications, such as diabetic kidney disease, may be assisted by pharmacological modulation of the effectors of glucotoxicity, such as TGF-ß1. This review focuses on describing how, through reductionist in vitro experimentation focusing on TGF-ß1-related responses to hyperglycaemia, we have identified induced in high glucose-1 (IHG-1), induced in high glucose-2 (IHG-2/Grem1) and the lipoxin-inducible microRNA let-7c as potential targets for harnessing new therapeutic approaches to limit the bioactivity of TGF-ß1 in diabetic kidney disease.

Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFßR1.

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-ß1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-ß1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-ß1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-ß1 signaling pathway, including the TGF-ß receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-ß receptor type 1 and the response to TGF-ß1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-ß1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.