Fibrotic Renal Disease Research Articles

Calcium-dependent dual oxidase 2 is a novel source of reactive oxygen species implicated in glomerular mesangial cell fibrotic response to angiotensin II and glucose

The vasoactive peptide angiotensin II (Ang II) and hyperglycemia contribute to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II or high concentrations of glucose (HG). Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression, along with its associated maturation factor, DuoxA2, are upregulated by Ang II and HG. Small interfering RNA (siRNA)-mediated downregulation of Duox2 significantly reduces Ang II-induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a-smooth muscle actin and fibronectin expression). To demonstrate that Duox2 activation is Ca2+-dependent, we show that the extracellular Ca2+ chelator BAPTA prevented Ang II-induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. Fura-2 fluorescence was utilized as well to show calcium mobilization in response to Ang II and this effect was abrogated with siDuox2 transfection. These data indicate that Ang II-stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury. In summary, we have identified a novel role for Duox2 and its cognate maturation factor, DuoxA2, as a major source of ROS in response to Ang II and HG and established the significance of Duox2 in Ang II-mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseases. Research reported in this poster was supported by the University of the Incarnate Word Offce of Research and Graduate Studies and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC2GM136569. The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification and Functional Mechanism Verification of Novel MicroRNAs Associated with the Fibrosis Progression in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a serious threat to human health worldwide, and its incidence is increasing annually. A growing amount of information is emerging about the role of micoRNAs (miRNAs) in the regulation of renal fibrosis, which has aroused interest in the development of drugs that block pathogenic miRNAs or restore protective miRNAs levels. To clarify the role of miRNAs in CKD, we selected patients with significant renal fibrotic disease (diabetic nephropathy (DN) and focal segmental glomerulosclerosis (FSGS)) as the disease group, and patients with little or no renal fibrotic disease (minimal change disease (MCD) and renal carcinoma adjacent to normal kidney) as controls. Significantly differentially expressed miRNAs were obtained by human kidney tissue sequencing, subsequently verified in mice models of DN and FSGS, and subsequently inhibited or overexpressed in human renal tubular epithelial cells (HK-2) stimulated by high glucose (HG) and TGF-β1 in vitro. Therefore, the mechanism of its action in renal fibrosis was further elaborated. Finally, the downstream target genes of the corresponding miRNAs were verified by bioinformatics analysis, qRT-PCR, western blot and double luciferase report analysis. Two novel miRNAs, hsa-miR-1470-3p (miR-1470) and hsa-miR-4483-3p (miR-4483), were detected by renal tissue sequencing in the disease group with significant renal fibrosis (DN and FSGS) and the control group with little or no renal fibrosis (MCD and normal renal tissue adjacent to renal carcinoma). Subsequent human renal tissue qRT-PCR verified that the expression of miR-1470 was significantly increased, while the expression of miR-4483 was markedly decreased in the disease group (p < 0.05). Moreover, in vivo DN and FSGS mice models, the expression levels of miR-1470 and miR-4483 were consistent with the results of human kidney tissue. In vitro, miR-4483 was suppressed, whereas miR-1470 was induced by treatment with TGF-β1 or HG. Inhibition of miR-1470 or overexpression of miR-4483 promoted HG or TGF-β1-induced fibrosis in HK-2 cells. Further study revealed that MMP-13 and TIMP1 were the target genes ofmiR-1470 and miR-4483, respectively. Our study identifies newly dysregulated miRNA profiles related to fibrosis kidneys. miR-1470 and miR-4483 are demonstrated to participate in kidney fibrosis by regulation of MMP-13, TIMP1 respectively. Our results may represent a promising research direction for renal disorders and help identify new biomarkers and therapeutic targets for CKD.

Attempt to Create a Drug for Treatment of Fibrotic Diseases Based on the Non-genomic Action of Nuclear Receptors

Nuclear receptors are ligand-dependent transcription factors that act as signal transducers by binding to and regulating the transcription of target DNA (genomic action). In recent years, nuclear receptors have also been found to exhibit a direct action on target proteins without affecting their transcription (non-genomic action). Independent complexes are expected to distinguish these two nuclear receptor actions. In this paper, I report the non-genomic action of peroxisome proliferator-activated receptor γ (PPARγ) agonists and propose a strategy for treatment of renal fibrotic diseases by PPARγ agonists with an emphasis on non-genomic actions.

Wheatgrass (Triticum aestivum) as an Efficient Phytoremediation Plant for Aristolochic Acid-Contaminated Water and Arable Soil

Dietary exposure to aristolochic acids (AAs) through AA-tainted wheat has long been identified as one of the main causes of Balkan endemic nephropathy (BEN), a slowly progressive renal fibrotic disease affecting numerous residents of farming villages alongside tributaries of the Danube River. Only recently though have we begun to appreciate that AAs, released from the decay of a common weed Aristolochia clematitis, are contaminating farmland soil and groundwater of endemic areas. To reduce the risk of human exposure to this class of highly potent phytotoxins, it is imperative to develop remediation methods for AA-polluted water and arable soil. In this study, we explored the potential use of some commonly used phytoremediation plants for the phytoextraction of AAs from AA-contaminated water and soil. Among the tested plants, results showed that wheatgrass is highly efficient in extracting AAs from both contaminated water and soil and metabolizing them to the less toxic aristolactams. Furthermore, both the phytoextraction and plant metabolism efficiencies increased in an acidic environment, which is typical of endemic villages, highlighting the applicability of the developed method for affected areas.

Calcium‐Dependent Dual Oxidase 2 is a Novel Source of Reactive Oxygen Species Implicated in Glomerular Mesangial Cell Fibrotic Response to Angiotensin II and Glucose

The vasoactive peptide angiotensin II (Ang II) and hyperglycemia contribute to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II or high concentrations of glucose (HG). Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression, along with its associated maturation factor, DuoxA2, are upregulated by Ang II and HG.Small interfering RNA (siRNA)‐mediated downregulation of Duox2 significantly reduces Ang II‐induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a‐smooth muscle actin and fibronectin expression).To demonstrate that Duox2 activation is Ca2+‐dependent, we show that the extracellular Ca2+ chelator BAPTA prevented Ang II‐induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. Fura‐2 fluorescence was utilized as well to show calcium mobilization in response to Ang II and this effect was abrogated with siDuox2 transfection. These data indicate that Ang II‐stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury.In summary, we have identified a novel role for Duox2 and its cognate maturation factor, DuoxA2, as a major source of ROS in response to Ang II and HG and established the significance of Duox2 in Ang II‐mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseases.

Inhibition of renal fibrosis with a human CXCL9-derived glycosaminoglycan-binding peptide.

ObjectivesRenal fibrosis accompanies all chronic kidney disorders, ultimately leading to end‐stage kidney disease and the need for dialysis or even renal replacement. As such, renal fibrosis poses a major threat to global health and the search for effective therapeutic strategies to prevent or treat fibrosis is highly needed. We evaluated the applicability of a highly positively charged human peptide derived from the COOH‐terminal domain of the chemokine CXCL9, namely CXCL9(74–103), for therapeutic intervention. Because of its high density of net positive charges at physiological pH, CXCL9(74–103) competes with full‐length chemokines for glycosaminoglycan (GAG) binding. Consequently, CXCL9(74–103) prevents recruitment of inflammatory leucocytes to sites of inflammation.MethodsCXCL9(74–103) was chemically synthesised and tested in vitro for anti‐fibrotic properties on human fibroblasts and in vivo in the unilateral ureteral obstruction (UUO) mouse model.ResultsCXCL9(74–103) significantly reduced the mRNA and/or protein expression of connective tissue growth factor (CTGF), alpha‐smooth muscle actin (α‐SMA) and collagen III by transforming growth factor (TGF)‐β1‐stimulated human fibroblasts. In addition, administration of CXCL9(74–103) inhibited fibroblast migration towards platelet‐derived growth factor (PDGF), without affecting cell viability. In the UUO model, CXCL9(74–103) treatment significantly decreased renal α‐SMA, vimentin, and fibronectin mRNA and protein expression. Compared with vehicle, CXCL9(74–103) attenuated mRNA expression of TGF‐β1 and the inflammatory markers/mediators MMP‐9, F4/80, CCL2, IL‐6 and TNF‐α. Finally, CXCL9(74–103) treatment resulted in reduced influx of leucocytes in the UUO model and preserved tubular morphology. The anti‐fibrotic and anti‐inflammatory effects of CXCL9(74–103) were mediated by competition with chemokines and growth factors for GAG binding.ConclusionsOur findings provide a scientific rationale for targeting GAG–protein interactions in renal fibrotic disease.

The Genomic Response to TGF-β1 Dictates Failed Repair and Progression of Fibrotic Disease in the Obstructed Kidney.

Tubulointerstitial fibrosis is a common and diagnostic hallmark of a spectrum of chronic renal disorders. While the etiology varies as to the causative nature of the underlying pathology, persistent TGF-β1 signaling drives the relentless progression of renal fibrotic disease. TGF-β1 orchestrates the multifaceted program of kidney fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery or re-differentiation, capillary collapse and subsequent interstitial fibrosis eventually leading to chronic and ultimately end-stage disease. An increasing complement of non-canonical elements function as co-factors in TGF-β1 signaling. p53 is a particularly prominent transcriptional co-regulator of several TGF-β1 fibrotic-response genes by complexing with TGF-β1 receptor-activated SMADs. This cooperative p53/TGF-β1 genomic cluster includes genes involved in cellular proliferative control, survival, apoptosis, senescence, and ECM remodeling. While the molecular basis for this co-dependency remains to be determined, a subset of TGF-β1-regulated genes possess both p53- and SMAD-binding motifs. Increases in p53 expression and phosphorylation, moreover, are evident in various forms of renal injury as well as kidney allograft rejection. Targeted reduction of p53 levels by pharmacologic and genetic approaches attenuates expression of the involved genes and mitigates the fibrotic response confirming a key role for p53 in renal disorders. This review focuses on mechanisms underlying TGF-β1-induced renal fibrosis largely in the context of ureteral obstruction, which mimics the pathophysiology of pediatric unilateral ureteropelvic junction obstruction, and the role of p53 as a transcriptional regulator within the TGF-β1 repertoire of fibrosis-promoting genes.

Calcium‐Dependent Dual Oxidase 2 is a Novel Source of Reactive Oxygen Species Implicated in Glomerular Mesangial Cell Fibrotic Response to Angiotensin II

The vasoactive peptide angiotensin II (Ang II) contributes to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II. Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression is upregulated by Ang II. Small interfering RNA (siRNA)-mediated downregulation of Duox2 significantly reduces Ang II-induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a-smooth muscle actin and fibronectin expression). To demonstrate that Duox2 activation is Ca2+-dependent, we show that the extracellular Ca2+ chelator BAPTA prevented Ang II-induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. Fura-2 fluorescence was utilized as well to show calcium mobilization in response to Ang II and this effect was abrogated with siDuox2 transfection. These data indicate that Ang II-stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury. In summary, we have identified a novel role for Duox2 as a major source of ROS in response to Ang II and established the significance of Duox2 in Ang II-mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseases.

Mir199a-3p and 5p Influence Fibrotic Disease States, And Increase In Expression After Radiation

The mir199a family, composed of 3p and 5p splice variants, are associated with fibrotic cardiac, lung and renal diseases of different etiologies. Mir199a-3p and 5p target vital negative regulators of fibrosis-inducing pathways including JAK/STAT via targeting of SOCS7, and caveolin-dependent internalization pathway through targeting of CAV1. The objective of this study was to investigate if mir199a 3p and 5p expression increase after irradiation (RT) and regulate radiation-induce fibrosis via the similar pathways. C57BL/6 were treated with focal 14Gy renal irradiation and kidneys and overlaying skin harvested after 24hrs, 10wks, or 20wks. Untreated mouse kidneys and skin served as age-matched controls. RNA was isolated from the skin and kidney tissue using a trizol extraction method, and quantitative PCR analysis was performed to assess gene expression changes in mir199a-3p and 5p and critical components of the JAK/STAT and caveolin pathways. Both mir199a-3p and 5p expression increased in kidneys at the 20wks, with increases over untreated controls of 2.1-fold (p<0.001) and 2.4-fold (p<0.001) respectively. A 10-fold increase in expression of mir199a precursor RNA was seen for skin at 20 wks, and this translated into a 3.1-fold increase of mir199a-3p (p = 0.046) but without a measurable increase in the mir199a-5p variant. SOCS7 expression levels decreased at 20wk consistent with mir199a targeting, although not significantly when comparing to untreated controls. Levels of CAV1 increased significantly over untreated controls at the 10wk (1.8-fold, p = 0.04). At 20 weeks, transcript levels of CAV1 decreased to a level no longer significantly different than in age-matched controls, consistent with mir199a targeting. We show that mir199a family members are elevated in the kidneys and overlaying skin after 14Gy of radiation and mechanistically-associated with fibrosis. On-going studies are evaluating mir199a-3p and 5p after more clinically-relevant doses of RT.

Adiponectin in renal fibrosis.

Renal fibrosis is an inevitable consequence of parenchymal scarring and is the common final pathway that mediates almost all progressive renal diseases. Adiponectin, a hormone produced by adipose tissue, possesses potent anti-insulin, anti-inflammatory, and anti-fibrotic properties. Reportedly, adiponectin serves as an important messenger that facilitates complex interactions between adipose tissue and other metabolically related organs. In recent years, a growing body of evidence supports adiponectin involvement in renal fibrosis. These studies provide a deeper understanding of the molecular mechanism of action of adiponectin in renal fibrosis and also offer a potential preventive and therapeutic target for renal fibrosis. In this review, the physiological role of adiponectin is briefly introduced, and then the mechanism of adiponectin-mediated renal fibrosis and the related signaling pathways are described. Finally, we summarize the findings regarding the clinical value of adiponectin in renal fibrotic diseases and prospected its application potential.

Thrombospondin 1 and Its Diverse Roles as a Regulator of Extracellular Matrix in Fibrotic Disease.

Thrombospondin 1 (TSP1) is a matricellular extracellular matrix protein that has diverse roles in regulating cellular processes important for the pathogenesis of fibrotic diseases. We will present evidence for the importance of TSP1 control of latent transforming growth factor beta activation in renal fibrosis with an emphasis on diabetic nephropathy. Other functions of TSP1 that affect renal fibrosis, including regulation of inflammation and capillary density, will be addressed. Emerging roles for TSP1 N-terminal domain regulation of collagen matrix assembly, direct effects of TSP1-collagen binding, and intracellular functions of TSP1 in mediating endoplasmic reticulum stress responses in extracellular matrix remodeling and fibrosis, which could potentially affect renal fibrogenesis, will also be discussed. Finally, we will address possible strategies for targeting TSP1 functions to treat fibrotic renal disease.

Macrophages in Renal Fibrosis.

Monocytes/macrophages are highly involved in the process of renal injury, repair and fibrosis in many aspects of experimental and human renal diseases. Monocyte-derived macrophages, characterized by high heterogeneity and plasticity, are recruited, activated, and polarized in the whole process of renal fibrotic diseases in response to local microenvironment. As classically activated M1 or CD11b+/Ly6Chigh macrophages accelerate renal injury by producing pro-inflammatory factors like tumor necrosis factor-alpha (TNFα) and interleukins, alternatively activated M2 or CD11b+/Ly6Cintermediate macrophages may contribute to kidney repair by exerting anti-inflammation and wound healing functions. However, uncontrolled M2 macrophages or CD11b+/Ly6Clow macrophages promote renal fibrosis via paracrine effects or direct transition to myofibroblast-like cells via the process of macrophage-to-myofibroblast transition (MMT). In this regard, therapeutic strategies targeting monocyte/macrophage recruitment, activation, and polarization should be emphasized in the treatment of renal fibrosis.

The MEK Inhibitor Trametinib Ameliorates Kidney Fibrosis by Suppressing ERK1/2 and mTORC1 Signaling.

During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored. We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis-mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects. Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-β1, the major profibrotic cytokine, thereby suppressing downstream mTORC1 pathway activation. Additionally, trametinib reduced the expression of myofibroblast marker α-smooth muscle actin and the proliferation of renal fibroblasts, corroborating our in vivo data. Crucially, trametinib also significantly ameliorated renal fibrosis progression when administered to animals subsequent to myofibroblast activation. Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.

NFE2L2 (Nrf2) attenuates Tgf‐β1 signaling by elevation of Smad7 in mouse mesangial MES‐13 cells

Renal fibrosis is the principal process underlying the progression of kidney diseases. Accumulating evidence indicates that oxidative stress plays a crucial role in renal fibrotic diseases. Transforming growth factor‐β1 (Tgf‐β1) is a potent stimulator of profibrotic responses, including expression of fibrosis markers such as extracellular matrix (ECM) and α‐smooth muscle actin (α‐SMA) through a Smad‐dependent pathway. Therefore, Smad‐dependent Tgf‐β1 signaling is known to play pivotal roles in renal fibrosis. In this regard, we investigated the potential involvement of nuclear factor (erythroid‐derived 2)‐like 2 (NFE2L2; Nrf2) in renal fibrosis with Tgf‐β1–Smad signaling. We previously reported that Smad7, a negative regulator of Smad signaling, may be a linker molecule between the Nrf2 pathway and Tgf‐β1 signaling in human renal tubular epithelial HK2 cell. In this study, we confirmed that elevated protein level of Smad7 attenuates Tgf‐β1 signaling by degradation of Tgf‐β1 Receptor 1 (TβR1) in Kelch‐like ECH‐associated protein 1 (Keap1) knockdown mouse mesangial MES‐13 cell, a cellular model of Nrf2 overexpression. As one of potential underlying molecular mechanisms of Smad7 regulation by Nrf2, reduced level of Smad‐ubiquitination‐regulatory factor 1 (Smurf1), an E3 ubiquitin ligase for Smad7, was remarkable. Further, we demonstrated that a Nrf2 inducer bardoxolone methyl ester (BARD), which is on clinical trials for kidney diseases, also elevated Smad7 level and attenuated Tgf‐β1 signaling in MES‐13. Collectively, these results indicate that Nrf2 can be an effective inhibitor of Smad‐dependent Tgf‐β1 signaling, and imply the beneficial role of Nrf2 in renal fibrosis.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF‐2015R1A2A1A10054384)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A three-dimensional (3D) bioprinted model of the renal proximal tubule for evaluation of drug-induced nephrotoxicity

Renal fibrosis, a characteristic of all chronic kidney diseases, lacks effective therapeutic drugs currently. Pirfenidone (PFD), a small molecule drug with good oral bioavailability, is widely used in idiopathic pulmonary fibrosis and exerts anti-fibrotic, anti-inflammatory, antioxidant, and anti-apoptotic effects. These effects have been attributed to the suppression of cell growth factors (in particular, but not exclusively, transforming growth factor-β) and the epithelial–mesenchymal transition, as well as the possible down-regulation of pro-inflammatory mediators (such as tumour necrosis factor-α), the protection of mitochondrial function, and the regulation of inflammatory cells. Considering the activation of similar anti-fibrotic pathways in lung and kidney disease and the broad activity of PFD, this drug has improved the treatment of the renal fibrotic disease. In this review, we briefly summarize the pharmacokinetics and safety of PFD as well as the mechanisms of PFD focusing on kidney disease. We summarize the effects of PFD on renal function and pathological alterations based on animal experiments, as well as changes in growth factors based on both animal and renal cell experiments. Moreover, given the activation of similar profibrotic pathways in pulmonary diseases and other disorders, we reviewed in-depth the possible signalling pathways targeted by PFD to attenuate renal fibrosis and protect renal function. Finally, we provide an overview of the current clinical trials of PFD for the treatment of renal fibrosis.

Elsholtzia ciliata (Thunb.) Hylander attenuates renal inflammation and interstitial fibrosis via regulation of TGF-ß and Smad3 expression on unilateral ureteral obstruction rat model

BackgroundRenal interstitial fibrosis is characterized by excessive accumulation of extracellular matrix, which leads to end-stage renal failure. PurposeThe aim of this study was to explore the effect of Elsholtzia ciliata (Thunb.) Hylander ethanol extract (ECE) on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Study designAfter quantitative analysis of ECE using the high performance liquid chromatography-photodiode array (HPLC-PDA) method, an in vitro study was performed to assess the anti-inflammatory and anti-fibrotic effects of ECE, using lipopolysaccharide (LPS) and transforming growth factor-ß (TGF-ß), respectively. MethodsFor in vivo study, all male Sprague Dawley (SD) rats (n=10/group), except for those in the control group, underwent UUO. The rats were orally treated with water (control), captopril (positive control, 200mg/kg), and ECE (300 and 500mg/kg) for 14 days. ResultsIn ECE, luteolin and rosmarinic acid were relatively abundant among the other flavonoids and phenolic acids. ECE treatment ameliorated LPS-induced overexpression of nuclear factor-κB, tumor necrosis factor (TNF-α), and interleukin-6 and improved oxidative stress in RAW 264.7 cells. Furthermore, ECE treatment suppressed TGF-ß-induced α-smooth muscle actin and matrix metalloproteinase 9 expression in human renal mesangial cells. In the UUO model, 14 consecutive days of ECE treatment improved UUO-induced renal damage and attenuated histopathological alterations and interstitial fibrosis. Moreover, the renal expression of TNF-α, TGF-ß, and Smad 3 were inhibited by ECE treatment. ConclusionTaken together, the effects of ECE may be mediated by blocking the activation of TGF-ß and inflammatory cytokines, leading subsequently to degradation of the ECM accumulation pathway. Based on these findings, ECE might serve as an improved treatment strategy for renal fibrotic disease.

Thrombospondin-1 Gene Polymorphism Is Associated with Estimated Pulmonary Artery Pressure in Patients with Sickle Cell Anemia

Thrombospondin-1 (TSP1) is a multifunctional glycoprotein present in platelet α-granules that contains domains for adhesive proteins, enzymes, and cell receptors. It is released by activated platelets and is increased in the plasma of patients with sickle cell anemia (SCA). TSP1 is known to mediate adherence of sickle reticulocytes to endothelial cells. Furthermore, TSP1 also inhibits the NO signaling pathway through its binding to the CD47 cell receptor on endothelial cells and platelets. Thus, we hypothesized that genetic variants in TSP1 may contribute to the marked phenotypic variation seen in SCA.We tested whether genetic variants of the TSP1 gene are associated with biomarkers of SCA severity and specific complications in a cohort of 452 patients with homozygous HbSS disease from the multicenter walk-PHaSST (Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy) trial. Genetic data from 406 patients were included in the analysis. Patients ranged in age from 12 to 70 years, and 51% were females. 307 patients were enrolled at various sites in the United States, while 99 patients were enrolled at Hammersmith Hospital in the UK. A total of 10 SNPs were selected, including haplotype tagged SNPs, coding variants, and SNPs potentially regulating gene expression and alternative splicing. DNA isolated from peripheral blood was used for genotyping.We found that the TSP1 SNPs rs1478604 and rs1478605 were significantly positively correlated with tricuspid regurgitation velocity (TRV) >2.5 m/sec, an echocardiographic marker of pulmonary hypertension associated with increased mortality, under a recessive model (r=0.10, p=0.0288 and r=0.13, p=0.0047 respectively). TRV was also analyzed as a categorical variable using regression analysis, and subjects were grouped based on the following clinically relevant cut-off points, 2.5 m/sec, 2.7 m/sec, and 3 m/sec. Univariate regression analyses showed TRV>2.5 m/sec was independently associated with the recessive genotypes of rs1478604 and rs1478605 (p=0.069 and 0.017 respectively). As previously published, we confirmed the association of TRV with age, gender, NT-proBNP and creatinine, and we adjusted for these variables in multivariate regression analyses for the association of TRV and each SNP. The results showed that the two SNPs were significantly associated with TRV>2.5 m/sec (rs1478604, OR 2.54, 95% CI (1, 6.44), p<0.05 and rs1478605, OR 4.20, 95% CI (1.41, 12.49), p<0.01) even after controlling for these variables.The results suggest that genetic variations in the TSP1 gene are associated with variations in estimated pulmonary artery pressure in patients with SCA. Both rs1478604 and rs1478605 are localized to the 5' untranslated region. Bioinformatic analysis of these SNPs for putative transcription factor binding sites demonstrated that the two SNPs are associated with both gain and loss of transcription factor binding compared to the wild type. Therefore, both SNPs are potentially functional in TSP1 regulation. These data also suggest possible translational implications in light of earlier pre-clinical reports that showed that mice lacking TSP1 were protected from hypoxia-mediate pulmonary and right ventricular remodeling and showed less alterations in PA pressure.In populations without SCA, these alleles have been independently linked to risk of malaria, corneal immune function, and gastric cancer. TSP1 activity has also been linked to fibrotic renal disease in a rat model and to IRI-mediated AKI in a murine model. The mechanism implicated in some of these cases involves disordered TSP1 regulation of transforming growth factor beta (TGF-beta), whose superfamily of receptors is dysregulated in the pathogenesis of pulmonary hypertension and kidney disease.Our findings support a hypothetical model of TSP1 involvement in the pathophysiology of SCA. Efforts to seek validation of the genetic findings from this study in an independent SCA cohort are currently underway. DisclosuresIsenberg:RCTI, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313; Vasculox, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313.

Heparanase: another renal player controlled by vitamin D.

Vitamin D deficiency is inevitable in chronic kidney diseases. Clinical and experimental therapies with vitamin D supplements or analogues have demonstrated nephroprotective effects, which vitamin D exerts partly by controlling the renin-angiotensin-aldosterone system, but also by modulating other signalling pathways. In recent work published in the Journal of Pathology, Garsen and colleagues identified heparanase as a novel target of vitamin D and its antiproteinuric activity. Heparanase is an endoglycosidase with a role in remodelling the extracellular matrix through its ability to degrade heparan sulphate, and is involved in the pathogenesis of several proteinuric and fibrotic renal diseases. The new evidence that vitamin D inhibits heparanase expression sets the stage for a better understanding of the vitamin's kidney-protecting effects and its possible application to proteinuric and non-proteinuric chronic kidney diseases.

MiR-21 promotes renal fibrosis in diabetic nephropathy by targeting PTEN and SMAD7.

The cytokine transforming growth factor (TGF)-β1 plays a central role in diabetic nephropathy (DN) with data implicating the miRNA (miR) miR-21 as a key modulator of its prosclerotic actions. In the present study, we demonstrate data indicating that miR-21 up-regulation positively correlates with the severity of fibrosis and rate of decline in renal function in human DN. Furthermore, concomitant analyses of various models of fibrotic renal disease and experimental DN, confirm tubular miR-21 up-regulation. The fibrotic changes associated with increased miR-21 levels are proposed to include the regulation of TGF-β1-mediated mothers against decapentaplegic homolog 3 (SMAD3)- and phosphoinositide 3-kinase (PI3K)-dependent signalling pathways via co-ordinated repression of mothers against decapentaplegic homolog 7 (SMAD7) and phosphatase and tensin homologue (PTEN) respectively. This represents a previously uncharacterized interaction axis between miR-21 and PTEN-SMAD7. Targeting of these proteins by miR-21 resulted in de-repression of the respective pathways as reflected by increases in SMAD3 and V-Akt murine thymoma viral oncogene homolog 1 (AKT) phosphorylation. Many of the changes typically induced by TGF-β1, including phosphorylation of signalling mediators, were further enhanced by miR-21. Collectively, these data present a unified model for a key role for miR-21 in the regulation of renal tubular extracellular matrix (ECM) synthesis and accumulation and provide important insights into the molecular pathways implicated in the progression of DN.

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology.

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-β), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.