Interstitial Matrix Deposition Research Articles

Astragaloside IV Alleviates Renal Tubular Epithelial-Mesenchymal Transition via CX3CL1-RAF/MEK/ERK Signaling Pathway in Diabetic Kidney Disease.

BackgroundEpithelial–mesenchymal transition (EMT) plays an important role in interstitial matrix deposition and renal fibrosis in diabetic kidney disease (DKD). It has been verified that Astragaloside IV (AS-IV) is beneficial for ameliorating DKD. However, the underlying mechanisms of AS-IV on regulating EMT in DKD are yet to be established. Accumulated evidence has suggested that C-X3-C motif ligand 1 (CX3CL1) plays a significant role in the progression of EMT.PurposeWe aimed to investigate whether AS-IV could alleviate EMT by regulating CX3CL1 in DKD and reveal its underlying mechanisms.MethodsFor the in vivo study, mice were divided into the following five groups (n=10): db/m+vehicle, db/db+vehicle, db/db+AS-IV-L (10mg/kg/d), db/db+AS-IV-M (20mg/kg/d), db/db+AS-IV-H (40mg/kg/d). After 12 weeks of treatment, the renal injuries were assessed based on the related parameters of urine, blood and histopathological examination. Immunohistochemistry and Western blotting were used to detect relative proteins levels. Then in HK-2 cells, the molecular mechanism of AS-IV attenuating the EMT in mice with DKD through the CX3CL1-RAF/MEK/ERK pathway was studied.ResultsIn the present study, we found that AS-IV reduced urinary protein levels and improved renal pathological damage in DKD mice. Moreover, AS-IV ameliorated the renal tubular EMT induced by hyperglycemia or high glucose (HG), and decreased the expression of CX3CL1 and inhibited the activation of the RAF/MEK/ERK pathway in vivo and in vitro. In HK-2 cells, downregulation of CX3CL1 suppressed the stimulation of the RAF/MEK/ERK pathway and EMT induced by HG. However, CX3CL1 overexpression eliminated the benefits of AS-IV on the RAF/MEK/ERK pathway and EMT.ConclusionIn summary, we indicated that AS-IV alleviates renal tubular EMT through the CX3CL1-RAF/MEK/ERK signaling pathway, indicating that CX3CL1 could be a potential therapeutic target of AS-IV in DKD.

PTEN-induced partial epithelial-mesenchymal transition drives diabetic kidney disease.

Epithelial-mesenchymal transition (EMT) contributes significantly to interstitial matrix deposition in diabetic kidney disease (DKD). However, detection of EMT in kidney tissue is impracticable, and anti-EMT therapies have long been hindered. We reported that phosphatase and tensin homolog (PTEN) promoted transforming growth factor beta 1 (TGF-β), sonic hedgehog (SHH), connective tissue growth factor (CTGF), interleukin 6 (IL-6), and hyperglycemia-induced EMT when PTEN was modified by a MEX3C-catalyzed K27-linked polyubiquitination at lysine 80 (referred to as PTENK27-polyUb). Genetic inhibition of PTENK27-polyUb alleviated Col4a3 knockout-, folic acid-, and streptozotocin-induced (STZ-induced) kidney injury. Serum and urine PTENK27-polyUb concentrations were negatively correlated with glomerular filtration rate (GFR) for diabetic patients. Mechanistically, PTENK27-polyUb facilitated dephosphorylation and protein stabilization of TWIST, SNAI1, and YAP in renal epithelial cells, leading to enhanced EMT. We identified that a small molecule, triptolide, inhibited MEX3C-catalyzed PTENK27-polyUb and EMT of renal epithelial cells. Treatment with triptolide reduced TWIST, SNAI1, and YAP concurrently and improved kidney health in Col4a3 knockout-, folic acid-injured disease models and STZ-induced, BTBR ob/ob diabetic nephropathy models. Hence, we demonstrated the important role of PTENK27-polyUb in DKD and a promising therapeutic strategy that inhibited the progression of DKD.

Effects of cell-type-specific expression of a pan-caspase inhibitor on renal fibrogenesis

The caspase family of enzymes is grouped into two major sub-families, namely apoptotic and inflammatory caspases, which play central roles in the induction of apoptosis, regulation of inflammation and immunity, and cellular differentiation. The role of caspase activation in tubular epithelium and interstitial cells of 3 lines of transgenic mice with obstructed nephropathy was examined: p35 mice bearing the pan-caspase inhibitor protein expressed by the p35 gene separated from the universal CAG promoter by a floxed STOP sequence were crossed with γGT.Cre and FSP1.Cre mice that express Cre recombinase in the cortical tubular epithelium and FSP1(+) interstitial cells, respectively. The γGT.Cre;p35, FSP1.Cre;p35 and p35 control mice were then challenged with unilateral ureter obstruction (UUO). Proinflammatory parameters such as protein levels of active IL-1β subunit and mRNA levels of TNF-α and NOD-like receptor pyrin domain containing-3, and profibrogenic parameters such as interstitial matrix deposition and mRNA levels of fibronectin EIIIA isoform and α1 chain of procollagen type I in the kidneys were significantly increased at 7days in the FSP1.Cre;p35- and p35-UUO mice, but not in the γGT.Cre;p35-UUO mice. These changes paralleled the numbers of apoptotic nuclei in tubules, but not in interstitial cells, and the protein levels of active caspase-3 subunit in the kidneys of FSP1.Cre;p35-, p35- and γGT.Cre;p35-UUO mice. This study provides evidence of the critical role of caspase activation in the tubular epithelium, but not in FSP1(+) interstitial cells, in apoptosis and inflammasome induction, leading to proinflammatory and profibrogenic processes in fibrous kidneys with UUO.

The Effect of Omega‐3 Polyunsaturated Fatty Acid and Soy Protein Isolate Supplementation on Kidney Function in Female Polycystic Kidney Diseased Rats

Polycystic kidney disease (PKD) is a genetic disorder characterized by multiple cyst formation that increases renal size, structural damage, and loss of function. Studies showed soy protein isolate (SPI) protective against chronic kidney disease and omega‐3 polyunsaturated fatty acids (n‐3 PUFAs) decrease inflammation. Few studies have examined the role of diet in PKD. The study objective was to investigate whether SPI and/or n‐3 PUFA supplementation attenuates PKD progression. Young (age 28 d) female pck rats were randomly assigned (n=12/group) to diets consisting of casein + corn oil (Casein + CO), casein + soybean oil (casein + SO), SPI + soybean oil (SPI + SO) or SPI + 1:1 soybean/salmon oil (SPI + BLEND) for 12‐weeks. Kidney weights were highest in the SPI + BLEND group. Histology showed interstitial inflammation, fibrotic changes, interstitial matrix deposition, and structural effacement due to cyst growth in all groups. Cortical cyst obstruction indicating more extensive nephron damage was only observed in the SPI + BLEND group. The SPI +BLEND group had the highest (11.78 + 0.60 mg/dL, P=0.01) and Casein + SO group the lowest (9.28 + 0.81, P=0.01) serum blood urea nitrogen. Urinary calcium and phosphorus was higher in the casein than SPI groups. There were no significant differences in serum calcium, phosphorus, uric acid, total protein or albumin. Based on the results, diet did not attenuate PKD progression.Grant Funding Source: WVU Hatch Grant H459

Genetic susceptibility to atrial fibrillation: does heart failure change our perspective?

This editorial refers to 'Genetic polymorphisms confer risk of atrial fibrillation in patients with heart failure: a population-based study', by J.G. Smith et al., published in this issue on pages 250-257.

Effects and mechanisms of angiotensin II receptor blockade with telmisartan in a normotensive model of mesangioproliferative nephritis

Renoprotective actions of angiotensin receptor blockers are not well established in normotensive, low-grade proteinuric glomerular diseases. We examined the effect of low-dose telmisartan (LT) and high-dose telmisartan (HT) versus conventional antihypertensive therapy in the rat anti-Thy1.1 model of glomerulonephritis. Rats were randomized on Day 4 after disease induction to no treatment (CT, control), LT or HT or hydrochlorothiazide + hydralazine (HCT + H). All rats remained normotensive: HT and HCT + H reduced blood pressure by 15-20%. LT, HT and HCT + H reduced glomerular endothelial cell proliferation and glomerular and interstitial matrix deposition on Day 14. Only HT reduced podocyte damage and tubular cell dedifferentiation on Day 9 and mesangial cell activation on Day 14. By gene expression analysis arrays, we identified discs-large homolog 1 and angiopoietin-like 4 as potential mediators of the HT effects. In addition, we identified several pathways possibly related to the pleiotropic effects of HT, including growth factor signalling, mammalian target of rapamycin signalling, protein ubiquitination, the Wnt-beta catenin pathway and hypoxia signaling. In summary, treatment with HT, initiated after the induction of disease, ameliorates glomerular and tubulointerstitial damage. We provide the first comprehensive insight into the mechanisms underlying the renoprotective effect of high-dose angiotensin II receptor blockers (ARBs). Our study lays the basis for future investigations on novel pathways affected by ARBs in renal disease.

Deletion of the Met receptor in the collecting duct decreases renal repair following ureteral obstruction

Hepatocyte growth factor and its receptor, Met, activate biological pathways necessary for repair and regeneration following kidney injury. The Met receptor is expressed in multiple cell types within the kidney, each of which is capable of regulating fibrotic responses. To specifically address the role of the Met receptor in the adult collecting duct during renal injury, a conditional knockout mouse (Met(fl/fl);HoxB7-Cre) was generated and tested using unilateral ureteral obstruction, a model of nephron injury, fibrosis, and repair. Following obstruction in these mice there was increased expression of collagens I and IV along with plasminogen activator inhibitor 1, a known regulator of matrix degradation, compared to ureteral obstructed non-flox littermates. There were trends toward increased interstitial fibrosis, infiltration of the interstitium, and acute tubular necrosis in the knockout mice despite similar degrees of hydronephrosis to the control littermates. The Met(fl/fl);HoxB7-Cre mice; however, had reduced tubular cell proliferation and kidney regenerative capacity after release of the obstruction, thus leading to diminished functional recovery. We suggest that Met receptor signaling in the collecting duct acts as a major regulator of cell survival and propagation of the repair process with a possible secondary role to diminish inflammatory and fibrotic responses.

Disease-Modifying Mutations in Familial Hypertrophic Cardiomyopathy

Cardiomyopathies are primary disorders of cardiac muscle associated with abnormalities of cardiac wall thickness, chamber size, contraction, relaxation, conduction, and rhythm. They are a major cause of morbidity and mortality at all ages and, like acquired forms of cardiovascular disease, frequently progress into heart failure.1 In contrast to dilated cardiomyopathy (DCM), which is characterized by left or biventricular dilation in association with depressed myocardial contractility,1 familial hypertrophic cardiomyopathy (FHC) is characterized by increased cardiac mass with myocyte and myofibrillar disarray. The disease is associated with electrical instability (ie, atrial and ventricular arrhythmias), which makes it the leading cause of death in young athletes. There is marked diversity in the morphological features and clinical manifestations of both DCM and FHC.2 Article p 1820 Family studies have demonstrated that hypertrophic cardiomyopathy is a heritable disorder that is transmitted as an autosomal-dominant trait or as a sporadic disease. There is neither a racial nor an ethnic predisposition to this condition. Hypertrophic cardiomyopathy is not a rare condition; several noninvasive studies have demonstrated echocardiographic criteria for this disease in 0.2% of young adults (reviewed by Maron3). Genetic linkage studies in FHC have found disease loci on at least 14 different genes, most of them encoding sarcomeric genes; hence, FHC has been called a disease of the sarcomere. In addition, several mutations within the Ras pathway have been shown to cause at least syndromic forms of hypertrophic cardiomyopathy,4,5 pointing to the presence of additional but poorly understood disease-causing and/or modifier genes. Thus far, however, most of the clinical heterogeneity of FHC has been accounted for by the substantial diversity of gene defects that cause the condition (ie, up to 45% of disease-causing mutations have been observed in the β-myosin heavy chain [MHC] gene myh7 and up to 35% in …

The great escape--myofibroblasts in fibrosis and the immune system

The extent of renal interstitial fibrosis is one of the most important prognostic factors in kidney biopsies and is a key component of almost every form of progressive renal failure [1]. Similarly, fibrosis is also a morphological correlate of progressive organ failure in liver, lung and heart. In all organs, fibrosis is the result of a process called fibrogenesis in which myofibroblasts (the name is due to the de novo expression of α-smooth muscle actin in these cells whose expression is normally restricted to vascular smooth muscle cells) are the key effector cells [2]. Myofibroblasts are formed via an intermediate form entitled the ‘protomyofibroblast’ characterized by the acquisition of contractile stress fibres [3]. In the kidney, myofibroblasts are derived mainly from activation of resident interstitial fibroblasts, albeit differentiation processes of periadventitial cells, mesenchymal stem cells or tubular epithelial cells may play a role as well [4]. Thus, myofibroblasts may have several precursors, and similar mechanisms of myofibroblast formation have been described in the liver [5] and lung [6]. However, it should be appreciated that not all matrix-synthesizing cells are myofibroblasts and vice versa that not all myofibroblasts contribute to interstitial matrix deposition pointing to a certain degree of heterogeneity of these cells [3].

Mice Lacking Neutrophil Elastase Are Resistant to Bleomycin-Induced Pulmonary Fibrosis

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.

Plasminogen Activator Inhibitor-1 in Chronic Kidney Disease

In 1984, Loskutoff et al. ([1][1]) purified plasminogen activator inhibitor-1 (PAI-1) from conditioned media of cultured endothelial cells. This 50-kd glycoprotein is the primary physiologic inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activators (tPA and uPA,

Renal injury in streptozotocin-diabetic Ren2-transgenic rats is mainly dependent on hypertension, not on diabetes

Induction of streptozotocin (STZ) diabetes in hypertensive rats transgenic for the mouse ren-2 gene (TGR) has been described as a model of progressive diabetic nephropathy. We investigated the long-term course of STZ diabetes in TGR and appropriate Sprague-Dawley control rats (SD) and tested the role of angiotensin-dependent hypertension by treating rats with the angiotensin II type 1 receptor blocker losartan (1 mg.kg(-1).day(-1)) via osmotic minipumps. Five weeks after STZ injection, diabetes developed in TGR and SD. Urinary albumin excretion was increased by diabetes and, to a much higher degree, by hypertension. The effects of hypertension and diabetes were not additive, and only the effects of hypertension were ameliorated by losartan. A similar pattern was observed for cell proliferation and macrophage infiltration in the kidney. In contrast, the effects of hypertension and diabetes on glomerular collagen IV accumulation were additive 5 wk after STZ injection. In a long-term study for 20 wk after STZ, survival was better in STZ-treated TGR than in normoglycemic TGR, whereas all SD survived. Impaired creatinine clearance and increased macrophage infiltration as well as glomerular and interstitial matrix deposition were prominent in TGR compared with SD, regardless of the presence or absence of diabetes. In conclusion, STZ diabetes in TGR may be useful to study glomerular and interstitial matrix deposition early in the course of diabetes. However, the long-term course of this animal model resembles severe hypertensive nephrosclerosis, rather than progressive diabetic nephropathy.

The role of mRNA stability in airway remodelling

As a consequence of long-term exposure to inflammatory mediators, the airways of asthmatics become remodelled. Airway fibrosis becomes apparent, with thickening of the lamina recticularis and increased interstitial matrix deposition being typical features of an asthmatic airway. Mucus hypersecretion occurs, airway smooth muscle mass is increased and neovascularization is evident in the subepithelial mucosa. As development of a remodelled airway is correlated with deterioration of lung function in asthmatics, there is an urgent need for therapies that reduce airway inflammation and reverse structural changes in a remodelled airway. However, in order to design efficacious anti-remodelling agents we first need a greater understanding of the molecular mechanism/s underlying the development of airway remodelling. To date, however, most studies have primarily focused on the transcriptional regulation of genes that promote airway remodelling. Post-transcriptional mechanisms, such as control of mRNA stability, remain largely unexplored. Levels of cellular mRNA transcripts are regulated by controlling the rate at which the mRNA decays, thus investigation into the mechanisms underlying mRNA stability in asthma are of critical importance. Therefore, this review will present an overview of the control of mRNA stability and examine how mRNA stability may play a role in the development of airway remodelling in asthma.

Bradykinin decreases plasminogen activator inhibitor-1 expression and facilitates matrix degradation in the renal tubulointerstitium under angiotensin-converting enzyme blockade.

A number of experimental and clinical investigations support the notion that angiotensin-converting enzyme inhibitor (ACEi) and angiotensin II type 1 receptor blocker (ARB) compounds attenuate renal fibrosis. Fibrosis can be attenuated by either suppressing matrix formation or facilitating matrix degradation. In this study, drugs of ACEi and ARB classes were tested for their ability to facilitate matrix degradation in the kidney. A murine model system in which cyclosporin A (CsA) treatment for a specified period caused interstitial matrix deposition in the kidney was used. CsA was then discontinued, and experimental procedures were initiated to investigate matrix degradation. Benazepril, an ACEi, facilitated matrix degradation via the bradykinin (BK) B2 receptor on tubular epithelial cells in the kidney, whereas CGP-48933, an ARB, did not. In this murine model of CsA nephropathy under ACE blockade, plasminogen activator inhibitor-1 (PAI-1) expression was decreased in tubular epithelial cells, possibly leading to conversion of plasminogen to plasmin by plasminogen activator and subsequent activation of matrix metalloproteinases. These findings were confirmed in this study by measurements of plasmin activity, collagenolytic activity, and matrix metalloproteinase activities in the kidneys. In tubular epithelial cells stimulated in vitro, BK suppressed PAI-1 gene expression. All of these results suggest that ACEi can decrease PAI-1 expression via BK, thereby facilitating matrix degradation via activation of degradative enzymes to reduce interstitial matrix deposition.

Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury.

The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses such as proliferation and apoptosis. To elucidate its role in the heart, we generated mice harboring a cardiomyocyte-restricted knockout of STAT3 using Cre/loxP-mediated recombination. STAT3-deficient mice developed reduced myocardial capillary density and increased interstitial fibrosis within the first 4 postnatal months, followed by dilated cardiomyopathy with impaired cardiac function and premature death. Conditioned medium from STAT3-deficient cardiomyocytes inhibited endothelial cell proliferation and increased fibroblast proliferation, suggesting the presence of paracrine factors attenuating angiogenesis and promoting fibrosis in vitro. STAT3-deficient mice showed enhanced susceptibility to myocardial ischemia/reperfusion injury and infarction with increased cardiac apoptosis, increased infarct sizes, and reduced cardiac function and survival. Our study establishes a novel role for STAT3 in controlling paracrine circuits in the heart essential for postnatal capillary vasculature maintenance, interstitial matrix deposition balance, and protection from ischemic injury and heart failure.

Angiotensin II type 1 receptor blockade attenuates renal fibrogenesis in an immune-mediated nephritic kidney through counter-activation of angiotensin II type 2 receptor

The relative roles of angiotensin II (Ang II) type 1 receptor (AT 1R) and Ang II type 2 receptor (AT 2R) in immune-mediated nephritis are unknown, and the effect of the blockade of AT 1R and its indirect counter-activation of AT 2R relative to the anti-fibrotic action in this disease is unclear. To address this question, we studied the role of AT 1R and AT 2R in anti-glomerular basement membrane nephritis in SJL mice. Groups of mice were treated with either an AT 1R antagonist (CGP-48933; CGP group), an AT 2R antagonist (PD-123319; PD group), both (CGP/PD group), or a vehicle (PCt group) from Day 29 to 56. At Day 56 post-treatment, fibrosis-related parameters such as interstitial matrix deposition, and the expression of genes of TGF-β1, plasminogen activator inhibitor-1, and type I collagen were significantly reduced in the kidney in the CGP group. There were no significant effects on these parameters in the PD group. However, this anti-fibrotic action by CGP-48933 was totally abolished by co-treatment with PD-123319 in the CGP/PD group. The gene expression of renin was significantly increased in the kidneys in the CGP and CGP/PD groups, suggesting that CGP-48933 had increased Ang II generation in those groups. In conclusion, counter-activation of AT 2R by increased Ang II under AT 1R blockade likely conferred an anti-fibrotic protection in this model.

Delayed administration of hepatocyte growth factor reduces renal fibrosis in obstructive nephropathy.

Hepatocyte growth factor (HGF) is a renotropic protein that elicits antifibrogenic activity by preventing the activation of matrix-producing myofibroblast cells in animal models of chronic renal diseases. However, whether a delayed administration of HGF can still attenuate renal fibrosis remains uncertain. In this study, we examined the therapeutic potential of exogenous HGF on an established renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Three days after UUO, the obstructed kidneys displayed interstitial fibrotic lesions with characteristic features of an established renal fibrosis, as manifested by myofibroblast activation, fibronectin overexpression, interstitial matrix deposition, and transforming growth factor-beta1 upregulation. Beginning at this time point, administration of recombinant HGF into mice by intravenous injections for 11 days markedly suppressed the progression of renal interstitial fibrosis. HGF significantly suppressed renal alpha-smooth muscle actin expression, total kidney collagen contents, interstitial matrix components, such as fibronectin, and renal expression of transforming growth factor-beta1 and its type I receptor. Compared with the starting point (3 days after UUO), HGF treatment largely blunted the progression of myofibroblast accumulation and collagen deposition but did not reverse it. Delayed administration of HGF also suppressed the myofibroblastic transdifferentiation from tubular epithelial cells in vitro, as demonstrated by a decline in alpha-smooth muscle actin and fibronectin expression. These results suggest that exogenous HGF exhibits potent therapeutic effects on retarding the progression of an established renal fibrosis.

Preventive effect of ACE inhibitor on interstitial myofibroblast formation and matrix deposition in a nephrotic model

The nephrotic mouse (ICGN strain) is a useful model for progressive nephrotic syndrome (NS). In the present study, we demonstrated the preventive effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor, on the progression of renal dysfunction and tubulo-interstitial fibrosis in the NS mice. Administration of enalapril (5 mg/dL in drinking water.) to the 4-week-old NS mice for a 4-week-period did not improve their nephrotic symptoms such as albuminuria and hypoalbuminemia, but significantly suppressed the increases in blood urea nitrogen and serum creatinine levels. Renal histopathology demonstrated that the administration of the ACE inhibitor significantly attenuated the progression of the tubular and interstitial lesions (tubular dilatation, luminal cast accumulation and interstitial expansion) rather than the glomerular sclerotic changes. The suppression of the increase in blood urea nitrogen level by enalapril depended on the attenuated tubular injury rather than on the unchanged glomerular matrix deposition. Immunohistochemical examination revealed that the administration of the ACE inhibitor suppressed the formation of myofibroblasts, identified by the alpha-smooth muscle actin-positive cells, in the interstitial spaces. Consequently, interstitial matrix deposition was significantly reduced in the NS mice treated with enalapril. From the results obtained with the spontaneous nephrotic model, we emphasize a possibility that ACE inhibitor may be effective for attenuating progression of renal dysfunction and fibrosis in human NS, even if the ACE inhibitor fails to improve nephrotic symptoms such as albuminuria and hypoalbuminemia.