Pathogenesis Of Renal Fibrosis Research Articles

MiR-192 prevents renal tubulointerstitial fibrosis in diabetic nephropathy by targeting Egr1.

Diabetic nephropathy (DN), as the most common and serious diabetic microvascular complication, has become the first cause of end-stage renal disease (ESRD) in many countries and regions. However, the pathogenesis of renal fibrosis during the development of DN remains unknown. The expression levels of miR-192 and early growth response factor 1 (Egr1) were determined by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western blotting in the renal tissues of Otsuka-Long-Evans-Tokushima-Fatty (OLETF) and Long-Evans-Tokushima-Otsuka (LETO) rats. The diabetic kidney environment was simulated by a high-sugar medium. The expression levels of miR-192 and Egr1 were further measured in the HK-2 cell line. Egr1 was verified as a potential target of miR-192 by using bioinformatics analysis and luciferase activity assay. The expression level of Egr1 was determined by overexpressing and knocking down the expression of miR-192. In addition, Western blotting was used to determine changes in Transforming growth factor-beta 1 (TGF-β1) and fibronectin (FN). Compared with the kidney tissue of LETO rats, the expression of miR-192 was decreased in OLETF rats, whereas the expression of Egr1 was increased. We found the same phenomenon in the HK-2 cell line cultured in the high-glucose medium. Next, miR-192 can act on Egr1 through 3'-UTR to reduce the expression of Egr1 verified by luciferase assay. In addition, the expression levels of TGF-β1 and FN changed significantly, as the expression level of Egr1 increased or decreased. MiR-192 causes degradation of TGF-β1 and FN through targeting Egr1 and affects the progression of TIF and even DN.

Antifibrotic role of PGC-1α-siRNA against TGF-β1-induced renal interstitial fibrosis

Peroxisome proliferator-activated receptor coactivator-1 alpha (PGC-1α) is a transcriptional coactivator that regulates energy metabolism and mitochondrial biogenesis. Recently, mitochondrial dysfunction has been indicated as an established risk factor for the development of renal fibrosis. However, whether PGC-1α is involved in the pathogenesis of renal fibrosis is unknown. In this study, we treated NRK-49F (normal rat kidney fibroblast) cells with transforming growth factor-beta 1 (TGF-β1) for 24 h to establish an in vitro fibrosis model. TGF-β1 induced the upregulation of type I collagen, fibronectin, TGF-β receptor I (TGFβ-RI), TGFβ-RII, Smad4, and pSmad2/3, as well as PGC-1α. NRK-49F cells transfected with pcDNA-PGC-1α showed significantly increased expression of fibronectin and type I collagen, as revealed by western blot assay. Interestingly, transfection with PGC-1α-siRNA caused a stark reversal of TGF-β1-induced cellular fibrosis, with concomitant suppression of fibronectin and type I collagen, as revealed by western blot and immunofluorescence assays. Moreover, SB431542 (TGFβ-RI), LY294002 (PI3K/Akt), and SB203580 (p38 MAPK), inhibitors of TGF-β-associated pathways, markedly suppressed TGF-β1-induced PGC-1α upregulation. These results implicate a role of PGC-1α in renal interstitial fibrosis mediated via the TGFβ-RI, PI3K/Akt, and p38 MAPK pathways. Our findings that PGC-1α-siRNA downregulates fibronectin and type I collagen suggest that it can be used as a novel molecular treatment for renal fibrosis.

Runt-Related Transcription Factor 1 (RUNX1) Promotes TGF-β-Induced Renal Tubular Epithelial-to-Mesenchymal Transition (EMT) and Renal Fibrosis through the PI3K Subunit p110δ

Renal fibrosis is widely considered a common mechanism leading to end-stage renal failure. Epithelial-to-mesenchymal transition (EMT) plays important roles in the pathogenesis of renal fibrosis. Runt-related transcription factor 1(RUNX1) plays a vital role in hematopoiesis via Endothelial-to-Hematopoietic Transition (EHT), a process that is conceptually similar to EMT, but its role in EMT and renal fibrosis is unclear. Here, we demonstrate that RUNX1 is overexpressed in the processes of TGF-β-induced partial EMT and renal fibrosis and that the expression level of RUNX1 is SMAD3-dependent. Knockdown of RUNX1 attenuated both TGF-β-induced phenotypic changes and the expression levels of EMT marker genes in renal tubular epithelial cells (RTECs). In addition, overexpression of RUNX1 promoted the expression of EMT marker genes in renal tubular epithelial cells. Moreover, RUNX1 promoted TGF-β-induced partial EMT by increasing transcription of the PI3K subunit p110δ, which mediated Akt activation. Specific deletion of Runx1 in mouse RTECs attenuated renal fibrosis, which was induced by both unilateral ureteral obstruction (UUO) and folic acid (FA) treatment. These findings suggest that RUNX1 is a potential target for preventing renal fibrosis.

Wnt9a Promotes Renal Fibrosis by Accelerating Cellular Senescence in Tubular Epithelial Cells.

Cellular senescence is associated with renal disease progression, and accelerated tubular cell senescence promotes the pathogenesis of renal fibrosis. However, the underlying mechanism is unknown. We assessed the potential role of Wnt9a in tubular cell senescence and renal fibrosis. Compared with tubular cells of normal subjects, tubular cells of humans with a variety of nephropathies and those of several mouse models of CKD expressed high levels of Wnt9a that colocalized with the senescence-related protein p16INK4A Wnt9a expression level correlated with the extent of renal fibrosis, decline of eGFR, and expression of p16INK4A Furthermore, ectopic expression of Wnt9a after ischemia-reperfusion injury (IRI) induced activation of β-catenin and exacerbated renal fibrosis. Overexpression of Wnt9a exacerbated tubular senescence, evidenced by increased detection of p16INK4A expression and senescence-associated β-galactosidase activity. Conversely, shRNA-mediated knockdown of Wnt9a repressed IRI-induced renal fibrosis in vivo and impeded the growth of senescent tubular epithelial cells in culture. Notably, Wnt9a-induced renal fibrosis was inhibited by shRNA-mediated silencing of p16INK4A in the IRI mouse model. In a human proximal tubular epithelial cell line and primary renal tubular cells, Wnt9a remarkably upregulated levels of senescence-related p16INK4A, p19ARF, p53, and p21 and decreased the phosphorylation of retinoblastoma protein. Wnt9a also induced senescent tubular cells to produce TGF-β1, which promoted proliferation and activation in normal rat kidney fibroblasts. Thus, Wnt9a drives tubular senescence and fibroblast activation. Furthermore, the Wnt9a-TGF-β pathway appears to create a reciprocal activation loop between senescent tubular cells and activated fibroblasts that promotes and accelerates the pathogenesis of renal fibrosis.

Adenovirus-mediated P311 ameliorates renal fibrosis through inhibition of epithelial-mesenchymal transition via TGF-β1-Smad-ILK pathway in unilateral ureteral obstruction rats

Epithelial-mesenchymal transition (EMT) is a critical step and key factor during renal fibrosis. Preventing renal tubular EMT is important for delaying the progression of chronic kidney disease (CKD). P311, a highly conserved 8-kDa intracellular protein, has been indicated as an important factor in myofibroblast transformation and in the progression of fibrosis. However, the related studies on P311 on renal fibrosis are limited and the mechanisms of P311 in the progression of renal tubulointerstitial fibrosis remain largely unknown. In the present study, we examined the effect of P311 on transforming growth factor-β1(TGF-β1)-mediated EMT in a rat model of unilateral ureteral occlusion(UUO) renal fibrosis. The recombinant adenovirus p311 (also called Ad-P311) was constructed and transferred it into UUO rats, the preventive effect and possible mechanism of P311 on TGF-β1-mediated EMT were explored. The UUO model was established successfully and Ad-P311 was administered into UUO rats each week for 4weeks, then the serum levels of Cr, blood urea nitrogen(BUN) and albumin(ALB) were evaluated. H&E staining and Masson staining were performed to observe the pathological changes of kidneys. Immunohistochemical staining and western blot analysis were used to examine the EMT markers [E-cadherin and α-smooth muscle actin (α-SMA)], and signal transducers (p-Smad2/3 and Smad7). Integrin linked kinase(ILK) as a keyintracellular mediator that controls TGF-β1-mediated-EMT was also assayed by western blot analysis. The results showed that P311 could alleviate renal tubular damage and interstitial fibrosis improving Cr, BUN and ALB serum levels in UUO kidneys. Furthermore, P311 attenuated TGF-β1-mediated EMT through Smad-ILK signaling pathway with an increase in α-SMA, pSmad2/3 and ILK expression, and a decrease in E-cadherin and Smad7 expression in UUO kidneys. In conclusion, P311 may be involved in the pathogenesis of renal fibrosis by blocking TGF-β1-mediated EMT via TGF-β1-Smad-ILK pathway in UUO kidneys. P311 may be a novel target for the control of renal fibrosis and the progression of CKD.

Role of artesunate in TGF‑β1‑induced renal tubular epithelial‑mesenchymal transdifferentiation in NRK‑52E cells.

The implications of epithelial-mesenchymal transdifferentiation (EMT) have extended beyond the confines of renal fibrosis to renal tubulointerstitial fibrosis. It has been proposed that EMT may be one of the mechanisms involved in the pathogenesis of renal fibrosis. However, the underlying mechanisms remain unknown. Transforming growth factor (TGF)-β1 is considered to be an important cytokine which regulates the transdifferentiation of tubular epithelial cells into myofibroblasts in renal tubulointerstitial fibrosis. In the present study, normal rat kidney tubular epithelial cells (NRK-52E) were treated for 48 h with TGF-β1 (5 ng/ml) and different concentrations of artesunate (ART; 0.01, 0.1 and 1 µg/ml). Western blotting, reverse transcription-semi quantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression of bone morphogenetic protein (BMP)-7, uterine sensitization-associated gene (USAG)-1, E-cadherin, α-smooth muscle actin (α-SMA) and extracellular matrix collagen type I (Col I) mRNA. ART was able to attenuate renal injury in a unilateral ureteral obstruction model. However, its anti-fibrotic effect remains to be elucidated. In the present study, it was observed that ART was able to ameliorate the TGF-β1-induced alterations in cellular morphology. In addition, ART inhibited the TGF-β1-induced USAG-1 increase and the decrease in BMP-7. Treatment with ART markedly attenuated the TGF-β1-induced upregulation of α-SMA and downregulation of E-cadherin. Additionally, ART was able to significantly attenuate the deposition of interstitial collagens, including Col I. The results of the present study further verified the therapeutic efficacy of ART in TGF-β1-induced renal interstitial fibrosis. These findings indicated that ART may hold the potential to prevent chronic kidney diseases via the suppression of USAG-1 expression or by increasing BMP-7 expression.

Apelin attenuates TGF-β1-induced epithelial to mesenchymal transition via activation of PKC-ε in human renal tubular epithelial cells

Epithelial to mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of renal fibrosis. Apelin, a bioactive peptide, has recently been recognized to protect against renal profibrotic activity, but the underlying mechanism has not yet been elucidated. In this study, we investigated the regulation of EMT in the presence of apelin-13 in vitro. Expression of the mesenchymal marker alpha-smooth muscle actin (α-SMA) and the epithelial marker E-cadherin was examined by immunofluorescence and western blotting in transforming growth factor beta 1 (TGF-β1)-stimulated human proximal tubular epithelial cells. Expression of extracellular matrix, fibronectin and collagen-I was examined by quantitative real-time PCR and ELISA. F13A, an antagonist of the apelin receptor APJ, and small interfering RNA targeting protein kinase C epsilon (PKC-ε) were used to explore the relevant signaling pathways. Apelin attenuated TGF-β1-induced EMT, and inhibited the EMT-associated increase in α-SMA, loss of E-cadherin, and secretion of extracellular matrix. Moreover, apelin activated PKC-ε in tubular epithelial cells, which in turn decreased phospho-Smad2/3 levels and increased Smad-7 levels. APJ inhibition or PKC-ε deletion diminished apelin-induced modulation of Smad signaling and suppression of tubular EMT. Our findings identify a novel PKC-ε-dependent mechanism in which apelin suppresses TGF-β1-mediated activation of Smad signaling pathways and thereby inhibits tubular EMT. These results suggest that apelin may be a new agent that can suppress renal fibrosis and retard chronic kidney disease progression.

Fibroblast growth factor 21 ameliorates high glucose-induced fibrogenesis in mesangial cells through inhibiting STAT5 signaling pathway

Fibroblast growth factor 21 (FGF21) is a member of the FGF family and acts as a potent regulator of glucose and lipid homeostasis, but its effect on renal fibrosis and the underlying mechanisms are totally unknown. The purpose of this study was designed to investigate whether FGF21 has effect on high glucose-induced fibrogenesis in human mesangial cells (HMCs) and the underlying mechanism. High glucose is well known to stimulate the expression of extracelluar matrix (ECM) in human mesangial cells. In humans, overexpression and deposition of ECM lead to renal fibrosis. Thus, in this study, HMCs were incubated in high glucose with or without various concentrations of FGF21. Results demonstrated that the expression of FN, Col, TGF β1 and α-SMA were significantly up-regulated in HMCs. Whereas, treatment with FGF21 down-regulated the expression of FN, Col, TGF β1 and α-SMA. In addition, growth factors are considered to be an important driving force for the pathogenesis of renal fibrosis. Significantly increased PDGF, VEGF and CTGF expression were found in HMCs induced by high glucose. FGF21 treatments significantly decreased these growth factors expression by down-regulating the phosphorylation level of STAT5. Here, we reported for the first time that FGF21 decreases ECM expression by inhibiting STAT5 signal pathway and consequently decreasing the expression of PDGF, VEGF and CTGF.

Shenqi detoxification granule combined with P311 inhibits epithelial-mesenchymal transition in renal fibrosis via TGF-β1-Smad-ILK pathway.

Shenqi detoxification granule (SDG), a traditional Chinese herbal formula, has been shown to have nephroprotective and anti-fibrotic activities in patients with chronic kidney disease (CKD). However, its mechanisms in renal fibrosis and the progression of CKD remain largely unknown. P311, a highly conserved 8-kDa intracellular protein, plays a key role in renal fibrosis by regulating epithelial-mesenchymal transition (EMT). Previously, we found P311 might be involved in the pathogenesis of renal fibrosis by inhibiting EMT via the TGF-β1-Smad-ILK pathway. We also found SDG combined with P311 could ameliorate renal fibrosis by regulating the expression of EMT markers. Here we further examined the effect and mechanism of SDG combined with P311 on TGF-β1-mediated EMT in a rat model of unilateral ureteral occlusion (UUO) renal fibrosis. After establishment of the UUO model successfully, the rats were gavaged with SDG daily and/or injected with recombinant adenovirus p311 (also called Ad-P311) through the tail vein each week for 4 weeks. Serum creatinine (Cr), blood urea nitrogen (BUN) and albumin (ALB) levels were tested to observe renal function, and hematoxylin eosin (HE) and Masson staining were performed to observe kidney histopathology. Furthermore, the expression of EMT markers (E-cadherin and α-smooth muscle actin (α-SMA)) and EMT-related molecules TGF-β1, pSmad2/3, Smad7 and ILK were observed using immunohistochemical staining and Western blot analysis. Treatment with SDG and P311 improved renal function and histopathological abnormalities, as well as reversing the changes of EMT markers and EMT-related molecules, which indicated SDG combined with P311 could attenuate renal fibrosis in UUO rats, and the underlying mechanism might involve TGF-β1-mediated EMT and the TGF-β1-Smad-ILK signaling pathway. Therefore, SDG might be a novel alternative therapy for treating renal fibrosis and delaying the progression of CKD. Furthermore, SDG combined with P311 might have a synergistic effect on attenuating renal fibrosis.

Estrogens do not protect, but androgens exacerbate, collagen accumulation in the female mouse kidney after ureteric obstruction

AimsControversy surrounds the gender basis of progression in chronic kidney disease. Unfortunately, most experimental studies addressing this question do not distinguish between direct effects of estrogen and indirect activation of estrogen receptors through conversion of testosterone to 17β-estradiol by aromatase. We examined the pathogenesis of renal fibrosis in female aromatase knockout (ArKO) mice, which lack circulating and stored estrogens, while having normal levels of testosterone. Main methodsArKO mice and their wild-type (ArWT) counterparts were subjected to unilateral ureteric obstruction (UUO), with kidney tissue collected at day(D) 0, 3 and 9 post-UUO. Effects of 5α-dihydrotestosterone (DHT) administration on each genotype were also studied. Tissue was assessed biochemically and histochemically for fibrosis. Western blot analysis was used to measure α-smooth muscle actin (α-SMA) expression and TGF-β1 signalling. Matrix metalloproteinase-2 (MMP-2) activity was measured by zymography. Key findingsUUO increased collagen content over time (p<0.05 (D3) and p<0.01 (D9) vs day 0), with no difference between genotypes in qualitative (collagen IV staining) and quantitative (hydroxyproline concentration) analyses. Systemic administration of non-aromatizable DHT increased collagen content after 3days of UUO in both genotypes. This was not paralleled by any change in α-SMA (myofibroblast burden) or TGF-β1 signalling but was commensurate with DHT reducing MMP2 activity in both genotypes (p<0.05 vs genotype controls). SignificancePhysiological concentrations of estrogens do not protect the injured kidney from fibrosis progression. Androgens rather than estrogens are the relevant factor involved in regulating disease-related renal scarring in this model.

MAD2B-mediated SnoN downregulation is implicated in fibroblast activation and tubulointerstitial fibrosis

MAD2B, an anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase ζ, is indispensible for mitotic checkpoint control and DNA repair. Previously, we established that MAD2B is expressed in glomerular and tubulointerstitial compartments and participates in high glucose-induced podocyte injury. However, its role in other renal diseases remains elusive. In the present study, we aim to illustrate the potential role of MAD2B in the pathogenesis of renal fibrosis. By immunofluorescence and Western blotting, we found MAD2B expression is obviously increased in tubulointerstitial fibrosis (TIF) patients and unilateral ureteral obstruction (UUO) mice. It is widely accepted that resident fibroblasts are the major source of collagen-producing myofibroblasts during TIF. Therefore, we evaluated the level of MAD2B in fibroblasts (NRK-49F) exposed to transforming growth factor (TGF)-β1 by immunoblotting and revealed that MAD2B is upregulated in a time-dependent manner. Intriguingly, SnoN, a transcriptional repressor of the TGF-β1/Smad signaling pathway, is decreased in TGF-β1-treated fibroblasts as well as the kidney cortex from TIF patients and UUO mice. Either in vitro or in vivo, local genetic depletion of MAD2B by lentiviral transfection could preserve SnoN abundance and suppress Smad3 phosphorylation, which finally dampens fibroblast activation, ECM accumulation, and alleviates the severity of TIF. However, the ubiquitin ligase APC/C is not involved in the MAD2B-mediated SnoN decline, although this process is ubiquitination dependent. In conclusion, our observation proposes that besides cell cycle management, MAD2B has a profibrotic role during fibroblast activation and TIF by suppressing SnoN expression. Targeting the MAD2B-SnoN pathway is a promising intervention for TIF.

Wnt6 regulates epithelial cell differentiation and is dysregulated in renal fibrosis.

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3β (Ser9), nuclear accumulation of β-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-β (TGF-β); Wnt6 reversed TGF-β-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-β-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/β(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.

Experimental inhibition of porcupine-mediated Wnt O-acylation attenuates kidney fibrosis

Activated Wnt signaling is critical in the pathogenesis of renal fibrosis, a final common pathway for most forms of chronic kidney disease. Therapeutic intervention by inhibition of individual Wnts or downstream Wnt/β-catenin signaling has been proposed, but these approaches do not interrupt the functions of all Wnts nor block non-canonical Wnt signaling pathways. Alternatively, an orally bioavailable small molecule, Wnt-C59, blocks the catalytic activity of the Wnt-acyl transferase porcupine, and thereby prevents secretion of all Wnt isoforms. We found that inhibiting porcupine dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Wnt-C59 treatment similarly blunts collagen mRNA expression in the obstructed kidney. Consistent with its actions to broadly arrest Wnt signaling, porcupine inhibition reduces expression of Wnt target genes and bolsters nuclear exclusion of β-catenin in the kidney following ureteral obstruction. Importantly, prevention of Wnt secretion by Wnt-C59 blunts expression of inflammatory cytokines in the obstructed kidney that otherwise provoke a positive feedback loop of Wnt expression in collagen-producing fibroblasts and epithelial cells. Thus, therapeutic targeting of porcupine abrogates kidney fibrosis not only by overcoming the redundancy of individual Wnt isoforms but also by preventing upstream cytokine-induced Wnt generation. These findings reveal a novel therapeutic maneuver to protect the kidney from fibrosis by interrupting a pathogenic crosstalk loop between locally generated inflammatory cytokines and the Wnt/β-catenin signaling pathway.

Role of Bone Marrow-Derived Fibroblasts in Renal Fibrosis

Renal fibrosis represents a common pathway leading to progression of chronic kidney disease. Renal interstitial fibrosis is characterized by extensive fibroblast activation and excessive production and deposition of extracellular matrix (ECM), which leads to progressive loss of kidney function. There is no effective therapy available clinically to halt or even reverse renal fibrosis. Although activated fibroblasts/myofibroblasts are responsible for the excessive production and deposition of ECM, their origin remains controversial. Recent evidence suggests that bone marrow-derived fibroblast precursors contribute significantly to the pathogenesis of renal fibrosis. Understanding the molecular signaling mechanisms underlying the recruitment and activation of the bone marrow-derived fibroblast precursors will lead to novel therapy for the treatment of chronic kidney disease. In this review, we summarize recent advances in our understanding of the recruitment and activation of bone marrow-derived fibroblast precursors in the kidney and the development of renal fibrosis and highlights new insights that may lead to novel therapies to prevent or reverse the development of renal fibrosis.

Depletion of CD8+ T Cells Exacerbates CD4+ T Cell-Induced Monocyte-to-Fibroblast Transition in Renal Fibrosis.

Bone marrow-derived monocyte-to-fibroblast transition is a key step in renal fibrosis pathogenesis, which is regulated by the inflammatory microenvironment. However, the mechanism by which the inflammatory microenvironment regulates this transition is not fully understood. In this study, we examined how the CD8(+) T cell/IFN-γ microenvironment regulates the monocyte-to-fibroblast transition in renal fibrosis. Genetic ablation of CD8 promoted a monocyte-to-fibroblast transition and increased renal interstitial fibrosis, whereas reconstitution of CD8 knockout (KO) mice with CD8(+) T cells decreased fibrosis. However, depletion of CD4(+) T cells in CD8 KO mice also reduced fibrosis. To elucidate the role of CD4(+) T cells in mediating CD8-regulated monocyte-to-fibroblast transition, CD4(+) T cells were isolated from obstructed kidneys of CD8 KO or wild-type mice. CD4(+) T cells isolated from CD8 KO obstructed kidney expressed more IL-4 and GATA3 and less IFN-γ and T-bet and showed increased monocyte-to-fibroblast transition in vitro compared with those isolated from wild-type obstructed kidney. To examine the role of IFN-γ-expressing CD8(+) T cells, we reconstituted CD8 KO mice with CD8(+) T cells isolated from IFN-γ KO mice. The IFN-γ KO CD8(+) cells had no effect on IL-4, GATA3, IFN-γ, and T-bet mRNA expression in obstructed kidneys or renal fibrosis. Taken together, our findings identify the axis of CD8(+) T cells and IFN-γ-CD4(+) T cells as an important microenvironment for the monocyte-to-fibroblast transition, which negatively regulates renal fibrosis.

Protein DJ-1 and its anti-oxidative stress function play an important role in renal cell mediated response to profibrotic agents.

In the pathogenesis of renal fibrosis, oxidative stress (OS) enhances the production of reactive oxygen species (ROS) leading to sustained cell growth, inflammation, excessive tissue remodelling and accumulation, which results in the development and acceleration of renal damage. In our previous work (Eltoweissy et al., 2011) we established protein DJ-1 (PARK7) as an important ROS scavenger and key player in renal cell response to OS. In the present study we investigated the impact of profibrogenic agonists on DJ-1 and shed light on the role of this protein in renal fibrosis. Treatment of renal fibroblasts and epithelial cells with the profibrogenic agonist ANG II or PDGF resulted in a significant up-regulation of DJ-1 expression parallel to an increase in the expression of fibrosis markers. Monitoring of DJ-1 expression in kidney extract and tissue sections from a renal fibrosis mouse model (Col4a3-deficient) revealed a disease grade dependent regulation of the protein. Overexpression of DJ-1 prompted cell resistance to OS in both fibroblasts and epithelial cells. Furthermore overexpression of DJ-1, involved in ROS scavenging, in which glutamic acid 18 (E18) is mutated to either to aspartic acid (D) or glutamine (Q) resulted in a significant increase in cell death under OS in the case of E18D mutation, whereas E18Q mutation did not impact significantly the cell response to OS, revealing the importance of the acidic group for the ROS scavenging activity of the DJ-1 protein more than the nature of the amino acid itself. Affinity precipitation of interaction partners of DJ-1 and its mutants revealed an important role of annexin A1 and A5 in the mechanism of action of DJ-1 in anti-oxidative stress response.

The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction

Transforming growth factor-β1 (TGF-β1)/Smad signaling has a central role in the pathogenesis of renal fibrosis. Smad3 and Smad4 are pro-fibrotic, while Smad2 is anti-fibrotic. However, these Smads form heterogeneous complexes, the functions of which are poorly understood. Here we studied Smad complex function in renal fibrosis using the mouse model of unilateral ureteric obstruction. Mice heterozygous for Smad3/4 (Smad3/4+/-) exhibited substantial protection from renal fibrosis through day 7 of obstruction, whereas Smad2/3+/- and Smad2/4+/- mice showed only modest protection. Formation of Smad3/Smad4/CDK9 complexes was an early event following obstruction in wild-type mice, which involved nuclear phosphorylation of the linker regions of Smad3. Significantly, Smad3 or Smad4 deficiency decreased the formation of Smad4/CDK9 or Smad3/CDK9 complex, Smad3 linker phosphorylation, and fibrosis but at different degrees. In vitro, TGF-β1 stimulation of collagen I promoter activity involved formation of Smad3/Smad4/CDK9 complexes, and overexpression of each component gave additive increases in collagen promoter activity. Co-administration of a CDK9 inhibitor and Smad3-specific inhibition achieved better protection from TGF-β1-induced fibrotic response in vitro and renal interstitial fibrosis in vivo. Thus formation of Smad3/Smad4/CDK9 complex drives renal fibrosis during ureteral obstruction. Formation of this complex represents a novel target for antifibrotic therapies.

The Role of Krüppel-like Factor 4 in Renal Fibrosis

Chronic kidney disease (CKD) caused by renal fibrosis is an important public health concern. It is therefore necessary to understand the molecular pathogenesis of renal fibrosis in order to develop novel therapeutic strategies. KLF4 is the most extensively studied factor among the various members of the Krüppel-like factor (KLF) family of zinc finger-containing transcription factors. Many studies have demonstrated that KLF4 inhibits the activation of myofibroblasts and exerts an inhibitory effect on fibrosis. However, other studies have indicated that KLF4 may promote renal fibrosis. These controversial results suggest that KLF4 may be crucially involved in the development of renal fibrosis, although the underlying mechanism(s) remain unclear. Here, we summarize the recent progress made in understanding the role of KLF4 in renal fibrosis. Together, these findings suggest that KLF4 may participate in the development of renal fibrosis, but that its inhibition of fibrosis is greater than its promotion of the condition, which suggests that KLF4 may serve as a novel therapeutic target for renal fibrosis.

Matrix-Stiffness–Regulated Inverse Expression of Krüppel-Like Factor 5 and Krüppel-Like Factor 4 in the Pathogenesis of Renal Fibrosis

The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor (Klf)5 was the most up-regulated transcription factor accompanied by the down-regulation of Klf4 when cells were on stiff matrix. These changes were reversed by soft matrix via extracellular signal-regulated kinase (ERK) inactivation. Knockdown of Klf5 or forced expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive and negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/cyclin D1 axis was up-regulated and Klf4 was down-regulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated unilateral ureteral obstruction-induced tubular dilatation and proliferation, preserved Klf4, and suppressed the ERK/YAP1/Klf5/cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiologic functions and fibrosis progression.

Renoprotective effects of incretin-based drugs: A novel pleiotropic effect of dipeptidyl peptidase-4 inhibitor.

Renoprotective effects of incretin-based drugs: A novel pleiotropic effect of dipeptidyl peptidase-4 inhibitor.