Pathogenesis Of Renal Fibrosis Research Articles

JAK3/STAT6 Stimulates Bone Marrow-Derived Fibroblast Activation in Renal Fibrosis.

Renal fibrosis is a final common manifestation of CKD resulting in progressive loss of kidney function. Bone marrow-derived fibroblast precursors contribute significantly to the pathogenesis of renal fibrosis. However, the signaling mechanisms underlying the activation of bone marrow-derived fibroblast precursors in the kidney are not fully understood. In this study, we investigated the role of the Janus kinase 3 (JAK3)/signal transducer and activator of transcription (STAT6) signaling pathway in the activation of bone marrow-derived fibroblasts. In cultured mouse monocytes, IL-4 or IL-13 activated STAT6 and induced expression of α-smooth muscle actin and extracellular matrix proteins (fibronectin and collagen I), which was abolished by a JAK3 inhibitor (CP690,550) in a dose-dependent manner or blocked in the absence of STAT6. In vivo, STAT6 was activated in interstitial cells of the obstructed kidney, an effect that was abolished by CP690,550. Mice treated with CP690,550 accumulated fewer bone marrow-derived fibroblasts in the obstructed kidneys compared with vehicle-treated mice. Treatment with CP690,550 also significantly reduced myofibroblast transformation, matrix protein expression, fibrosis development, and apoptosis in obstructed kidneys. Furthermore, STAT6-deficient mice accumulated fewer bone marrow-derived fibroblasts in the obstructed kidneys, produced less extracellular matrix protein, and developed much less fibrosis. Finally, wild-type mice engrafted with STAT6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the obstructed kidneys and showed less severe renal fibrosis compared with wild-type mice engrafted with STAT6(+/+) bone marrow cells. Our results demonstrate that JAK3/STAT6 has an important role in bone marrow-derived fibroblast activation, extracellular matrix production, and interstitial fibrosis development.

Conditional Knockout of Src Homology 2 Domain-containing Protein Tyrosine Phosphatase-2 in Myeloid Cells Attenuates Renal Fibrosis after Unilateral Ureter Obstruction.

Background:Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase. Studies have revealed its roles in various disease, however, whether SHP-2 involves in renal fibrosis remains unclear. The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.Methods:Myeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system, and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO). The total collagen deposition in the renal interstitium was assessed using picrosirius red stain. F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium. Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney. Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.Results:Src homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO. Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice. Meanwhile, the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice. However, no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.Conclusions:Our observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis, and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

Adenovirus-mediated P311 inhibits TGF-β1-induced epithelial-mesenchymal transition in NRK-52E cells via TGF-β1-Smad-ILK pathway.

P311, a highly conserved 8-kDa intracellular protein, has been indicated as an important factor in myofibroblast transformation and in the progression of fibrosis. In the present study, we constructed a recombinant adenovirus vector of p311 (called Ad-P311) and transferred it into rat renal proximal tubular epithelial cells (NRK-52E) to explore the effect of P311 on epithelial-mesenchymal transition (EMT) of NRK-52E cells induced by TGF-β1 and to elucidate its underlying mechanism against EMT. After successfully construction of Ad-P311 and transfer into NRK-52E cells, the proliferation and growth of P311-expressing cells was detected by MTT assay. TGF-β1 was used to induce NRK-52E cells and Western blot analysis was used to examine the EMT markers (E-cadherin and α-smooth muscle actin (α-SMA)), signal transducers (p-Smad2/3 and Smad7). Integrin Linked Kinase (ILK) as a key intracellular mediator that controls TGF-β1-induced-EMT was also assayed by Western blot analysis. The results showed that P311 transfection could significantly inhibit the proliferation and growth of TGF-β1 induced NRK-52E cells. The results also showed that TGF-β1 could induce EMT in NRK-52E cells 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. However, P311 efficiently blocked Smad-ILK pathway activation and attenuated all these EMT changes induced by TGF-β1. These findings suggest that P311 might be involved in the pathogenesis of renal fibrosis by inhibiting the EMT process via TGF-β1-Smad-ILK pathway. P311 might be a novel target for the control of renal fibrosis and the progression of CKD.

Proinflammatory Cytokines and Potassium Channels in the Kidney

Proinflammatory cytokines affect several cell functions via receptor-mediated processes. In the kidney, functions of transporters and ion channels along the nephron are also affected by some cytokines. Among these, alteration of activity of potassium ion (K+) channels induces changes in transepithelial transport of solutes and water in the kidney, since K+ channels in tubule cells are indispensable for formation of membrane potential which serves as a driving force for the transepithelial transport. Altered K+ channel activity may be involved in renal cell dysfunction during inflammation. Although little information was available regarding the effects of proinflammatory cytokines on renal K+ channels, reports have emerged during the last decade. In human proximal tubule cells, interferon-γ showed a time-dependent biphasic effect on a 40 pS K+ channel, that is, delayed suppression and acute stimulation, and interleukin-1β acutely suppressed the channel activity. Transforming growth factor-β1 activated KCa3.1 K+ channel in immortalized human proximal tubule cells, which would be involved in the pathogenesis of renal fibrosis. This review discusses the effects of proinflammatory cytokines on renal K+ channels and the causal relationship between the cytokine-induced changes in K+ channel activity and renal dysfunction.

Myeloid HIF-1 attenuates the progression of renal fibrosis in murine obstructive nephropathy

Hypoxia-inducible factors (HIFs) play an important role in the pathogenesis of renal fibrosis. Although the role of macrophage infiltration in the progression of renal fibrosis is well known, the role of macrophage HIF-1 remains to be revealed. To address this question, myeloid specific conditional HIF-1 knock out mice were subjected to unilateral ureteral obstruction (UUO). Renal interstitial deposition of collagen Ⅲ and mRNA expressions of collagen Ⅰ and collagen Ⅲ were markedly increased at 7 days after UUO and myeloid HIF-1 depletion significantly accelerated these increases. Immunohistochemistry and flow cytometric analysis revealed that renal infiltrating macrophages were increased with duration of UUO but myeloid HIF-1 depletion did not affect these changes. Myeloid HIF-1 depletion did not affect M1 and M2 macrophage phenotype polarization in obstructed kidneys. Renal connective tissue growth factor (CTGF) expression was markedly increased and myeloid HIF-1 depletion further enhanced this increase. Immunomagnetic separation of renal cells revealed that renal CTGF was expressed predominantly in renal cells other than macrophages. It is suggested that myeloid HIF-1 attenuates the progression of renal fibrosis in murine obstructive kidney. Alteration of CTGF expression in renal cells other than macrophages is one of possible mechanisms by which myeloid HIF-1 protected renal fibrosis.

Chemokine receptor Cxcr4 contributes to kidney fibrosis via multiple effectors.

Kidney fibrosis is the final common pathway for virtually every type of chronic kidney disease and is a consequence of a prolonged healing response that follows tissue inflammation. Chronic kidney inflammation ultimately leads to progressive tissue injury and scarring/fibrosis. Several pathways have been implicated in the progression of kidney fibrosis. In the present study, we demonstrate that G protein-coupled chemokine (C-X-C motif) receptor (CXCR)4 was significantly upregulated after renal injury and that sustained activation of Cxcr4 expression augmented the fibrotic response. We demonstrate that after unilateral ureteral obstruction (UUO), both gene and protein expression of Cxcr4 were highly upregulated in tubular cells of the nephron. The increased Cxcr4 expression in tubules correlated with their increased dedifferentiated state, leading to increased mRNA expression of platelet-derived growth factor (PDGF)-α, transforming growth factor (TGF)-β1, and concurrent loss of bone morphogenetic protein 7 (Bmp7). Ablation of tubular Cxcr4 attenuated UUO-mediated fibrotic responses, which correlated with a significant reduction in PDGF-α and TGF-β1 levels and preservation of Bmp7 expression after UUO. Furthermore, Cxcr4(+) immune cells infiltrated the obstructed kidney and further upregulate their Cxcr4 expression. Genetic ablation of Cxcr4 from macrophages was protective against UUO-induced fibrosis. There was also reduced total kidney TGF-β1, which correlated with reduced Smad activation and α-smooth muscle actin levels. We conclude that chronic high Cxcr4 expression in multiple effector cell types can contribute to the pathogenesis of renal fibrosis by altering their biological profile. This study uncovered a novel cross-talk between Cxcr4-TGF-β1 and Bmp7 pathways and may provide novel targets for interrupting the progression of fibrosis.

MiR-141 regulates TGF-β1-induced epithelial-mesenchymal transition through repression of HIPK2 expression in renal tubular epithelial cells.

Epithelial-mesenchymal transition (EMT) plays a critical role in embryonic development, wound healing, tissue regeneration, cancer progression and organ fibrosis. The proximal tubular epithelial cells undergo EMT, resulting in matrix-producing fibroblasts and thereby contribute to the pathogenesis of renal fibrosis. The profibrotic cytokine, TGF-β, is now recognized as the main pathogenic driver that has been shown to induce EMT in tubular epithelial cells. Increasing evidence indicate that HIPK2 dysfunction may play a role in fibroblasts behavior, and therefore, HIPK2 may be considered as a novel potential target for anti-fibrosis therapy. Recently, members of the miR-200 family (miR-200a, b and c and miR-141) have been shown to inhibit EMT. However, the steps of the multifactorial renal fibrosis progression that these miRNAs regulate, particularly miR-141, are unclear. To study the functional importance of miR-141 in EMT, a well-established in vitro EMT assay was used to demonstrate renal tubulointerstitial fibrosis; transforming growth factor-β1-induced EMT in HK-2 cells. Overexpression of miR-141 in HK-2 cells, either with or without TGF-β1 treatment, hindered EMT by enhancing E-cadherin and decreasing vimentin and fibroblast-specific protein 1 expression. miR-141 expression was repressed during EMT in a dose- and time-dependent manner through upregulation of HIPK2 expression. Ectopic expression of HIPK2 promoted EMT by decreasing E-cadherin. Furthermore, co-transfection of miR-141 with the HIPK2 ORF clone partially inhibited EMT by restoring E-cadherin expression. miR-141 downregulated the expression of HIPK2 via direct interaction with the 3′-untranslated region of HIPK2. Taken together, these findings aid in the understanding of the role and mechanism of miR-141 in regulating renal fibrosis via the TGF-β1/miR-141/HIPK2/EMT axis, and miR-141 may represent novel biomarkers and therapeutic targets in the treatment of renal fibrosis.

GQ5 Hinders Renal Fibrosis in Obstructive Nephropathy by Selectively Inhibiting TGF-β-Induced Smad3 Phosphorylation.

TGF-β1, via Smad-dependent or Smad-independent signaling, has a central role in the pathogenesis of renal fibrosis. This pathway has been recognized as a potential target for antifibrotic therapy. Here, we identified GQ5, a small molecular phenolic compound isolated from the dried resin of Toxicodendron vernicifluum, as a potent and selective inhibitor of TGF-β1-induced Smad3 phosphorylation. In TGF-β1-stimulated renal tubular epithelial cells and interstitial fibroblast cells, GQ5 inhibited the interaction of Smad3 with TGF-β type I receptor (TβRI) by blocking binding of Smad3 to SARA, suppressed subsequent phosphorylation of Smad3, reduced nuclear translocation of Smad2, Smad3, and Smad4, and downregulated the transcription of major fibrotic genes such as α-smooth muscle actin (α-SMA), collagen I, and fibronectin. Notably, intraperitoneal administration of GQ5 in rats immediately after unilateral ureteral obstruction (UUO) selectively inhibited Smad3 phosphorylation in UUO kidneys, suppressed renal expression of α-SMA, collagen I, and fibronectin, and resulted in impressive renal protection after obstructive injury. Late administration of GQ5 also effectively attenuated fibrotic lesions in obstructive nephropathy. In conclusion, our results suggest that GQ5 hinders renal fibrosis in rats by selective inhibition of TGF-β1-induced Smad3 phosphorylation.

Toll-like receptor activation: from renal inflammation to fibrosis

Toll-like receptors (TLRs) are a conserved family of pattern recognition receptors that play a fundamental role in the innate immune system by triggering proinflammatory signaling pathways in response to microbial pathogens through exogenous pathogen-associated molecular patterns or tissue injury through endogenous danger-associated molecular patterns. In the kidney, TLRs are widely expressed in a variety of cell types. Emerging evidence demonstrates the participation of TLRs in the activation of these cells during renal fibrosis. This review highlights the role of TLRs and their endogenous ligands in the pathogenesis of renal fibrosis using ureteral obstruction and diabetic nephropathy as models of chronic kidney disease.

Prostaglandin E2 EP1 receptor enhances TGF-β1-induced mesangial cell injury.

Increasing evidence indicates that transforming growth factor-β1 (TGF-β1) is a pivotal mediator in the pathogenesis of renal fibrosis. Mesangial cells (MCs) are important for glomerular function under both physiological and pathological conditions. Studies have found that the expression level of prostaglandin E2 (PGE2) in MCs increases under high glucose conditions, that PGE2 affects the proliferation and hypertrophy of MCs mainly through the EP1 pathway, and that the proliferation of MCs and the accumulation of extracellular matrix are the main events leading to glomerular fibrosis. In this study, we investigated the effects and mechanisms of action of the EP1 receptor, which is induced by transforming growth factor (TGF)-β1, on the proliferation of mouse MCs, the accumulation of extracellular matrix and the expression of PGE2 synthase. Primary mouse glomerular MCs were isolated from EP1 receptor-deficient mice (EP1-/- mice, in which the EP1 receptor was knocked down) and wild-type (WT) mice (WT MCs). In our preliminary experiments, we found that cell proliferation, as well as the mRNA and protein expression of cyclin D1, proliferating cell nuclear antigen (PCNA), fibronectin (FN), collagen I (ColI), membrane-associated PGE2 synthase-1 (mPGES-1) and cyclooxygenase-2 (COX-2) in the WT MCs were significantly increased following treatment with 10 ng/ml TGF-β1 for 24 h. Compared with the WT MCs, following the knockdown of the EP1 gene, the TGF-β1-induced MC injury was markedly suppressed. The aforementioned changes were notably enhanced following treatment with the EP1 agonist, 17-phenyl trinor PGE2 ethyl amide. Additionally, TGF-β1 induced extracellular signal-regulated kinase (ERK) phosphorylation. We found that the TGF-β1-induced ERK phosphorylation was alleviated by EP1 knockdown and promoted by EP1 expression. These results suggest that the EP1 receptor plays a role in the proliferation of mouse MCs, in the accumulation of extracellular matrix and in the expression of mPGES-1 induced by TGF-β1. Its mechanisms of action are possibly related to the reinforcement of ERK phosphorylation.

IL-17A produced by both γδ T and Th17 cells promotes renal fibrosis via RANTES-mediated leukocyte infiltration after renal obstruction.

IL-17A-producing T lymphocytes play a crucial role in inflammatory kidney diseases, but their role in renal fibrosis remains to be explored. Here, we demonstrated that up-regulation of IL-17A was associated with the development of obstructive kidney injury. The primary source of IL-17A production in obstructed kidneys was infiltrating γδ T lymphocytes and CD4(+) T cells. IL-17A-deficient mice were protected from myofibroblast activation and extracellular matrix deposition, leading to reduced kidney fibrosis in response to obstructive injury. Mechanistically, IL-17A deficiency suppressed the expression of the chemokine RANTES in infiltrated CD3(+) T cells and peritubular inflammation following renal obstruction. Administration of RANTES-neutralizing antibody significantly reduced the accumulation of T cells and macrophages, and of collagen deposition in obstructed kidneys. Taken together, our results indicate that IL-17A contributes significantly to the pathogenesis of renal fibrosis by regulating RANTES-mediated inflammatory cell infiltration.

The role of adenosine receptors A2A and A2B signaling in renal fibrosis.

Renal fibrosis, the key histopathological lesion in the development and progression of chronic kidney disease (CKD), has been the focus of much research in recent decades. The growing burden of CKD in both developed and developing nations highlights a need for novel therapies to halt the progression of renal disease. Insights into the pathogenesis of renal fibrosis and the key cellular and molecular mediators have been critical in the process of identifying potential targets of therapy. Adenosine signaling is an innate biological autocrine and paracrine cellular signaling pathway involving several key mediators: ectonucleotidases, adenosine, and adenosine receptors. Short-term activation of the adenosine A2A and A2B receptors decreases inflammation, which precedes renal fibrosis. However, in conditions of persistent, excessive adenosine exposure, such as in patients born with adenosine deaminase (ADA) deficiency, adenosine signaling via A2B receptor promotes renal fibrosis, as seen in chronic inflammation. This review will describe the increasingly recognized complex role of adenosine signaling in the development of renal fibrosis. We will speculate how the knowledge gained may be employed in the search for more effective therapies based on these complex signaling pathways.

Smad3 signaling activates bone marrow-derived fibroblasts in renal fibrosis

Recent studies have demonstrated that bone marrow-derived fibroblasts contribute significantly to the pathogenesis of renal fibrosis. However, the signaling mechanisms underlying the activation of bone marrow-derived fibroblasts in the kidney are incompletely understood. As TGF-β1/Smad3 signaling has been shown to have an important role in the pathogenesis of kidney fibrosis, we investigated the role of Smad3 in the activation of bone marrow-derived fibroblasts in the kidney following obstructive injury using Smad3-knockout mice and Smad3-null monocytes. Compared with wild-type mice, Smad3-knockout mice accumulated significantly fewer bone marrow-derived fibroblasts in the kidney after obstructive injury. Furthermore, Smad3-knockout mice exhibited less myofibroblast activation and expressed less α-SMA in the obstructed kidney. Consistent with these findings, genetic deletion of Smad3 reduced total collagen deposition and suppressed the expression of extracellular matrix proteins. Moreover, wild-type mice engrafted with Smad3−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidney with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with Smad3+/+ bone marrow cells. In cultured monocytes, TGF-β1 induced phosphorylation of Smad3 and Smad3 deficiency abolished TGF-β1-induced expression of α-SMA and extracellular matrix proteins. Taken together, our results demonstrate that Smad3 signaling has an essential role in the activation of bone marrow-derived fibroblasts in the kidney during the pathogenesis of renal fibrosis.

The chemokine receptor CXCR6 contributes to recruitment of bone marrow–derived fibroblast precursors in renal fibrosis

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. Since chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly fewer bone marrow-derived fibroblasts accumulated in the kidney of CXCR6 knockout mice in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type mice. CXCR6 deficiency inhibited total collagen deposition and suppressed expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, wild type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Transplant of wild type bone marrow into CXCR6−/− recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may play important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

Protection from renal fibrosis, putative role of TRIB3 gene silencing

BackgroundRenal fibrosis is thought to be the common pathway in most cases of chronic kidney disease. Recently, TRIB3 was found to play an important role in progression of cardiac fibrosis in an insulin-resistant state. We investigated whether TRIB3 might participate in the pathogenesis of renal fibrosis in insulin-resistant rats. MethodsWe randomly separated 40 male Sprague–Dawley into 4 groups for treatment (n=10 each): control and high-fat diet (HFD) with TRIB3 siRNA adenovirus transfection, vehicle transfection or HFD alone. Insulin resistance markers were measured. Renal tissues were stained with hematoxylin and eosin, Masson's trichrome and periodic acid-Schiff. ResultsRats with HFD showed insulin resistance and TRIB3 overexpression. Upregulated TRIB3 expression could induce renal fibrosis accompanied by increased phosphorylation of extracellular signal-regulated kinase (ERK). Also, TRIB3 siRNA knockdown could ameliorate renal fibrosis, which was accompanied by decreased phosphorylation of ERK. ConclusionsTRIB3 gene silencing can attenuate renal fibrosis for beneficial effect on the development of renal fibrosis in chronic kidney disease in rat.

Protective effects of angiotensin‐(1–7) administrated with an angiotensin‐receptor blocker in a rat model of chronic kidney disease

Angiotensin-(1-7) (Ang-(1-7)) opposes angiotensin-II-induced cell growth, matrix accumulation and fibrosis in cardiac tissue. However, the role of Ang-(1-7) in the pathogenesis of renal fibrosis is uncertain. This study observed the effects of Ang-(1-7), on its own or in combination with losartan, an angiotensin-receptor blocker, on five-sixths nephrectomized rats. Male Sprague-Dawley rats underwent five-sixths nephrectomy, and then were either untreated, treated with Ang-(1-7), treated with losartan, or treated with a combination therapy of Ang-(1-7) and losartan. After 8 weeks, renal function was assessed by measuring systolic blood pressure, serum creatinine and proteinuria. The effect of nephrectomy on the renin-angiotensin system was examined by measuring plasma levels of Ang-II and Ang-(1-7). The extent of glomerulosclerosis and tubulointerstitial fibrosis was assessed by periodic acid-Schiff staining and Masson-trichrome staining. The expression of plasminogen activator inhibitor-1, fibronectin and angiopoietins-Tie-2 was investigated by immunohistochemistry and western blot. In the groups of treated rats, serum creatinine, proteinuria and markers of glomerulosclerosis, such as fibronectin and plasminogen activator inhibitor-1, were ameliorated compared with the untreated, nephrectomized rats. Plasma Ang-(1-7) levels were elevated in all treatment groups, but the plasma Ang-II levels were reduced in the Ang-(1-7)-treated group and the combination therapy group. The ratio of Ang-1/Ang-2 was increased in the combination therapy group compared with two other treatment groups. Ang-(1-7) ameliorated the renal injury of nephrectomized rats. The combination of Ang-(1-7) treatment alongside losartan exerted a superior effect to that of Ang-(1-7) alone on regression of glomerulosclerosis.

CCR2 Regulates the Uptake of Bone Marrow-Derived Fibroblasts in Renal Fibrosis

Recent studies have shown that bone marrow-derived fibroblasts contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying the recruitment of bone marrow-derived fibroblasts into the kidney are incompletely understood. Bone marrow-derived fibroblasts express the chemokine receptor - CCR2. In this study, we tested the hypothesis that CCR2 participates in the recruitment of fibroblasts into the kidney during the development of renal fibrosis. Bone marrow-derived collagen-expressing GFP+ fibroblasts were detected in the obstructed kidneys of chimeric mice transplanted with donor bone marrow from collagen α1(I)-GFP reporter mice. These bone marrow-derived fibroblasts expressed PDGFR-β and CCR2. CCR2 knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors expressing the hematopoietic marker-CD45 and the mesenchymal markers-PDGFR-β or procollagen I in the obstructed kidneys compared with wild-type mice. Furthermore, CCR2 knockout mice displayed fewer bone marrow-derived myofibroblasts and expressed less α-SMA or FSP-1 in the obstructed kidneys compared with wild-type mice. Consistent with these findings, genetic deletion of CCR2 inhibited total collagen deposition and suppressed expression of collagen I and fibronectin. Moreover, genetic deletion of CCR2 inhibits MCP-1 and CXCL16 gene expression associated with a reduction of inflammatory cytokine expression and macrophage infiltration, suggesting a linear interaction between two chemokines/ligand receptors in tubular epithelial cells. Taken together, our results demonstrate that CCR2 signaling plays an important role in the pathogenesis of renal fibrosis through regulation of bone marrow-derived fibroblasts. These data suggest that inhibition of CCR2 signaling could constitute a novel therapeutic approach for fibrotic kidney disease.

Complement 3 activates the renal renin-angiotensin system by induction of epithelial-to-mesenchymal transition of the nephrotubulus in mice

We have demonstrated that mesenchymal cells from spontaneously hypertensive rats genetically express complement 3 (C3). Mature tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT) that is linked to the pathogenesis of renal fibrosis and injury. In this study, we investigated the contribution of C3 in EMT and in the renal renin-angiotensin (RA) systems associated with hypertension. C3a induced EMT in mouse TCMK-1 epithelial cells, which displayed increased expression of renin and Krüppel-like factor 5 (KLF5) and nuclear localization of liver X receptor α (LXRα). C3 and renin were strongly stained in the degenerated nephrotubulus and colocalized with LXRα and prorenin receptor in unilateral ureteral obstruction (UUO) kidneys from wild-type mice. In C3-deficient mice, hydronephrus and EMT were suppressed, with no expression of renin and C3. After UUO, systolic blood pressure was increased in wild-type but not C3-deficient mice. In wild-type mice, intrarenal angiotensin II (ANG II) levels were markedly higher in UUO kidneys than normal kidneys and decreased with aliskiren. There were no increases in intrarenal ANG II levels after UUO in C3-deficient mice. Thus C3 induces EMT and dedifferentiation of epithelial cells, which produce renin through induction of LXRα. These data indicate for the first time that C3 may be a primary factor to activate the renal RA systems to induce hypertension.

Adiponectin Promotes Monocyte-to-Fibroblast Transition in Renal Fibrosis

Bone marrow-derived fibroblasts may contribute substantially to the pathogenesis of renal fibrosis through the excessive production and deposition of extracellular matrix. However, the mechanisms underlying the accumulation and activation of these fibroblasts are not understood. Here, we used a mouse model of tubulointerstitial fibrosis to determine whether adiponectin, which is elevated in CKD and is associated with disease progression, regulates monocyte-to-fibroblast transition and fibroblast activation in injured kidneys. In wild-type mice, the expression of adiponectin and the number of bone marrow-derived fibroblasts in the kidney increased after renal obstruction. In contrast, the obstructed kidneys of adiponectin-knockout mice had fewer bone marrow-derived fibroblasts. Adiponectin deficiency also led to a reduction in the number of myofibroblasts, the expression of profibrotic chemokines and cytokines, and the number of procollagen-expressing M2 macrophages in injured kidneys. Consistent with these findings, adiponectin-deficiency reduced the expression of collagen I and fibronectin. Similar results were observed in wild-type and adiponectin-knockout mice after ischemia-reperfusion injury. In cultured bone marrow-derived monocytes, adiponectin stimulated the expression of α-smooth muscle actin (SMA) and extracellular matrix proteins and activated AMP-activated protein kinase (AMPK) in a time- and dose-dependent manner. Furthermore, specific activation of AMPK increased the expression of α-SMA and extracellular matrix proteins, while inhibition of AMPK attenuated these responses. Taken together, these findings identify adiponectin as a critical regulator of monocyte-to-fibroblast transition and renal fibrosis, suggesting that inhibition of adiponectin/AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.

Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial–mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0–3, 6–9 or 10–13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.