Curtailing Endothelial TGF-β Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD

Excessive TGF-β signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-β signaling on development of fibrosis, there is a lack of information on ablating TGF-β signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-β receptor knockout (TβRII^endo+/−) mice to explore whether curtailed TGF-β signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TβRII^endo+/+ mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TβRII^endo+/− mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TβRII^endo+/+ counterparts. In addition, partial deletion of TβRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-β–induced canonical Smad2 signaling was reduced in TβRII^+/− ECs; however, activin receptor-like kinase 1 (ALK1)–mediated Smad1/5 phosphorylation in TβRII^+/− ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TβRII^+/− ECs compared with TβRII^+/+ ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-β signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-β signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.     

MicroRNA-21 in Glomerular Injury

TGF-β₁ is a pleotropic growth factor that mediates glomerulosclerosis and podocyte apoptosis, hallmarks of glomerular diseases. The expression of microRNA-21 (miR-21) is regulated by TGF-β₁, and miR-21 inhibits apoptosis in cancer cells. TGF-β₁–transgenic mice exhibit accelerated podocyte loss and glomerulosclerosis. We determined that miR-21 expression increases rapidly in cultured murine podocytes after exposure to TGF-β₁ and is higher in kidneys of TGF-β₁–transgenic mice than wild-type mice. miR-21–deficient TGF-β₁–transgenic mice showed increased proteinuria and glomerular extracellular matrix deposition and fewer podocytes per glomerular tuft compared with miR-21 wild-type TGF-β₁–transgenic littermates. Similarly, miR-21 expression was increased in streptozotocin-induced diabetic mice, and loss of miR-21 in these mice was associated with increased albuminuria, podocyte depletion, and mesangial expansion. In cultured podocytes, inhibition of miR-21 was accompanied by increases in the rate of cell death, TGF-β/Smad3-signaling activity, and expression of known proapoptotic miR-21 target genes p53, Pdcd4, Smad7, Tgfbr2, and Timp3. In American-Indian patients with diabetic nephropathy (n=48), albumin-to-creatinine ratio was positively associated with miR-21 expression in glomerular fractions (r=0.6; P<0.001) but not tubulointerstitial fractions (P=0.80). These findings suggest that miR-21 ameliorates TGF-β1 and hyperglycemia-induced glomerular injury through repression of proapoptotic signals, thereby inhibiting podocyte loss. This finding is in contrast to observations in murine models of tubulointerstitial kidney injury but consistent with findings in cancer models. The aggravation of glomerular disease in miR-21–deficient mice and the positive association with albumin-to-creatinine ratio in patients with diabetic nephropathy support miR-21 as a feedback inhibitor of TGF-β signaling and functions.     

Zinc-α2-Glycoprotein Exerts Antifibrotic Effects in Kidney and Heart

Zinc-α2-glycoprotein (AZGP1) is a secreted protein synthesized by epithelial cells and adipocytes that has roles in lipid metabolism, cell cycling, and cancer progression. Our previous findings in AKI indicated a new role for AZGP1 in the regulation of fibrosis, which is a unifying feature of CKD. Using two models of chronic kidney injury, we now show that mice with genetic AZGP1 deletion develop significantly more kidney fibrosis. This destructive phenotype was rescued by injection of recombinant AZGP1. Exposure of AZGP1-deficient mice to cardiac stress by thoracic aortic constriction revealed that antifibrotic effects were not restricted to the kidney but were cardioprotective. In vitro, recombinant AZGP1 inhibited kidney epithelial dedifferentiation and antagonized fibroblast activation by negatively regulating TGF-β signaling. Patient sera with high levels of AZGP1 similarly attenuated TGF-β signaling in fibroblasts. Taken together, these findings indicate a novel role for AZGP1 as a negative regulator of fibrosis progression, suggesting that recombinant AZGP1 may have translational effect for treating fibrotic disease.     

Combination therapy with DHA and BMSCs suppressed podocyte injury and attenuated renal fibrosis by modulating the TGF-β1/Smad pathway in MN mice

Artemisinin has immunomodulatory, anti-inflammatory, and antifibrotic effects. Some studies have demonstrated that artemisinins have a protective effect on the kidney. DHA is a derivative of artemisinin and has effects similar to those of artemisinin. Human bone marrow-derived mesenchymal stem cells (BMSCs) accelerate renal repair following acute injury. In the study, we investigated the effects of combination therapy with DHA and BMSCs on membranous nephropathy (MN) mice. The 24-h urinary protein, serum total cholesterol (TC) and triglyceride (TG) levels, and renal histopathology, were measured to evaluate kidney damage. Anti-PLA2R, IgG, and complement 3 (C3) were detected by ELISA. The expression levels of the podocyte injury-related proteins were analyzed by immunohistochemistry. The protein expression levels of α-SMA, ED-1, TGF-β1, p-Smad2, and p-Smad3 were detected by western blot to analyze renal fibrosis and its regulatory mechanism. Results showed that combination therapy with DHA and BMSCs significantly ameliorated kidney damage in MN model mice by decreasing the levels of 24 h urinary protein, TC and TG. This combination therapy also improved renal histology and reduced the expression of IgG and C3 in the glomerulus. In addition, this combination therapy decreased the expression of podocin and nephrin and relieved renal fibrosis by downregulating α-SMA and ED-1. Furthermore, this combination therapy suppressed TGF-β1 expression and Smad2/3 phosphorylation. This result (i.e., this combination therapy inhibited the TGF-β1/Smad pathway) was also supported in vitro. Taken together, combination therapy with DHA and BMSCs ameliorated podocyte injury and renal fibrosis in MN mice by downregulating the TGFβ1/Smad pathway.     

Blocking TGF-β1 protects the peritoneal membrane from dialysate-induced damage

During peritoneal dialysis (PD), mesothelial cells undergo mesothelial-to-mesenchymal transition (MMT), a process associated with peritoneal-membrane dysfunction. Because TGF-β1 can induce MMT, we evaluated the efficacy of TGF-β1-blocking peptides in modulating MMT and ameliorating peritoneal damage in a mouse model of PD. Exposure of the peritoneum to PD fluid induced fibrosis, angiogenesis, functional impairment, and the accumulation of fibroblasts. In addition to expressing fibroblast-specific protein-1 (FSP-1), some fibroblasts co-expressed cytokeratin, indicating their mesothelial origin. These intermediate-phenotype (Cyto(+)/FSP-1(+)) fibroblasts had features of myofibroblasts with fibrogenic capacity. PD fluid treatment triggered the appearance of CD31(+)/FSP-1(+) and CD45(+)/FSP-1(+) cells, suggesting that fibroblasts also originate from endothelial cells and from cells recruited from bone marrow. Administration of blocking peptides significantly ameliorated fibrosis and angiogenesis, improved peritoneal function, and reduced the number of FSP-1(+) cells, especially in the Cyto(+)/FSP-1(+) subpopulation. Conversely, overexpression of TGF-β1 in the peritoneum by adenovirus-mediated gene transfer led to a marked accumulation of fibroblasts, most of which derived from the mesothelium. Taken together, these results demonstrate that TGF-β1 drives the peritoneal deterioration induced by dialysis fluid and highlights a role of TGF-β1-mediated MMT in the pathophysiology of peritoneal-membrane dysfunction.     

Autophagy Regulates TGF-β Expression and Suppresses Kidney Fibrosis Induced by Unilateral Ureteral Obstruction

Autophagy is an evolutionarily conserved process that cells use to degrade and recycle cellular proteins and remove damaged organelles. During the past decade, there has been a growing interest in defining the basic cellular mechanism of autophagy and its roles in health and disease. However, the functional role of autophagy in kidney fibrosis remains poorly understood. Here, using GFP-LC3 transgenic mice, we show that autophagy is induced in renal tubular epithelial cells (RTECs) of obstructed kidneys after unilateral ureteral obstruction (UUO). Deletion of LC3B (LC3^−/− mice) resulted in increased collagen deposition and increased mature profibrotic factor TGF-β levels in obstructed kidneys. Beclin 1 heterozygous (beclin 1^+/−) mice also displayed increased collagen deposition in the obstructed kidneys after UUO. We also show that TGF-β1 induces autophagy in primary mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. LC3 deficiency resulted in increased levels of mature TGF-β in primary RTECs. Under conditions of TGF-β1 stimulation and autoinduction, inhibition of autolysosomal protein degradation by bafilomycin A1 increased mature TGF-β protein levels without alterations in TGF-β1 mRNA. These data suggest a novel intracellular mechanism by which mature TGF-β1 protein levels may be regulated in RTECs through autophagic degradation, which suppresses kidney fibrosis induced by UUO. The dual functions of TGF-β1, as an inducer of TGF-β1 autoinduction and an inducer of autophagy and TGF-β degradation, underscore the multifunctionality of TGF-β1.In the kidney, fibrosis is responsible for chronic progressive kidney failure, and the prevalence of CKD is increasing worldwide.^1,2 Extracellular matrix (ECM) protein production and progressive accumulation are hallmarks of renal tubulointerstitial fibrosis in progressive kidney disease. Collagens are the main components of the ECM in the kidney, and type I collagen (Col-I) is the major type associated with disease states.^3,4 The cellular mechanisms that facilitate tubulointerstitial fibrosis after injury remain incompletely defined. Recent lineage tracing or genetic fate mapping studies have strongly challenged the theory that renal tubular epithelial cells (RTECs) traverse the tubular basement membrane to become myofibroblasts in a process of epithelial-to-mesenchymal transition (EMT), but rather, that interstitial pericytes/perivascular fibroblasts are the myofibroblast progenitor cells.^5–7 It also has been proposed that profibrotic factors, such as TGF-β1, are upregulated in the tubular interstitial area on injury, leading to kidney fibrosis.⁸ TGF-β1 induces production of ECM proteins, including fibronectin and collagens, and inhibits degradation of ECM proteins mainly by matrix metalloproteinases.^9–11 Given the recent evidence that casts doubts about the role of EMT in vivo, how RTECs contribute to the development of renal tubulointerstitial fibrosis is not entirely clear.TGF-β is synthesized as a single polypeptide precursor that includes a preregion signal peptide, which is removed by proteolytic cleavage, and pro–TGF-β, containing a proregion called the latency-associated peptide and a mature TGF-β, and it converts to homodimeric pro–TGF-β through disulfide bonds.¹² After cleavage by proprotein convertases, such as furin, latency-associated peptide remains noncovalently associated with the dimeric form of mature TGF-β as the small latent complex (SLC).¹³ SLC formation occurs in the Golgi apparatus, and mature TGF-β is secreted as part of SLC and associated with latent TGF-β–binding protein to form TGF-β large latent complex, which interacts with ECM. On stimulus, the dimeric form of mature TGF-β is dissociated from large latent complex and becomes the bioactive mature TGF-β ligand, which can then bind TGF-β receptors to trigger downstream Smad-dependent or -independent signaling pathways.^12,13 Thus, the availability of mature TGF-β is the limiting factor of TGF-β activity and not TGF-β synthesis per se, because the body generates more pro–TGF-β than necessary. Whereas TGF-β/TGF-β receptor downstream signaling pathways have been extensively investigated, the regulation of TGF-β maturation and bioavailability has not been well studied but may serve as an important target for fibrotic diseases that alter TGF-β signaling.Macroautophagy, hereafter referred to as autophagy, is a fundamental cellular homeostatic process that cells use to degrade and recycle cellular proteins and remove damaged organelles. The process of autophagy involves the formation of double membrane–bound vesicles called autophagosomes that envelop and sequester cytoplasmic components, including macromolecular aggregates and cellular organelles, for bulk degradation by a lysosomal degradative pathway.¹⁴ Autophagy can be induced in response to either intracellular or extracellular factors, such as amino acid or growth factor deprivation, hypoxia, low cellular energy state, endoplasmic reticulum stress or oxidative stress, organelle damage, and pathogen infection.^15–22 To date, over 30 genes involved in autophagy have been identified in yeast, and they have been termed autophagy-related genes (Atgs). The mammalian ortholog of Atg8 is comprised of a family of proteins known as microtubule-associated protein 1 light chain 3 (LC3) that functions as a structural component in the formation of autophagosomes.²³ LC3B (herein referred to as LC3) is the best characterized form and the most widely used as an autophagic marker. The conversion of the cytosolic form of LC3 (LC3-I) to lipidated form (LC3-II) indicates autophagosome formation. In contrast to LC3, Beclin 1, encoded by the beclin 1 gene, is the mammalian ortholog of yeast Atg6 that is required for the initiation of autophagy through its interaction with Vps34. Homozygous deletion of beclin 1 (beclin 1^−/−) exhibits early embryonic lethality, whereas heterozygous deletion (beclin 1^+/−) results in increased incidence of spontaneous tumorigenesis, abnormal proliferation of mammary epithelial cells and germinal center B lymphocytes, and increased susceptibility to neurodegeneration.^24–27We previously reported that autophagy promotes intracellular degradation of Col-I induced by TGF-β1 in glomerular mesangial cells.²⁸ In the present study, we explored the functional role of autophagy in an in vivo model of progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) in autophagy-deficient LC3 null (LC3^−/−) and heterozygous (beclin 1^+/−) mice and green fluorescent protein (GFP)-LC3 transgenic mice. We also performed functional studies in primary cultured mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. We hypothesized that induction of autophagy in RTECs promotes TGF-β degradation and thereby reduces TGF-β secretion and suppresses development of kidney fibrosis.     

Kielin/Chordin-Like Protein Attenuates both Acute and Chronic Renal Injury

The secreted kielin/chordin-like (KCP) protein, one of a family of cysteine-rich proteins, suppresses TGF-β signaling by sequestering the ligand from its receptor, but it enhances bone morphogenetic protein (BMP) signaling by promoting ligand-receptor interactions. Given the critical roles for TGF-β and BMP proteins in enhancing or suppressing renal interstitial fibrosis, respectively, we examined whether secreted KCP could attenuate renal fibrosis in mouse models of chronic and acute disease. Transgenic mice that express KCP in adult kidneys showed significantly less expression of collagen IV, α-smooth muscle actin, and other markers of disease progression in the unilateral ureteral obstruction model of renal interstitial fibrosis. In the folic acid nephrotoxicity model of acute tubular necrosis, mice expressing KCP survived high doses of folic acid that were lethal for wild-type mice. With a lower dose of folic acid, mice expressing KCP exhibited improved renal recovery compared with wild-type mice. Thus, these data suggest that extracellular regulation of the TGF-β/BMP signaling axis by KCP, and by extension possibly other cysteine-rich domain proteins, can attenuate both acute and chronic renal injury.Progressive renal diseases, which can lead to end stage and ultimately require dialysis and transplantation, are increasing in frequency and correlate with the rise of diabetes, obesity, and hypertension among populations in the United States and Europe. Renal interstitial and/or glomerular fibrosis is common to most all progressive renal diseases, allograft nephropathy, and aging.¹ Yet effective therapies for chronic fibrosis are still not forthcoming.Among the most well studied signaling pathways in renal fibrotic disease are those of the TGF-β superfamily,² the most relevant of which are the TGF-βs, bone morphogenetic proteins (BMPs), and Activins. In animal models, increased renal fibrosis correlates with increased expression of TGF-β.³ In mice that overexpress a TGF-β transgene⁴ or are treated with recombinant TGF-β,⁵ increased tubular interstitial fibrosis and tubular atrophy were observed over time. In loss-of-function studies, the severity of renal histopathology was reduced in experimental glomerular nephritis models by treating animals with antibodies against TGF-β⁶ or a soluble type II TGF-β receptor.⁷ Significantly, genetic deletion of the downstream TGF-β signaling molecule Smad3 also results in protection against renal interstitial fibrosis in the unilateral ureteral obstruction (UUO) model,^8,9 suggesting that TGF-β and its effectors drive the initiation and progression of fibrotic disease. BMPs are thought to counteract the profibrotic effects of TGF-β in animal models of renal disease. In the kidney, BMP7 has been studied in detail and was shown to alleviate and even reverse fibrosis, either through direct application or by stimulation of the BMP receptor ALK3.^10,11Regulation of TGF-β superfamily signaling occurs at multiple levels, within and outside of the cell, and offers multiple potential avenues of intervention. The active TGF-β family ligand is a disulfide-linked homo- or heterodimer that is processed from large inactive precursors. A diverse family of secreted proteins can inhibit TGF-β superfamily signaling by sequestering ligands away from receptors. In the Golgi, processing of the TGF-β proprotein results in the formation of a latent complex.^12,13 The latency associated protein LAP1, which is the cleaved amino terminus of the TGF-β proprotein, together with latent TGF-β binding protein 1 (LTBP) and the active TGF-β homodimer constitute the large latent complex, which associates with the extracellular matrix and can be released and activated by proteolytic cleavage. Extracellular inhibitors of BMP signaling include vertebrate chordin, which binds directly to BMPs through the cysteine-rich (CR) domains containing CXXCXC and CCXXC motifs.^14–16 Whereas chordin blocks BMP/receptor interactions, the CR domain protein KCP enhances BMP/receptor interactions to increase the efficacy of signaling.¹⁷ Similarly, the CR domain protein connective-tissue growth factor also enhances TGF-β–mediated signaling while suppressing the BMP-dependent pathway.¹⁸We previously identified KCP (Crim2) as a protein with 18 CR domains that is expressed in the developing kidney at both early and late stages.¹⁷ KCP expression corresponds to the formation of epithelial structures within the intermediate mesoderm and to the formation of the proximal tubules in the more developed metanephric kidney. Unlike chordin or connective-tissue growth factor, KCP enhances BMP-mediated signaling by facilitating the binding of BMP7 to BMP receptor 1A.¹⁷ Conversely, KCP is able to inhibit TGF-β signaling by blocking ligand-receptor interactions.¹⁹ Mice homozygous for a mutant KCP allele show no gross developmental abnormalities but KCP mutations enhance the renal developmental phenotype in mutants of CV2,²⁰ another gene that encodes a multi-CR domain activator of BMPs. However, Kcp^−/− mice exhibit enhanced susceptibility to developing renal interstitial fibrosis in two different animal models,¹⁷ a process regulated by both BMPs and TGF-β. Such animals exhibit lower BMP signaling and higher TGF-β signaling upon renal injury.In this report, we address whether altering the balance between the TGF-β and BMP signaling pathways can be achieved by ectopic or overexpression of the secreted KCP protein to change the course of renal fibrosis. Transgenic mice were engineered to express KCP protein in renal proximal tubule cells and subjected to UUO or acute tubular necrosis. Although KCP expression by itself had few measurable deleterious affect, KCP transgenic mice were significantly more resistant to interstitial fibrosis and renal injury. These studies point to a novel renal protective function for KCP. That the TGF/BMP signaling cascades can be shifted by a secreted protein opens new therapeutic avenues of intervention for both acute and chronic renal disease.     

Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA₄) attenuated TGF-β1–induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-β1 suppressed expression of let-7c. In cells pretreated with LXA₄, upregulation of let-7c persisted despite subsequent stimulation with TGF-β1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA₄ analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-β1 signaling pathway, including the TGF-β receptor type 1. Consistent with this, LXA₄-induced upregulation of let-7c inhibited both the expression of TGF-β receptor type 1 and the response to TGF-β1. Overexpression of let-7c mimicked the antifibrotic effects of LXA₄ in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA₄. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA₄-mediated upregulation of let-7c suppresses TGF-β1–induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.There is a growing appreciation of the role of endogenously produced lipid mediators including lipoxins, resolvins, and PGD synthase metabolites in promoting the resolution of inflammatory responses.^1–4 We and others recently described distinct proresolution and antifibrotic properties of lipoxins in renal fibrosis.^5,6 TGF-β1 is implicated in numerous fibrotic conditions including tubulointerstitial fibrosis and diabetic kidney disease. The development of fibrosis in this context may reflect activation of parenchymal fibroblasts, recruitment of circulating fibrocytes, and de-differentiation of epithelia and pericytes.^7,8 In this study, we investigated the effect of lipoxin A4 (LXA₄) on TGF-β1–induced fibrotic responses of renal epithelial cells and the mechanism underlying attenuation of this fibrotic injury pattern by lipoxins.MicroRNAs (miRNAs) comprise a class of small noncoding RNAs that negatively regulate gene expression by base-pairing to partially complementary sites in the 3′ untranslated regions (UTRs) of target mRNA, preventing translation. miRNAs are implicated in the development and progression of a wide range of complex human diseases,^9–12 including diabetic nephropathy.^13–15 Here we report that LXA₄ upregulates expression of the miRNA let-7c in HK-2 cells and attenuates response to TGF-β1. Lipoxins are protective in unilateral ureteral obstruction (UUO)–induced renal fibrosis and we report that this is associated with increased let-7c expression. Conversely, TGF-β1 decreases let-7c expression and this is associated with increased expression of let-7c targets, including TGFβ receptor type 1 (TGFβR1), collagens (COL1A1, COL1A2), and thrombospondin (THBS1). Overexpression of let-7c mimics the fibrosuppressant effects of LXA₄, whereas suppression of let-7c mimics responses to TGF-β1. The importance of regulation of let-7c targets is further supported by evidence from human renal biopsy material where we report upregulation of several let-7c targets in CKD.     

EGF Receptor Inhibition Alleviates Hyperuricemic Nephropathy

Hyperuricemia is an independent risk factor for CKD and contributes to kidney fibrosis. In this study, we investigated the effect of EGF receptor (EGFR) inhibition on the development of hyperuricemic nephropathy (HN) and the mechanisms involved. In a rat model of HN induced by feeding a mixture of adenine and potassium oxonate, increased EGFR phosphorylation and severe glomerular sclerosis and renal interstitial fibrosis were evident, accompanied by renal dysfunction and increased urine microalbumin excretion. Administration of gefitinib, a highly selective EGFR inhibitor, prevented renal dysfunction, reduced urine microalbumin, and inhibited activation of renal interstitial fibroblasts and expression of extracellular proteins. Gefitinib treatment also inhibited hyperuricemia-induced activation of the TGF-β1 and NF-κB signaling pathways and expression of multiple profibrogenic cytokines/chemokines in the kidney. Furthermore, gefitinib treatment suppressed xanthine oxidase activity, which mediates uric acid production, and preserved expression of organic anion transporters 1 and 3, which promotes uric acid excretion in the kidney of hyperuricemic rats. Thus, blocking EGFR can attenuate development of HN via suppression of TGF-β1 signaling and inflammation and promotion of the molecular processes that reduce uric acid accumulation in the body.     

Lineage Tracing Reveals Distinctive Fates for Mesothelial Cells and Submesothelial Fibroblasts during Peritoneal Injury

Fibrosis of the peritoneal cavity remains a serious, life-threatening problem in the treatment of kidney failure with peritoneal dialysis. The mechanism of fibrosis remains unclear partly because the fibrogenic cells have not been identified with certainty. Recent studies have proposed mesothelial cells to be an important source of myofibroblasts through the epithelial–mesenchymal transition; however, confirmatory studies in vivo are lacking. Here, we show by inducible genetic fate mapping that type I collagen–producing submesothelial fibroblasts are specific progenitors of α-smooth muscle actin–positive myofibroblasts that accumulate progressively in models of peritoneal fibrosis induced by sodium hypochlorite, hyperglycemic dialysis solutions, or TGF-β1. Similar genetic mapping of Wilms’ tumor-1–positive mesothelial cells indicated that peritoneal membrane disruption is repaired and replaced by surviving mesothelial cells in peritoneal injury, and not by submesothelial fibroblasts. Although primary cultures of mesothelial cells or submesothelial fibroblasts each expressed α-smooth muscle actin under the influence of TGF-β1, only submesothelial fibroblasts expressed α-smooth muscle actin after induction of peritoneal fibrosis in mice. Furthermore, pharmacologic inhibition of the PDGF receptor, which is expressed by submesothelial fibroblasts but not mesothelial cells, attenuated the peritoneal fibrosis but not the remesothelialization induced by hypochlorite. Thus, our data identify distinctive fates for injured mesothelial cells and submesothelial fibroblasts during peritoneal injury and fibrosis.Many patients with kidney failure rely on the peritoneal membrane to perform life-saving dialysis.^1–3 In addition to changes in permeability of the peritoneal membrane, the dialysis process itself frequently triggers a fibrosing process that progressively reduces membrane function resulting in dialysis failure, sometimes with high patient mortality.^4–7 In a small percentage of patients, severe fibrosis occurs primarily in the visceral peritoneum, resulting in encapsulating peritoneal sclerosis (EPS), a catastrophic complication with obscure pathogenesis and a high mortality rate.^6,7 Such dialysis failure is characterized by progressive peritoneal fibrosis that can be seen as with thickening of basal lamina and accumulation of α-smooth muscle actin (αSMA)⁺ myofibroblasts.^4,5,8–10The peritoneum is composed of mesothelium, basal lamina, and submesothelial (SM) connective tissue.^11–13 The mesothelium consists of a single layer of flattened mesothelial cells (MCs) that lines the peritoneal cavity and internal organs.^12,14–16 In many circumstances such as organ development or tissue injury repair, MCs are the cellular source of growth factors including TGF-β1, PDGF, and vascular endothelial growth factor, which support cell proliferation and differentiation of parenchymal and stromal cells as well as angiogenesis.^8,17–22 By contrast, SM connective tissue containing plexuses of blood vessels, lymphatic channels, and scattered fibroblasts has drawn much less attention.^22–24 A number of studies suggested MCs as the major source of myofibroblasts through the epithelial–mesenchymal transition (EMT) during peritoneal fibrosis.^8,18,25–27 However, these studies relied predominantly on in vitro experiments to show that MCs can be stimulated to express αSMA and produce matrix proteins outside of the body under the influence of profibrotic agents such as TGF-β1.^8,25–28 Nevertheless, confirmatory studies of mesothelial EMT in vivo are lacking even though costaining of cytokeratin and αSMA was previously shown.^8,18 The contribution of SM fibroblasts to myofibroblasts in vivo is not clear despite some studies in vitro have suggested.^22–24A conditional cell lineage analysis using WT1^CreERT2/+ mice demonstrated that Wilms’ tumor-1 (WT1)⁺ septum transversum mesenchyme gives rise to MCs, SM fibroblasts covering the liver, and hepatic stellate cells within the liver during hepatic development.^12,13 Using WT1^CreERT2/+ mice, a recent study reported that WT1⁺ MCs may differentiate into myofibroblasts in liver injury.²⁹ WT1 expression in MCs is observed in both embryonic development and adult peritoneum; however, the expression of WT1 by SM fibroblasts is not clearly defined in the adult peritoneum.^{12,13,21,30–32} Hence, the progenitors of myofibroblasts in the injured liver and the peritoneum remain controversial despite these studies.Because efforts to design new antifibrotic therapies require a rigorous understanding of the cellular origin of myofibroblasts in vivo, we performed lineage tracing of both MCs and SM fibroblasts in models of peritoneal fibrosis induced by sodium hypochlorite solution, hyperglycemic dialysis solution, or adenovirus-expressing TGF-β1 (AdTGF-β1). Although these models are more akin to EPS than the progressive thickening peritoneum seen in humans on peritoneal dialysis, they represent robust tools to study the pathogenesis of peritoneal fibrosis in the laboratory.^33–36 Contrary to the prevailing model, our findings indicate that peritoneal myofibroblasts derive from SM fibroblasts and peritoneal membrane disruption is repaired by surviving MCs.     

EGF Receptor Deletion in Podocytes Attenuates Diabetic Nephropathy

The generation of reactive oxygen species (ROS), particularly superoxide, by damaged or dysfunctional mitochondria has been postulated to be an initiating event in the development of diabetes complications. The glomerulus is a primary site of diabetic injury, and podocyte injury is a classic hallmark of diabetic glomerular lesions. In streptozotocin-induced type 1 diabetes, podocyte-specific EGF receptor (EGFR) knockout mice (EGFR^podKO) and their wild-type (WT) littermates had similar levels of hyperglycemia and polyuria, but EGFR^podKO mice had significantly less albuminuria and less podocyte loss compared with WT diabetic mice. Furthermore, EGFR^podKO diabetic mice had less TGF-β1 expression, Smad2/3 phosphorylation, and glomerular fibronectin deposition. Immunoblotting of isolated glomerular lysates revealed that the upregulation of cleaved caspase 3 and downregulation of Bcl2 in WT diabetic mice were attenuated in EGFR^podKO diabetic mice. Administration of the SOD mimetic mito-tempol or the NADPH oxidase inhibitor apocynin attenuated the upregulation of p-c-Src, p-EGFR, p-ERK1/2, p-Smad2/3, and TGF-β1 expression and prevented the alteration of cleaved caspase 3 and Bcl2 expression in glomeruli of WT diabetic mice. High-glucose treatment of cultured mouse podocytes induced similar alterations in the production of ROS; phosphorylation of c-Src, EGFR, and Smad2/3; and expression of TGF-β1, cleaved caspase 3, and Bcl2. These alterations were inhibited by treatment with mito-tempol or apocynin or by inhibiting EGFR expression or activity. Thus, results of our studies utilizing mice with podocyte-specific EGFR deletion demonstrate that EGFR activation has a major role in activating pathways that mediate podocyte injury and loss in diabetic nephropathy.     

Klotho and Phosphate Are Modulators of Pathologic Uremic Cardiac Remodeling

Cardiac dysfunction in CKD is characterized by aberrant cardiac remodeling with hypertrophy and fibrosis. CKD is a state of severe systemic Klotho deficiency, and restoration of Klotho attenuates vascular calcification associated with CKD. We examined the role of Klotho in cardiac remodeling in models of Klotho deficiency—genetic Klotho hypomorphism, high dietary phosphate intake, aging, and CKD. Klotho-deficient mice exhibited cardiac dysfunction and hypertrophy before 12 weeks of age followed by fibrosis. In wild-type mice, the induction of CKD led to severe cardiovascular changes not observed in control mice. Notably, non-CKD mice fed a high-phosphate diet had lower Klotho levels and greatly accelerated cardiac remodeling associated with normal aging compared with those on a normal diet. Chronic elevation of circulating Klotho because of global overexpression alleviated the cardiac remodeling induced by either high-phosphate diet or CKD. Regardless of the cause of Klotho deficiency, the extent of cardiac hypertrophy and fibrosis correlated tightly with plasma phosphate concentration and inversely with plasma Klotho concentration, even when adjusted for all other covariables. High-fibroblast growth factor–23 concentration positively correlated with cardiac remodeling in a Klotho-deficient state but not a Klotho-replete state. In vitro, Klotho inhibited TGF-β1–, angiotensin II–, or high phosphate–induced fibrosis and abolished TGF-β1– or angiotensin II–induced hypertrophy of cardiomyocytes. In conclusion, Klotho deficiency is a novel intermediate mediator of pathologic cardiac remodeling, and fibroblast growth factor–23 may contribute to cardiac remodeling in concert with Klotho deficiency in CKD, phosphotoxicity, and aging.     

MiR-122-5p promotes peritoneal fibrosis in a rat model of peritoneal dialysis by targeting Smad5 to activate Wnt/β-catenin pathway

Peritoneal fibrosis (PF) is the main reason leading to declining efficiency and ultrafiltration failure of peritoneum, which restricts the application of peritoneal dialysis (PD). We aimed to investigate the effects and mechanisms of miR-122-5p on the PF. Sprague-Dawley (SD) rats were infused with glucose-based standard PD fluid to establish PF model. HE staining was performed to evaluate the extent of PF. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization (FISH) were performed to measure the expression level of miR-122-5p. Western blot was used to test the expression of transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF)-A, Fibronectin 1 (FN1), extracellular matrix protein 1 (ECM1), Smad5, α-smooth muscle actin (SMA), collagen type 1(COL-1), Vimentin, E-Cadherin, Wnt1, β-catenin, p-β-catenin, c-Myc, c-Jun, and Cyclin D1. Immunohistochemistry (IHC) staining was used to detect type I collagen alpha 1 (Col1α1), α-SMA, and E-Cadherin expression. We found PF was glucose concentration-dependently enhanced in peritoneum of PD rat. The PD rats showed increased miR-122-5p and decreased Smad5 expression. MiR-122-5p silencing improved PF and epithelial–mesenchymal transition (EMT) process in PD rats. MiR-122-5p silencing attenuated the activity of the Wnt/β-catenin signaling pathway. Importantly, dual-luciferase reporter assay showed Smad5 was a target gene of miR-122-5p. Smad5 overexpression significantly reversed the increases of PF and EMT progression induced by miR-122-5p overexpression. Moreover, miR-122-5p mimic activated Wnt/β-catenin activity, which was blocked by Smad5 overexpression. Overall, present results demonstrated that miR-122-5p overexpression showed a deterioration effect on PD-related PF by targeting Smad5 to activate Wnt/β-catenin pathway.     

Multiple Genes of the Renin-Angiotensin System Are Novel Targets of Wnt/β-Catenin Signaling

Activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of CKD and cardiovascular disease. However, current anti-RAS therapy only has limited efficacy, partly because of compensatory upregulation of renin expression. Therefore, a treatment strategy to simultaneously target multiple RAS genes is necessary to achieve greater efficacy. By bioinformatics analyses, we discovered that the promoter regions of all RAS genes contained putative T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding sites, and β-catenin induced the binding of LEF-1 to these sites in kidney tubular cells. Overexpression of either β-catenin or different Wnt ligands induced the expression of all RAS genes. Conversely, a small-molecule β-catenin inhibitor ICG-001 abolished RAS induction. In a mouse model of nephropathy induced by adriamycin, either transient therapy or late administration of ICG-001 abolished established proteinuria and kidney lesions. ICG-001 inhibited renal expression of multiple RAS genes in vivo and abolished the expression of other Wnt/β-catenin target genes. Moreover, ICG-001 therapy restored expression of nephrin, podocin, and Wilms’ tumor 1, attenuated interstitial myofibroblast activation, repressed matrix expression, and inhibited renal inflammation and fibrosis. Collectively, these studies identify all RAS genes as novel downstream targets of Wnt/β-catenin. Our results indicate that blockade of Wnt/β-catenin signaling can simultaneously repress multiple RAS genes, thereby leading to the reversal of established proteinuria and kidney injury.     

TGF-β1 Promotes Lymphangiogenesis during Peritoneal Fibrosis

Peritoneal fibrosis (PF) causes ultrafiltration failure (UFF) and is a complicating factor in long-term peritoneal dialysis. Lymphatic reabsorption also may contribute to UFF, but little is known about lymphangiogenesis in patients with UFF and peritonitis. We studied the role of the lymphangiogenesis mediator vascular endothelial growth factor-C (VEGF-C) in human dialysate effluents, peritoneal tissues, and peritoneal mesothelial cells (HPMCs). Dialysate VEGF-C concentration correlated positively with the dialysate-to-plasma ratio of creatinine (D/P Cr) and the dialysate TGF-β1 concentration. Peritoneal tissue from patients with UFF expressed higher levels of VEGF-C, lymphatic endothelial hyaluronan receptor-1 (LYVE-1), and podoplanin mRNA and contained more lymphatic vessels than tissue from patients without UFF. Furthermore, mesothelial cell and macrophage expression of VEGF-C increased in the peritoneal membranes of patients with UFF and peritonitis. In cultured mesothelial cells, TGF-β1 upregulated the expression of VEGF-C mRNA and protein, and this upregulation was suppressed by a TGF-β type I receptor (TGFβR-I) inhibitor. TGF-β1–induced upregulation of VEGF-C mRNA expression in cultured HPMCs correlated with the D/P Cr of the patient from whom the HPMCs were derived (P<0.001). Moreover, treatment with a TGFβR-I inhibitor suppressed the enhanced lymphangiogenesis and VEGF-C expression associated with fibrosis in a rat model of PF. These results suggest that lymphangiogenesis associates with fibrosis through the TGF-β–VEGF-C pathway.The decrease in ultrafiltration capacity that is associated with the high peritoneal solute transport that is observed after prolonged peritoneal dialysis (PD) treatment is a major reason for its discontinuation.^1–4 Several studies have shown that a higher peritoneal solute transport rate is associated with reduced survival of PD patients.^1,2,5 The characteristic features of chronic peritoneal damage in PD treatment are associated with submesothelial fibrosis and neoangiogenesis.^6,7 Analyses of the surface peritoneum showed no significant changes in vessel density with duration of PD.^6,8 In addition, the vessel density in patients with ultrafiltration failure (UFF) was significantly higher than the vessel density in normal individuals or non-PD patients, but it was not higher than the vessel density in patients undergoing PD.⁶ These findings suggest that factors other than increased vascular density may be involved in disease states associated with increased transport of peritoneal membranes. In addition, the relationship between peritoneal fibrosis and UFF remains obscure.Blood capillaries have a continuous basal lamina with tight interendothelial junctions and are supported by pericytes and smooth muscle cells. In contrast, lymphatic capillaries are thin-walled with a wide lumen and do not contain pericytes or basement membrane. The structures of lymphatic vessels are suitable for the removal of tissue fluid, cells, and macromolecules from the interstitium.^9–11 If lymphangiogenesis develops in the peritoneal membrane, absorption of the PD fluid could be increased and lead to UFF. An increase in the number of lymphatic vessels has recently been reported in several disease conditions, including tumor metastasis,^12–15 chronic respiratory inflammatory diseases,^16–18 wound healing,¹⁹ and renal transplant rejection.^20,21 We recently reported that lymphangiogenesis had developed in tubulointerstitial fibrosis of human renal biopsy specimens,²² and we also reported the mechanisms of lymphangiogenesis in rat unilateral ureteral obstruction models.²³The lymphatic absorption rate, which is measured by the rate at which intraperitoneally administered radioactive serum albumin or macromolecule dextran 70 disappears, is significantly higher in patients with UFF, and lymphatic reabsorption is considered to be one of the causes of UFF.^24–27 However, the results from these clinical approaches have been controversial.^28,29 In addition, little is known about the pathology and the process of lymphangiogenesis in patients with UFF and peritonitis.In this study, we investigated lymphangiogenesis and the expression of vascular endothelial growth factor-C (VEGF-C), which is a potentially important mediator of lymphangiogenesis, in human peritoneal tissues, PD effluent, and peritoneal mesothelial cells. We also explored VEGF-C induction by TGF-β1 in the human mesothelial cell line (Met-5A) and cultured human peritoneal mesothelial cells (HPMCs) from the spent PD effluent of patients with varying rates of peritoneal transport. Finally, we explored the relationship between peritoneal fibrosis and lymphangiogenesis in rats that were administered chlorhexidine gluconate (CG) into the abdominal cavity, which provides a model of chemically induced peritoneal inflammation/fibrosis.^30–32 This work is the first report to show that lymphangiogenesis is linked to the peritoneal fibrosis that is often associated with a high peritoneal transport rate.