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.     

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.     

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.     

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.     

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.     

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.     

Metabolomics Reveals a Key Role for Fumarate in Mediating the Effects of NADPH Oxidase 4 in Diabetic Kidney Disease

The NADPH oxidase (NOX) isoform NOX4 has been linked with diabetic kidney disease (DKD). However, a mechanistic understanding of the downstream effects of NOX4 remains to be established. We report that podocyte-specific induction of NOX4 in vivo was sufficient to recapitulate the characteristic glomerular changes noted with DKD, including glomerular hypertrophy, mesangial matrix accumulation, glomerular basement membrane thickening, albuminuria, and podocyte dropout. Intervention with a NOX1/NOX4 inhibitor reduced albuminuria, glomerular hypertrophy, and mesangial matrix accumulation in the F1 Akita model of DKD. Metabolomic analyses from these mouse studies revealed that tricarboxylic acid (TCA) cycle–related urinary metabolites were increased in DKD, but fumarate levels were uniquely reduced by the NOX1/NOX4 inhibitor. Expression of fumarate hydratase (FH), which regulates urine fumarate accumulation, was reduced in the diabetic kidney (in mouse and human tissue), and administration of the NOX1/NOX4 inhibitor increased glomerular FH levels in diabetic mice. Induction of Nox4 in vitro and in the podocyte-specific NOX4 transgenic mouse led to reduced FH levels. In vitro, fumarate stimulated endoplasmic reticulum stress, matrix gene expression, and expression of hypoxia-inducible factor-1α (HIF-1α) and TGF-β. Similar upregulation of renal HIF-1α and TGF-β expression was observed in NOX4 transgenic mice and diabetic mice and was attenuated by NOX1/NOX4 inhibition in diabetic mice. In conclusion, NOX4 is a major mediator of diabetes-associated glomerular dysfunction through targeting of renal FH, which increases fumarate levels. Fumarate is therefore a key link connecting metabolic pathways to DKD pathogenesis, and measuring urinary fumarate levels may have application for monitoring renal NOX4 activity.     

Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation

BackgroundAKI is characterized by abrupt and reversible kidney dysfunction, and incomplete recovery leads to chronic kidney injury. Previous studies by us and others have indicated that macrophage infiltration and polarization play key roles in recovery from AKI. The role in AKI recovery played by IFN regulatory factor 4 (IRF4), a mediator of polarization of macrophages to the M2 phenotype, is unclear.MethodsWe used mice with myeloid or macrophage cell–specific deletion of Irf4 (MΦ Irf4 ^−/−) to evaluate Irf4’s role in renal macrophage polarization and development of fibrosis after severe AKI.ResultsSurprisingly, although macrophage Irf4 deletion had a minimal effect on early renal functional recovery from AKI, it resulted in decreased renal fibrosis 4 weeks after severe AKI, in association with less-activated macrophages. Macrophage Irf4 deletion also protected against renal fibrosis in unilateral ureteral obstruction. Bone marrow–derived monocytes (BMDMs) from MΦ Irf4 ^−/− mice had diminished chemotactic responses to macrophage chemoattractants, with decreased activation of AKT and PI3 kinase and increased PTEN expression. PI3K and AKT inhibitors markedly decreased chemotaxis in wild-type BMDMs, and in a cultured macrophage cell line. There was significant inhibition of homing of labeled Irf4 ^−/− BMDMs to postischemic kidneys. Renal macrophage infiltration in response to AKI was markedly decreased in MΦ Irf4 ^−/− mice or in wild-type mice with inhibition of AKT activity.ConclusionsDeletion of Irf4 from myeloid cells protected against development of tubulointerstitial fibrosis after severe ischemic renal injury in mice, due primarily to inhibition of AKT-mediated monocyte recruitment to the injured kidney and reduced activation and subsequent polarization into a profibrotic M2 phenotype.     

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.     

Reduced prostate branching morphogenesis in stromal fibroblast,but not in epithelial,estrogen receptor α knockout mice

Early studies suggested that estrogen receptor alpha (ERα) is involved in estrogen-mediated imprinting effects in prostate development. We recently reported a more complete ERα knockout (KO) mouse model via mating β-actin Cre transgenic mice with floxed ERα mice. These ACTB-ERαKO male mice showed defects in prostatic branching morphogenesis, which demonstrates that ERα is necessary to maintain proliferative events in the prostate. However, within which prostate cell type ERα exerts those important functions remains to be elucidated. To address this, we have bred floxed ERα mice with either fibroblast-specific protein (FSP)-Cre or probasin-Cre transgenic mice to generate a mouse model that has deleted ERα gene in either stromal fibroblast (FSP-ERαKO) or epithelial (pes-ERαKO) prostate cells. We found that circulating testosterone and fertility were not altered in FSP-ERαKO and pes-ERαKO male mice. Prostates of FSP-ERαKO mice have less branching morphogenesis compared to that of wild-type littermates. Further analyses indicated that loss of stromal ERα leads to increased stromal apoptosis, reduced expression of insulin-like growth factor-1 (IGF-1) and FGF10, and increased expression of BMP4. Collectively, we have established the first in vivo prostate stromal and epithelial selective ERαKO mouse models and the results from these mice indicated that stromal fibroblast ERα plays important roles in prostatic branching morphogenesis via a paracrine fashion. Selective deletion of the ERα gene in mouse prostate epithelial cells by probasin-Cre does not affect the regular prostate development and homeostasis.     

Four-and-a-Half LIM Domains Protein 2 Is a Coactivator of Wnt Signaling in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a microvascular complication that leads to kidney dysfunction and ESRD, but the underlying mechanisms remain unclear. Podocyte Wnt-pathway activation has been demonstrated to be a trigger mechanism for various proteinuric diseases. Notably, four-and-a-half LIM domains protein 2 (FHL2) is highly expressed in urogenital systems and has been implicated in Wnt/β-catenin signaling. Here, we used in vitro podocyte culture experiments and a streptozotocin-induced DKD model in FHL2 gene-knockout mice to determine the possible role of FHL2 in DKD and to clarify its association with the Wnt pathway. In human and mouse kidney tissues, FHL2 protein was abundantly expressed in podocytes but not in renal tubular cells. Treatment with high glucose or diabetes-related cytokines, including angiotensin II and TGF-β1, activated FHL2 protein and Wnt/β-catenin signaling in cultured podocytes. This activation also upregulated FHL2 expression and promoted FHL2 translocation from cytosol to nucleus. Genetic deletion of the FHL2 gene mitigated the podocyte dedifferentiation caused by activated Wnt/β-catenin signaling under Wnt-On, but not under Wnt-Off, conditions. Diabetic FHL2^+/+ mice developed markedly increased albuminuria and thickening of the glomerular basement membrane compared with nondiabetic FHL2^+/+ mice. However, FHL2 knockout significantly attenuated these DKD-induced changes. Furthermore, kidney samples from patients with diabetes had a higher degree of FHL2 podocyte nuclear translocation, which was positively associated with albuminuria and progressive renal function deterioration. Therefore, we conclude that FHL2 has both structural and functional protein-protein interactions with β-catenin in the podocyte nucleus and that FHL2 protein inhibition can mitigate Wnt/β-catenin–induced podocytopathy.     

Pifithrin-α ameliorates glycerol induced rhabdomyolysis and acute kidney injury by reducing p53 activation

ObjectivesRhabdomyolysis is a series of symptoms caused by the dissolution of striped muscle, and acute kidney injury (AKI) is a potential complication of severe rhabdomyolysis. The underlying causes of AKI are remarkably complex and diverse. Here, we aim to investigate whether pifithrin-α protected against rhabdomyolysis-induced AKI and to determine the involved mechanisms.MethodsIntramuscular injection in the right thigh caudal muscle of C57BL/6J mice with 7.5 ml/kg saline (Group A) or of the same volume 50% glycerol was used to induce rhabdomyolysis and subsequent AKI (Group B). Pifithrin-α was injected intraperitoneally 4 h before (Group C) or 4 h after (Group D) the glycerol injection. Serum creatine kinase, blood urea nitrogen, and creatinine were determined, and the renal cortex was histologically analyzed. Renal expression levels of interested mRNAs and proteins were determined and compared, too.ResultsIntramuscular injection of glycerol induced rhabdomyolysis and subsequent AKI in mice (Groups B–D). Renal function reduction and histologic injury of renal tubular epithelial cells were associated with increased p53 activation, oxidative stress, and inflammation. Notably, compared with pifithrin-α rescue therapy (Group D), pretreatment of pifithrin-α (Group C) protected the mice from severe injury more effectively.ConclusionsOur present study suggests that p53 may be a therapeutic target of AKI caused by glycerol, and the inhibition of p53 can block glycerol-mediated AKI by using pharmacological agents instead of genetic inhibitory approaches, which further supports that p53 played a pivotal role in renal tubular injury when challenged with glycerol.     

Antiaging Gene Klotho Attenuates Pancreatic β-Cell Apoptosis in Type 1 Diabetes

Apoptosis is the major cause of death of insulin-producing β-cells in type 1 diabetes mellitus (T1DM). Klotho is a recently discovered antiaging gene. We found that the Klotho gene is expressed in pancreatic β-cells. Interestingly, halplodeficiency of Klotho (KL^+/−) exacerbated streptozotocin (STZ)-induced diabetes (a model of T1DM), including hyperglycemia, glucose intolerance, diminished islet insulin storage, and increased apoptotic β-cells. Conversely, in vivo β-cell–specific expression of mouse Klotho gene (mKL) attenuated β-cell apoptosis and prevented STZ-induced diabetes. mKL promoted cell adhesion to collagen IV, increased FAK and Akt phosphorylation, and inhibited caspase 3 cleavage in cultured MIN6 β-cells. mKL abolished STZ- and TNFα-induced inhibition of FAK and Akt phosphorylation, caspase 3 cleavage, and β-cell apoptosis. These promoting effects of Klotho can be abolished by blocking integrin β1. Therefore, these cell-based studies indicated that Klotho protected β-cells by inhibiting β-cell apoptosis through activation of the integrin β1-FAK/Akt pathway, leading to inhibition of caspase 3 cleavage. In an autoimmune T1DM model (NOD), we showed that in vivo β-cell–specific expression of mKL improved glucose tolerance, attenuated β-cell apoptosis, enhanced insulin storage in β-cells, and increased plasma insulin levels. The beneficial effect of Klotho gene delivery is likely due to attenuation of T-cell infiltration in pancreatic islets in NOD mice. Overall, our results demonstrate for the first time that Klotho protected β-cells in T1DM via attenuating apoptosis.     

Deletion of Lkb1 in Renal Tubular Epithelial Cells Leads to CKD by Altering Metabolism

Renal tubule epithelial cells are high-energy demanding polarized epithelial cells. Liver kinase B1 (LKB1) is a key regulator of polarity, proliferation, and cell metabolism in epithelial cells, but the function of LKB1 in the kidney is unclear. Our unbiased gene expression studies of human control and CKD kidney samples identified lower expression of LKB1 and regulatory proteins in CKD. Mice with distal tubule epithelial-specific Lkb1 deletion (Ksp-Cre/Lkb1^flox/flox) exhibited progressive kidney disease characterized by flattened dedifferentiated tubule epithelial cells, interstitial matrix accumulation, and dilated cystic-appearing tubules. Expression of epithelial polarity markers β-catenin and E-cadherin was not altered even at later stages. However, expression levels of key regulators of metabolism, AMP-activated protein kinase (Ampk), peroxisome proliferative activated receptor gamma coactivator 1-α (Ppargc1a), and Ppara, were significantly lower than those in controls and correlated with fibrosis development. Loss of Lkb1 in cultured epithelial cells resulted in energy depletion, apoptosis, less fatty acid oxidation and glycolysis, and a profibrotic phenotype. Treatment of Lkb1-deficient cells with an AMP-activated protein kinase (AMPK) agonist (A769662) or a peroxisome proliferative activated receptor alpha agonist (fenofibrate) restored the fatty oxidation defect and reduced apoptosis. In conclusion, we show that loss of LKB1 in renal tubular epithelial cells has an important role in kidney disease development by influencing intracellular metabolism.