Restoration of the ER stress response protein TDAG51 in hepatocytes mitigates NAFLD in mice.

Endoplasmic reticulum stress is associated with insulin resistance and the development of nonalcoholic fatty liver disease. Deficiency of the endoplasmic reticulum stress response T-cell death-associated gene 51 (TDAG51) (TDAG51-/-) in mice promotes the development of high-fat diet (HFD)-induced obesity, fatty liver, and hepatic insulin resistance. However, whether this effect is due specifically to hepatic TDAG51 deficiency is unknown. Here, we report that hepatic TDAG51 protein levels are consistently reduced in multiple mouse models of liver steatosis and injury as well as in liver biopsies from patients with liver disease compared to normal controls. Delivery of a liver-specific adeno-associated virus (AAV) increased hepatic expression of a TDAG51-GFP fusion protein in WT, TDAG51-/-, and leptin-deficient (ob/ob) mice. Restoration of hepatic TDAG51 protein was sufficient to increase insulin sensitivity while reducing body weight and fatty liver in HFD fed TDAG51-/- mice and in ob/ob mice. TDAG51-/- mice expressing ectopic TDAG51 display improved Akt (Ser473) phosphorylation, post-insulin stimulation. HFD-fed TDAG51-/- mice treated with AAV-TDAG51-GFP displayed reduced lipogenic gene expression, increased beta-oxidation and lowered hepatic and serum triglycerides, findings consistent with reduced liver weight. Further, AAV-TDAG51-GFP-treated TDAG51-/- mice exhibited reduced hepatic precursor and cleaved sterol regulatory-element binding proteins (SREBP-1 and SREBP-2). In vitro studies confirmed the lipid-lowering effect of TDAG51 overexpression in oleic acid-treated Huh7 cells. These studies suggest that maintaining hepatic TDAG51 protein levels represents a viable therapeutic approach for the treatment of obesity and insulin resistance associated with nonalcoholic fatty liver disease.

Both sexes develop DKD in the CD1 uninephrectomized streptozotocin mouse model.

Diabetic kidney disease (DKD) is characterized by a progressive increase in albuminuria and typical pathologic features. Recent studies have shown that sex is an important factor to consider in the pathogenesis of DKD. Presently, the hallmarks of this disease have primarily been studied in male rodent models. Here we explored the influence of sex in a murine model of DKD. CD1 mice underwent a right nephrectomy followed by intraperitoneal injection with 200 mg/kg streptozotocin to induce type 1 diabetes. Due to a high mortality rate, females required a reduction in streptozotocin to 150 mg/kg. Mice were followed for 12 weeks. Both sexes developed comparable hyperglycemia, while albuminuria and glomerular volume were increased to a greater degree in females and kidney hypertrophy was only seen in females. Males had a greater increase in blood pressure and glomerular basement membrane thickening, and a greater decrease in endpoint weight. Serum TGFß1 levels were increased only in females. However, both sexes showed a similar increase in induction of kidney fibrosis. T cell and macrophage infiltration were also increased in both sexes. While some differences were observed, overall, both sexes developed clinical and pathologic characteristics of early DKD. Future studies evaluating therapeutic interventions can thus be assessed in both sexes of this DKD model.

A novel multi-ancestry proteome-wide Mendelian randomization study implicates extracellular proteins, tubular cells, and fibroblasts in estimated glomerular filtration rate regulation.

Estimated glomerular filtration rate (eGFR) impacts the concentration of plasma biomarkers confounding biomarker association studies of eGFR with reverse causation. To identify biomarkers causally associated with eGFR, we performed a proteome-wide Mendelian randomization study. Genetic variants nearby biomarker coding genes were tested for association with plasma concentration of 1,161 biomarkers in a multi-ancestry sample of 12,066 participants from the Prospective Urban and Rural Epidemiological (PURE) study. Using two-sample Mendelian randomization, individual variants' effects on biomarker concentration were correlated with their effects on eGFR and kidney traits from published genome-wide association studies (GWAS). Genetically altered concentrations of 22 biomarkers were associated with eGFR above a Bonferroni-corrected significance threshold. Five biomarkers were previously identified by GWAS (UMOD, FGF5, LGALS7, NINJ1, COL18A1). Nine biomarkers were within 1 Mb of the lead GWAS variant but the gene for the biomarker was unidentified as the candidate for the GWAS signal (INHBC, TNFRSF11A, TCN2, PXN1, PRTN3, PSMD9, TFPI, ITGB6, CA3). Single-cell transcriptomic data indicated the 22 biomarkers are expressed in kidney tubules, collecting duct, fibroblasts, and immune cells. Pathway analysis showed significant enrichment of identified biomarkers in the extracellular kidney parenchyma. Thus, using genetic regulators of biomarker concentration via proteome-wide Mendelian randomization, we identified 22 biomarkers that appear to causally impact eGFR in either a beneficial or adverse manner. The current study provides rationale for novel therapeutic targets for eGFR and emphasized a role for extracellular proteins produced by tubular cells and fibroblasts for impacting eGFR.

Cell surface GRP78 regulates TGFß1-mediated profibrotic responses via TSP1 in diabetic kidney disease.

Introduction: Diabetic kidney disease (DKD) is the leading cause of kidney failure in North America, characterized by glomerular accumulation of extracellular matrix (ECM) proteins. High glucose (HG) induction of glomerular mesangial cell (MC) profibrotic responses plays a central role in its pathogenesis. We previously showed that the endoplasmic reticulum resident GRP78 translocates to the cell surface in response to HG, where it mediates Akt activation and downstream profibrotic responses in MC. Transforming growth factor ß1 (TGFß1) is recognized as a central mediator of HG-induced profibrotic responses, but whether its activation is regulated by cell surface GRP78 (csGRP78) is unknown. TGFß1 is stored in the ECM in a latent form, requiring release for biological activity. The matrix glycoprotein thrombospondin 1 (TSP1), known to be increased in DKD and by HG in MC, is an important factor in TGFß1 activation. Here we determined whether csGRP78 regulates TSP1 expression and thereby TGFß1 activation by HG. Methods: Primary mouse MC were used. TSP1 and TGFß1 were assessed using standard molecular biology techniques. Inhibitors of csGRP78 were: 1) vaspin, 2) the C-terminal targeting antibody C38, 3) siRNA downregulation of its transport co-chaperone MTJ-1 to prevent GRP78 translocation to the cell surface, and 4) prevention of csGRP78 activation by its ligand, active α2-macroglobulin (α2M*), with the neutralizing antibody Fα2M or an inhibitory peptide. Results: TSP1 transcript and promoter activity were increased by HG, as were cellular and ECM TSP1, and these required PI3K/Akt activity. Inhibition of csGRP78 prevented HG-induced TSP1 upregulation and deposition into the ECM. The HG-induced increase in active TGFß1 in the medium was also inhibited, which was associated with reduced intracellular Smad3 activation and signaling. Overexpression of csGRP78 increased TSP-1, and this was further augmented in HG. Discussion: These data support an important role for csGRP78 in regulating HG-induced TSP1 transcriptional induction via PI3K/Akt signaling. Functionally, this enables TGFß1 activation in response to HG, with consequent increase in ECM proteins. Means of inhibiting csGRP78 signaling represent a novel approach to preventing fibrosis in DKD.

A therapeutic target for CKD: activin A facilitates TGFß1 profibrotic signaling.

BACKGROUND: TGFß1 is a major profibrotic mediator in chronic kidney disease (CKD). Its direct inhibition, however, is limited by adverse effects. Inhibition of activins, also members of the TGFß superfamily, blocks TGFß1 profibrotic effects, but the mechanism underlying this and the specific activin(s) involved are unknown. METHODS: Cells were treated with TGFß1 or activins A/B. Activins were inhibited generally with follistatin, or specifically with neutralizing antibodies or type I receptor downregulation. Cytokine levels, signaling and profibrotic responses were assessed with ELISA, immunofluorescence, immunoblotting and promoter luciferase reporters. Wild-type or TGFß1-overexpressing mice with unilateral ureteral obstruction (UUO) were treated with an activin A neutralizing antibody. RESULTS: In primary mesangial cells, TGFß1 induces secretion primarily of activin A, which enables longer-term profibrotic effects by enhancing Smad3 phosphorylation and transcriptional activity. This results from lack of cell refractoriness to activin A, unlike that for TGFß1, and promotion of TGFß type II receptor expression. Activin A also supports transcription through regulating non-canonical MRTF-A activation. TGFß1 additionally induces secretion of activin A, but not B, from tubular cells, and activin A neutralization prevents the TGFß1 profibrotic response in renal fibroblasts. Fibrosis induced by UUO is inhibited by activin A neutralization in wild-type mice. Worsened fibrosis in TGFß1-overexpressing mice is associated with increased renal activin A expression and is inhibited to wild-type levels with activin A neutralization. CONCLUSIONS: Activin A facilitates TGFß1 profibrotic effects through regulation of both canonical (Smad3) and non-canonical (MRTF-A) signaling, suggesting it may be a novel therapeutic target for preventing fibrosis in CKD.

Integrin ß1/Cell Surface GRP78 Complex Regulates TGFß1 and Its Profibrotic Effects in Response to High Glucose.

Diabetic kidney disease (DKD) is the leading cause of kidney failure worldwide. Characterized by overproduction and accumulation of extracellular matrix (ECM) proteins, glomerular sclerosis is its earliest manifestation. High glucose (HG) plays a central role by increasing matrix production by glomerular mesangial cells (MC). We previously showed that HG induces translocation of GRP78 from the endoplasmic reticulum to the cell surface (csGRP78), where it acts as a signaling molecule to promote intracellular profibrotic FAK/Akt activation. Here, we identify integrin ß1 as a key transmembrane signaling partner for csGRP78. We show that it is required for csGRP78-regulated FAK/Akt activation in response to HG, as well as downstream production, secretion and activity of the well characterized profibrotic cytokine transforming growth factor ß1 (TGFß1). Intriguingly, integrin ß1 also itself promotes csGRP78 translocation. Furthermore, integrin ß1 effects on cytoskeletal organization are not required for its function in csGRP78 translocation and signaling. These data together support an important pathologic role for csGRP78/integrin ß1 in mediating key profibrotic responses to HG in kidney cells. Inhibition of their interaction will be further evaluated as a therapeutic target to limit fibrosis progression in DKD.

Inhibitory Antibodies against PCSK9 Reduce Surface CD36 and Mitigate Diet-Induced Renal Lipotoxicity.

Background: PCSK9 modulates the uptake of circulating lipids through a range of receptors, including the low-density lipoprotein receptor (LDLR) and CD36. In the kidney, CD36 is known to contribute to renal injury through pro-inflammatory and -fibrotic pathways. In this study, we sought to investigate the role of PCSK9 in modulating renal lipid accumulation and injury through CD36 using a high fat diet (HFD)-induced murine model. Methods: The effect of PCSK9 on the expression of CD36 and intracellular accumulation of lipid was examined in cultured renal cells and in the kidneys of male C57BL/6J mice. The effect of these findings was subsequently explored in a model of HFD-induced renal injury in Pcsk9 -/- and Pcsk9 +/+ littermate control mice on a C57BL/6J background. Results: In the absence of PCSK9, we observed heightened CD36 expression levels, which increased free fatty acid (FFA) uptake in cultured renal tubular cells. As a result, PCSK9 deficiency was associated with an increase in long-chain saturated FFA-induced ER stress. Consistent with these observations, Pcsk9-/- mice fed a HFD displayed elevated ER stress, inflammation, fibrosis, and renal injury relative to HFD-fed control mice. In contrast to Pcsk9-/- mice, pretreatment of WT C57BL/6J mice with evolocumab, an anti-PCSK9 monoclonal antibody (mAb) that binds to and inhibits the function of circulating PCSK9, protected against HFD-induced renal injury in association with reducing cell surface CD36 expression on renal epithelia. Conclusions: We report that circulating PCSK9 modulates renal lipid uptake in a manner dependent on renal CD36. In the context of increased dietary fat consumption, the absence of circulating PCSK9 may promote renal lipid accumulation and subsequent renal injury. However, although the administration of evolocumab blocks the interaction of PCSK9 with the LDLR, this evolocumab/PCSK9 complex can still bind CD36, thereby protecting against HFD-induced renal lipotoxicity.

ACLY and CKD: A Mendelian Randomization Analysis.

Introduction: Adenosine triphosphate-citrate lyase (ACLY) inhibition is a therapeutic strategy under investigation for atherosclerotic cardiovascular disease, nonalcoholic steatohepatitis, and metabolic syndrome. Mouse models suggest that ACLY inhibition could reduce inflammation and kidney fibrosis. Genetic analysis of ACLY in chronic kidney disease (CKD) has not been performed. Methods: We constructed a genetic instrument by selecting variants associated with ACLY expression in the expression quantitative trait loci genetics consortium (eQTLGen) from blood samples from 31,684 participants. In a 2-sample Mendelian randomization analysis, we evaluated the effect of genetically predicted ACLY expression on the risk of CKD, estimated glomerular filtration rate (eGFR), and albumin-to-creatinine ratio (ACR) using the CKD Genetics (CKDGen) consortium, UK Biobank, and the Finnish Genetics (FinnGen) consortium totaling 66,396 CKD cases and 958,517 controls. Results: ACLY is constitutively expressed in all cell types including in whole blood. The genetic instrument included 13 variants and explained 1.5% of the variation in whole blood ACLY gene expression. A 34% reduction in ACLY expression score was associated with a 0.04 mmol/l reduced low-density lipoprotein (LDL) cholesterol (P = 3.4 × 10-4) and a 9% reduced risk of CKD (stages 3, 4, 5, dialysis, or eGFR < 60 ml/min per 1.73 m2) (odds ratio [OR] = 0.91, 95% CI: 0.85-0.98, P = 0.008), but no association was observed with either eGFR or ACR. Conclusion: Mendelian randomization analyses revealed that genetically reduced ACLY expression was associated with reduced risk of CKD but had no effect on either eGFR or ACR. Further evaluation of ACLY in kidney disease is warranted.

Activin B, a new player in kidney fibrosis?†.

Interest has been growing in the role of activin A in both acute and chronic kidney disease. The role of other activins, however, remains relatively unexplored. In a recent issue of The Journal of Pathology, an elegant study by Sun et al identified upregulation of INHBB, the subunit of activin B, in three different models of kidney fibrosis, as well as in human kidneys with fibrosis. This increase was shown to be mediated by upregulation of the transcription factor Sox9. Using overexpression and inhibition strategies, the importance of INHBB to kidney interstitial fibroblast activation and kidney fibrosis was clearly shown. Importantly, INHBB and Sox9 are not appreciably expressed in normal tissue. These studies lay important groundwork for the further investigation of activin B targeting as a potential therapeutic approach to attenuate kidney fibrosis. © 2021 The Pathological Society of Great Britain and Ireland.

Activated Alpha 2-Macroglobulin Is a Novel Mediator of Mesangial Cell Profibrotic Signaling in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is caused by the overproduction of extracellular matrix proteins (ECM) by glomerular mesangial cells (MCs). We previously showed that high glucose (HG) induces cell surface translocation of GRP78 (csGRP78), mediating PI3K/Akt activation and downstream ECM production. Activated alpha 2-macroglobulin (α2M*) is a ligand known to initiate this signaling cascade. Importantly, increased α2M was observed in diabetic patients' serum, saliva, and glomeruli. Primary MCs were used to assess HG responses. The role of α2M* was assessed using siRNA, a neutralizing antibody and inhibitory peptide. Kidneys from type 1 diabetic Akita and CD1 mice and human DKD patients were stained for α2M/α2M*. α2M transcript and protein were significantly increased with HG in vitro and in vivo in diabetic kidneys. A similar increase in α2M* was seen in media and kidneys, where it localized to the mesangium. No appreciable α2M* was seen in normal kidneys. Knockdown or neutralization of α2M/α2M* inhibited HG-induced profibrotic signaling (Akt activation) and matrix/cytokine upregulation (collagen IV, fibronectin, CTGF, and TGFß1). In patients with established DKD, urinary α2M* and TGFß1 levels were correlated. These data reveal an important role for α2M* in the pathogenesis of DKD and support further investigation as a potential novel therapeutic target.

Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.

miR299a-5p promotes renal fibrosis by suppressing the antifibrotic actions of follistatin.

Caveolin-1 (cav-1), an integral protein of the membrane microdomains caveolae, is required for synthesis of matrix proteins by glomerular mesangial cells (MC). Previously, we demonstrated that the antifibrotic protein follistatin (FST) is transcriptionally upregulated in cav-1 knockout MC and that its administration is protective against renal fibrosis. Here, we screened cav-1 wild-type and knockout MC for FST-targeting microRNAs in order to identity novel antifibrotic therapeutic targets. We identified that miR299a-5p was significantly suppressed in cav-1 knockout MC, and this was associated with stabilization of the FST 3'UTR. Overexpression and inhibition studies confirmed the role of miR299a-5p in regulating FST expression. Furthermore, the profibrotic cytokine TGFß1 was found to stimulate the expression of miR299a-5p and, in turn, downregulate FST. Through inhibition of FST, miR299a-5p overexpression augmented, while miR299a-5p inhibition diminished TGFß1 profibrotic responses, whereas miR299a-5p overexpression re-enabled cav-1 knockout MC to respond to TGFß1. In vivo, miR299a-5p was upregulated in the kidneys of mice with chronic kidney disease (CKD). miR299a-5p inhibition protected these mice against renal fibrosis and CKD severity. Our data demonstrate that miR299a-5p is an important post-transcriptional regulator of FST, with its upregulation an important pathogenic contributor to renal fibrosis. Thus, miR299a-5p inhibition offers a potential novel therapeutic approach for CKD.

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

The emerging role of activins in renal disease.

PURPOSE OF REVIEW: This review highlights recent discoveries and advances that have been made in understanding the role of the TGFß superfamily members activins, and in particular, activin A (ActA), in renal disease. RECENT FINDINGS: A deleterious role for ActA in renal disease and its complications has begun to emerge. We summarize data supporting an important contribution of ActA to kidney fibrosis and inflammation of varying causes, as well as its role in the development of a particular bone mineral disorder seen in chronic kidney disease (CKD) called mineral bone disorder (MBD), including vascular calcification. Finally, we discuss ActA in the context of anemia associated with chronic kidney disease and review potential approaches to treatment based on ActA blockade. SUMMARY: ActA is an important contributor to the pathogenesis of acute and chronic kidney disease of varying causes. Preclinical studies show that ActA inhibition, through various approaches, is protective in rodent models of kidney disease. The potential adverse effects of some of these approaches can be attributed to their targeting of other TGFß family ligands. Further preclinical and clinical investigations testing the therapeutic efficacy of more selective ActA inhibition on the progression of acute and chronic kidney disease and its impact on bone-mineral disorder would more definitively establish its role in renal disease.

The caveolin-1 regulated protein follistatin protects against diabetic kidney disease.

Glomerular matrix protein accumulation, mediated largely by mesangial cells, is central to the pathogenesis of diabetic kidney disease. Our previous studies showed that the membrane microdomains caveolae and their marker protein caveolin-1 regulate matrix protein synthesis in mesangial cells in response to diabetogenic stimuli, and that caveolin-1 knockout mice are protected against diabetic kidney disease. In a screen to identify the molecular mechanism underlying this protection, we also established that secreted antifibrotic glycoprotein follistatin is significantly upregulated by caveolin-1 deletion. Follistatin potently neutralizes activins, members of the transforming growth factor-ß superfamily. A role for activins in diabetic kidney disease has not yet been established. Therefore, in vitro, we confirmed the regulation of follistatin by caveolin-1 in primary mesangial cells and showed that follistatin controls both basal and glucose-induced matrix production through activin inhibition. In vivo, we found activin A upregulation by immunohistochemistry in both mouse and human diabetic kidney disease. Importantly, administration of follistatin to type 1 diabetic Akita mice attenuated early diabetic kidney disease, characterized by albuminuria, hyperfiltration, basement membrane thickening, loss of endothelial glycocalyx and podocyte nephrin, and glomerular matrix accumulation. Thus, activin A is an important mediator of high glucose-induced profibrotic responses in mesangial cells, and follistatin may be a potential novel therapy for the prevention of diabetic kidney disease.

GDF10 blocks hepatic PPARγ activation to protect against diet-induced liver injury.

OBJECTIVE: Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor ß (TGFß) superfamily and are known to play a central role in the growth and differentiation of developing tissues. Accumulating evidence, however, demonstrates that many of these factors, such as BMP-2 and -4, as well as GDF15, also regulate lipid metabolism. GDF10 is a divergent member of the TGFß superfamily with a unique structure and is abundantly expressed in brain and adipose tissue; it is also secreted by the latter into the circulation. Although previous studies have demonstrated that overexpression of GDF10 reduces adiposity in mice, the role of circulating GDF10 on other tissues known to regulate lipid, like the liver, has not yet been examined. METHODS: Accordingly, GDF10-/- mice and age-matched GDF10+/+ control mice were fed either normal control diet (NCD) or high-fat diet (HFD) for 12 weeks and examined for changes in liver lipid homeostasis. Additional studies were also carried out in primary and immortalized human hepatocytes treated with recombinant human (rh)GDF10. RESULTS: Here, we show that circulating GDF10 levels are increased in conditions of diet-induced hepatic steatosis and, in turn, that secreted GDF10 can prevent excessive lipid accumulation in hepatocytes. We also report that GDF10-/- mice develop an obese phenotype as well as increased liver triglyceride accumulation when fed a NCD. Furthermore, HFD-fed GDF10-/- mice develop increased steatosis, endoplasmic reticulum (ER) stress, fibrosis, and injury of the liver compared to HFD-fed GDF10+/+ mice. To explain these observations, studies in cultured hepatocytes led to the observation that GDF10 attenuates nuclear peroxisome proliferator-activated receptor γ (PPARγ) activity; a transcription factor known to induce de novo lipogenesis. CONCLUSION: Our work delineates a hepatoprotective role of GDF10 as an adipokine capable of regulating hepatic lipid levels by blocking de novo lipogenesis to protect against ER stress and liver injury.

Follistatin Protects Against Glomerular Mesangial Cell Apoptosis and Oxidative Stress to Ameliorate Chronic Kidney Disease.

Aims: Interventions to inhibit oxidative stress and apoptosis, important pathogenic contributors toward the progression of chronic kidney disease (CKD), are not well established. Here, we investigated the role of a transforming growth factor beta (TGFß) superfamily neutralizing protein, follistatin (FST), in the regulation of apoptosis and oxidative stress in glomerular mesangial cells (MCs) and in the progression of CKD. Results: The endoplasmic reticulum (ER) stress inducer thapsigargin (Tg), known to cause MC apoptosis, led to a post-translational increase in the expression of FST. Recombinant FST protected, whereas FST downregulation augmented, Tg-induced apoptosis without affecting Ca2+ release or ER stress induction. Although activins are the primary ligands neutralized by FST, their inhibition with neutralizing antibodies did not affect Tg-induced apoptosis. Instead, FST protected against Tg-induced apoptosis through neutralization of reactive oxygen species (ROS) independently of its ability to neutralize activins. Importantly, administration of FST to mice with CKD protected against renal cell apoptosis and oxidative stress. This was associated with improved kidney function, reduced albuminuria, and attenuation of fibrosis. Innovation and Conclusion: Independent of its activin neutralizing ability, FST protected against Tg-induced apoptosis through neutralization of ROS and consequent suppression of oxidative stress, seen both in vitro and in vivo. Importantly, FST also ameliorated fibrosis and improved kidney function in CKD. FST is, thus, a novel potential therapeutic agent for delaying the progression of CKD. Antioxid. Redox Signal. 31, 551-571.

Caveolin-1 regulation of Sp1 controls production of the antifibrotic protein follistatin in kidney mesangial cells.

BACKGROUND: We previously showed that caveolin-1 (cav-1), an integral membrane protein, is required for the synthesis of matrix proteins by glomerular mesangial cells (MC). In a previous study to understand how cav-1 is involved in regulating matrix production, we had identified significant upregulation of the antifibrotic protein follistatin in cav-1 knockout MC. Follistatin inhibits the profibrotic effects of several members of the transforming growth factor beta superfamily, in particular the activins. Here, we characterize the molecular mechanism through which cav-1 regulates the expression of follistatin. METHODS: Kidneys from cav-1 wild type and knockout (KO) mice were analyzed and primary cultures of MC from cav-1 wild-type and KO mice were utilized. FST promoter deletion constructs were generated to determine the region of the promoter important for mediating FST upregulation in cav-1 KO MC. siRNA-mediated down-regulation and overexpression of Sp1 in conjunction with luciferase activity assays, immunoprecipitation, western blotting and ChiP was used to assess the role of Sp1 in transcriptionally regulating FST expression. Pharmacologic kinase inhibitors and specific siRNA were used to determine the post-translational mechanism through which cav-1 affects Sp1 activity. RESULTS: Our results establish that follistatin upregulation occurs at the transcript level. We identified Sp1 as the critical transcription factor regulating activation of the FST promoter in cav-1 KO MC through binding to a region within 123 bp of the transcription start site. We further determined that the lack of cav-1 increases Sp1 nuclear levels and transcriptional activity. This occurred through increased phosphoinositide 3-kinase (PI3K) activity and downstream protein kinase C (PKC) zeta-mediated phosphorylation and activation of Sp1. CONCLUSIONS: These findings shed light on the transcriptional mechanism by which cav-1 represses the expression of a major antifibrotic protein, and can inform the development of novel antifibrotic treatment strategies.

Parathyroid hormone-related protein induces fibronectin up-regulation in rat mesangial cells through reactive oxygen species/Src/EGFR signaling.

Parathyroid hormone-related protein (PTHrP) is known to be up-regulated in both glomeruli and tubules in patients with diabetic kidney disease (DKD), but its role remains unclear. Previous studies show that PTHrP-induced hypertrophic response in mesangial cells (MCs) and epithelial-mesenchymal transition (EMT) in tubuloepithelial cells can be mediated by TGF-ß1. In the present study, although long-term PHTrP (1-34) treatment increased the mRNA and protein level of TGF-ß1 in primary rat MCs, fibronectin up-regulation occurred earlier, suggesting that fibronectin induction is independent of TGF-ß1/Smad signaling. We thus evaluated the involvement of epidermal growth factor receptor (EGFR) signaling and found that nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species mediates PTHrP (1-34)-induced Src kinase activation. Src phosphorylates EGFR at tyrosine 845 and then transactive EGFR. Subsequent PI3K activation mediates Akt and ERK1/2 activation. Akt and ERK1/2 discretely lead to excessive protein synthesis of fibronectin. Our study thus demonstrates the new role of PTHrP in fibronectin up-regulation for the first time in glomerular MCs. These data also provided new insights to guide development of therapy for glomerular sclerosis.

Cell surface expression of 78-kDa glucose-regulated protein (GRP78) mediates diabetic nephropathy.

The 78-kDa glucose-regulated protein (GRP78) is a well-established endoplasmic reticulum (ER)-resident chaperone that maintains protein homeostasis and regulates the unfolded protein response. Under conditions of ER stress, GRP78 is also expressed at the cell surface and implicated in tumorigenesis, immunity, and cellular signaling events. The role of cell surface-associated GRP78 (csGRP78) in the pathogenesis of diabetic nephropathy has not yet been defined. Here we explored the role of csGRP78 in regulating high glucose (HG)-induced profibrotic AKT Ser/Thr kinase (AKT) signaling and up-regulation of extracellular matrix proteins. Using primary kidney mesangial cells, we show that HG treatment, but not the osmotic control mannitol, induces csGRP78 expression through an ER stress-dependent mechanism. We found that csGRP78, known to be located on the outer membrane leaflet, interacts with the transmembrane protein integrin ß1 and activates focal adhesion kinase and downstream PI3K/AKT signaling. Localization of GRP78 at the cell surface and its interaction with integrin ß1 were also required for extracellular matrix protein synthesis in response to HG. Surprisingly, both the N and C termini of csGRP78 were necessary for this profibrotic response. Increased localization of GRP78 at the plasma membrane was also found in the glomerular mesangial area of type 1 diabetic mice in two different models (streptozotocin-induced and Akita). In freshly isolated glomeruli from Akita mice, csGRP78 co-localized with the mesangial cell surface marker α8-integrin. In conclusion, our work reveals a role for csGRP78 in HG-induced profibrotic responses in mesangial cells, informing a potential approach to treating diabetic nephropathy.