Negative Modulation of B Cell Activation by Melanocortin 1 Receptor Signaling Protects against Membranous Nephropathy.

SIGNIFICANCE STATEMENT: Emerging evidence suggests that melanocortin neuropeptides-specifically adrenocorticotropic hormone-offer a novel, steroidogenic-independent therapeutic modality for membranous nephropathy (MN). The molecular mechanism underlying this beneficial effect, however, remains largely elusive. To investigate whether melanocortins modulate humoral immunity, the authors induced passive Heymann nephritis, a model of human MN, in wild-type and melanocortin 1 receptor (MC1R) knockout rats and treated them with melanocortin agents. Additional rats received adoptive transfer of bone marrow-derived cells beforehand from wild-type or MC1R knockout rats. The findings indicate that MC1R signaling plays a key role in negative modulation of B-cell activation and thereby suppresses humoral immune responses in passive Heymann nephritis, and suggest that MC1R signaling might offer a novel B cell-targeted therapeutic strategy for MN. BACKGROUND: Emerging evidence suggests that the pituitary neuropeptide melanocortins-specifically, adrenocorticotropic hormone-offer a novel nonsteroidogenic therapeutic modality for membranous nephropathy (MN). However, the mechanism(s) of action remains elusive. METHODS: To investigate whether melanocortins modulate humoral immunity, we induced passive Heymann nephritis (PHN), a model of MN, in wild-type (WT) and melanocortin 1 receptor (MC1R) knockout (KO) rats. We treated the animals with melanocortin agents-repository corticotropin injection, the nonsteroidogenic pan-melanocortin receptor agonist [Nle 4 , DPhe 7 ]-α-melanocyte stimulating hormone, the selective MC1R agonist MS05, vehicle gel, or phosphate-buffered saline-and evaluated kidney function, histology, and molecular changes. Additional rats received adoptive transfer of syngeneic bone marrow-derived cells beforehand from WT or MC1R KO rats. RESULTS: KO of MC1R worsened PHN and this was associated with increased deposition of autologous immunoglobulin G (IgG) and complement C5b-9 in glomeruli and higher circulating levels of autologous IgG-evidence of a sensitized humoral immune response. Melanocortin therapy ameliorated PHN in WT rats, coinciding with reduced glomerular deposition of autologous IgG and C5b -9. The beneficial efficacy of melanocortins was blunted in KO rats but restored by adoptive transfer of syngeneic bone marrow-derived cells derived from WT rats. Mechanistically, MC1R was expressed in B lymphocytes and was negatively associated with B cell activation. MC1R agonism triggered the expression of microphthalmia-associated transcription factor in activated B cells in a cAMP-dependent mode and also repressed the expression of interferon regulatory factor 4 (a lymphoid transcription factor essential for B-cell development and maturation), resulting in suppressed plasma cell differentiation and IgG production. CONCLUSIONS: MC1R signaling negatively modulates B cell activation and suppresses humoral immune responses in PHN, suggesting that MC1R signaling might offer a novel therapeutic target for MN.

Hematopoietic-specific melanocortin 1 receptor signaling protects against nephrotoxic serum nephritis and mediates the beneficial effect of melanocortin therapy.

The melanocortin hormone system has emerged as a novel therapeutic target for treating refractory glomerular diseases. However, the role of hematopoietic melanocortin 1 receptor (MC1R) signaling remains unknown. Upon insult by rabbit nephrotoxic serum, MC1R null-mutant mice developed more severe crescentic glomerulonephritis than wild-type mice, marked by aggravated proteinuria, kidney dysfunction and histologic lesions. Melanocortin therapy, using Repository Corticotropin Injection (Acthar Gel), the pan-melanocortin receptor agonist NDP-MSH, or the MC1R agonist MS05, ameliorated experimental nephritis in wild-type mice but this effect was blunted in null mice. Exacerbated experimental nephritis in null mice was associated with increased glomerular deposition of autologous IgG and C5b-9, in parallel with higher circulating levels of autologous IgG2c and IgG3. Additionally, the Th1 immune response was potentiated in null mice with experimental nephritis, accompanied by diminished kidney FoxP3+ regulatory T cells. Kidney infiltration of macrophages was also augmented by MC1R deficiency with an enhanced M1 polarization. Moreover, adoptive transfer of syngeneic bone marrow-derived cells from wild-type mice mitigated experimental nephritis in null mice and restored the beneficial efficacy of melanocortins. Mechanistically, MC1R was expressed by diverse subsets of kidney leukocytes, including macrophages, T and B lymphocytes, and was inversely associated with the NFκB pathway, a key player in immune responses. MS05 attenuated the production of rabbit IgG-specific IgG2c and IgG3 in cultured wild-type splenocytes, and promoted M2 polarization in M1-primed wild-type macrophages, associated with NFκB inhibition. In contrast, in null splenocytes or macrophages, this effect of MS05 was barely detectable, but was mimicked by an NFκB inhibitor. Thus, hematopoietic MC1R signaling attenuates experimental nephritis and mediates the beneficial effect of melanocortin therapy via, in part, regulating the immune response.

Hemopexin accumulates in kidneys and worsens acute kidney injury by causing hemoglobin deposition and exacerbation of iron toxicity in proximal tubules.

Hemopexin, a heme scavenging protein, accumulates in the kidneys during acute kidney injury (AKI). However, the function of this accumulated hemopexin in the kidney is unclear. In both the cisplatin-induced and the unilateral kidney ischemia-reperfusion injury models of AKI, we found accumulation of hemoglobin and hemopexin in the kidneys localized to the proximal tubules. Next, hemopexin wild-type and knockout mice were compared in both AKI models and hemopexin wild type mice had significantly worse kidney injury. Furthermore, there was increased kidney expression of kidney injury molecule-1 (a biomarker of AKI) and heme oxygenase-1 (an indicator of oxidative stress) in hemopexin wild type compared with knockout mice in both models of AKI. Next, the interaction of hemopexin and hemoglobin in vitro was investigated using cultured proximal tubular cells. Co-incubation of hemopexin with hemoglobin resulted in hemoglobin deposition and exaggerated hemoglobin-induced injury. Deferoxamine, an iron chelator, and ferrostatin-1, a ferroptosis inhibitor, inhibited this deleterious effect of hemoglobin and hemopexin in proximal tubular cells, implicating iron toxicity in the mechanism of hemopexin mediated injury. Furthermore, the protective effect of deferoxamine in cisplatin-induced AKI was apparent in hemopexin wild type, but not in hemopexin knockout mice, further implicating hemopexin as a mediator of iron toxicity in AKI. Thus, our findings demonstrate that hemopexin accumulates in the kidneys and worsens kidney injury in AKI by increasing hemoglobin deposition on proximal tubular cells to exaggerate hemoglobin-induced cell injury.

The ketone body ß-hydroxybutyrate mitigates the senescence response of glomerular podocytes to diabetic insults.

Diabetic kidney disease (DKD) is one of the most common complications of diabetes and is clinically featured by progressive albuminuria, consequent to glomerular destruction that involves podocyte senescence. Burgeoning evidence suggests that ketosis, in particular ß-hydroxybutyrate, exerts a beneficial effect on aging and on myriad metabolic or chronic diseases, including obesity, diabetes and chronic kidney diseases. Its effect on DKD is largely unknown. In vitro in podocytes exposed to a diabetic milieu, ß-hydroxybutyrate treatment substantially mitigated cellular senescence and injury, as evidenced by reduced formation of γH2AX foci, reduced staining for senescence-associated-ß-galactosidase activity, diminished expression of key mediators of senescence signaling like p16INK4A and p21, and preserved expression of synaptopodin. This beneficial action of ß-hydroxybutyrate coincided with a reinforced transcription factor Nrf2 antioxidant response. Mechanistically, ß-hydroxybutyrate inhibition of glycogen synthase kinase 3ß (GSK3ß), a convergent point for myriad signaling pathways regulating Nrf2 activity, seems to contribute. Indeed, trigonelline, a selective inhibitor of Nrf2, or ectopic expression of constitutively active mutant GSK3ß abolished, whereas selective activation of Nrf2 was sufficient for the anti-senescent and podocyte protective effects of ß-hydroxybutyrate. Moreover, molecular modeling and docking analysis revealed that ß-hydroxybutyrate is able to directly target the ATP-binding pocket of GSK3ß and thereby block its kinase activity. In murine models of streptozotocin-elicited DKD, ß-hydroxybutyrate therapy inhibited GSK3ß and reinforced Nrf2 activation in glomerular podocytes, resulting in lessened podocyte senescence and injury and improved diabetic glomerulopathy and albuminuria. Thus, our findings may pave the way for developing a ß-hydroxybutyrate-based novel approach of therapeutic ketosis for treating DKD.

Melanocortin 5 receptor signaling pathway in health and disease.

Melanocortin hormone system plays a key role in maintaining the homeostasis of our body via their neuro-immune-endocrine activities and regulates a diverse array of physiological functions, including melanogenesis, inflammation, immunomodulation, adrenocortical steroidogenesis, hemodynamics, natriuresis, energy homeostasis, sexual function, and exocrine secretion. The pathobiologic actions of all melanocortins are conveyed by melanocortin receptors. As the last melanocortin receptor to be cloned and characterized, melanocortin receptor 5 (MC5R) is widely expressed in both central nervous system and a number of peripheral organ systems in man. However, the exact effect of the MC5R mediated melanocortinergic signaling remains largely uncertain. Owing to the recent advances in developing highly selective peptidomimetic agonists and antagonists of MC5R and also to studies in MC5R knockout animals, our understanding of MC5R pathobiology has been greatly expanded and strengthened. Evidence suggests that MC5R plays a key role in governing immune reaction and inflammatory response, and is pivotal for the regulation of sexual behavior, thermoregulation, and exocrine secretion, like sebogenesis, lacrimal secretion and release of sex pheromones. As such, recent translational efforts have focused on developing novel sebum-suppressive therapies for seborrhoea and acne vulgaris based on antagonizing MC5R. Conversely, selective MC5R agonists have demonstrated promising beneficial effects in immune-mediated diseases, metabolic endocrinopathies and other disease conditions, such as glomerular diseases and dry eyes, skin and mouth. Thus, MC5R-mediated signaling is essential for health. Therapeutic targeting of MC5R represents a promising and pragmatic therapeutic strategy for diverse diseases. This review article delineates the biophysiology of MC5R-mediated biophysiology of the melanocortin hormone system, discusses the existing data on MC5R-targeted therapy in experimental disease models, and envisages the translational potential for treating human diseases.

Glycogen synthase kinase 3ß hyperactivity in urinary exfoliated cells predicts progression of diabetic kidney disease.

Burgeoning evidence points to glycogen synthase kinase (GSK)3ß as a key player in diverse kidney diseases. However, as a pivotal transducer of the insulin signaling pathway, the role of GSK3ß in diabetic kidney disease remains uncertain. In db/db mice, renal expression of total and activated GSK3ß was increasingly elevated. This preceded the development of diabetic kidney disease, and correlated with the progression of signs of diabetic kidney injury, including albuminuria and extracellular matrix accumulation in glomeruli and tubulointerstitia. In vitro, exposure of glomerular podocytes, mesangial cells, and renal tubular cells to a diabetic milieu induced GSK3ß overexpression and hyperactivity, which seem essential and sufficient for eliciting diabetic cellular damages in kidney cells, because the cytopathic effect of the diabetic milieu was mitigated by GSK3ß knockdown, but was mimicked by ectopic expression of constitutively active GSK3ß even in the normal milieu. In consistency, kidney biopsy specimens procured from patients with varying stages of diabetic nephropathy revealed an amplified expression of total and activated GSK3ß in glomeruli and renal tubules, associated with the severity of diabetic nephropathy. Moreover, in retrospective cohorts of type 2 diabetic patients that were followed for over five years, the relative activity of GSK3ß in banked urinary exfoliated cells represented an independent risk factor for development or progression of renal impairment. Furthermore, receiver operating characteristic curve analysis demonstrated that GSK3ß activity in urinary exfoliated cells provided much better power than albuminuria in discriminating diabetic patients with progressive renal impairment from those with stable kidney function. Thus, renal expression and activity of GSK3ß are amplified in experimental and clinical diabetic nephropathy. Hence, GSK3ß in urinary exfoliated cells may serve as a novel biomarker for predicting diabetic kidney disease progression.

Relapse of Nephrotic Syndrome after Adrenocorticotropic Hormone-Induced Remission: Implications of Adrenocorticotropic Hormone Antibodies.

BACKGROUND: Prolonged use of corticosteroids continues to be the mainstay in the management of most proteinuric glomerulopathies, but is limited by extensive side effects. Alternative medications such as adrenocorticotropic hormone (ACTH) have been recently used to treat refractory glomerulopathies and have shown superior outcomes when compared with steroids. However, the clinical responsiveness to ACTH therapy varies considerably with a number of patients exhibiting de novo or acquired resistance. The underlying mechanism remains unknown. METHODS: A patient with steroid-dependent focal segmental glomerulosclerosis (FSGS) developed severe steroid side effects impacting quality of life and was converted to repository porcine ACTH therapy. Immediate response in the form of remission of nephrotic syndrome was noted followed by relapse in 10 weeks. Suspecting the role of some ACTH-antagonizing factors, the patient's serum was examined. RESULTS: Immunoblot-based antibody assay revealed high titers of de novo IgG antibodies in the patient's serum that were reactive to the porcine corticotropin with negligible cross-reactivity to human corticotropin. In vitro, in cultured B16 melanoma cells that express abundant melanocortin receptors, addition of the patient's serum substantially abrogated the porcine corticotropin triggered signaling activity of the melanocortinergic pathway, marked by phosphorylation of glycogen synthase kinase 3ß, thus suggesting a mitigating effect on the biological functionality of porcine corticotropin. CONCLUSION: ACTH is a useful alternative therapeutic modality for refractory proteinuric glomerulopathies like FSGS. However, as quintessential therapeutic biologics, natural ACTH, regardless of purity and origin, is inevitably antigenic and may cause the formation of neutralizing antibodies in some sensitive patients, followed by resistance to ACTH therapy. It is imperative to develop ACTH analogues with less immunogenicity for improving its responsiveness in patients with glomerular diseases.

Melanocortin therapy ameliorates podocytopathy and proteinuria in experimental focal segmental glomerulosclerosis involving a podocyte specific non-MC1R-mediated melanocortinergic signaling.

The clinical effectiveness of adrenocorticotropin in inducing remission of steroid-resistant nephrotic syndrome points to a steroidogenic-independent anti-proteinuric activity of melanocortins. However, which melanocortin receptors (MCR) convey this beneficial effect and if systemic or podocyte-specific mechanisms are involved remain uncertain. In vivo, wild-type (WT) mice developed heavy proteinuria and kidney dysfunction following Adriamycin insult, concomitant with focal segmental glomerulosclerosis (FSGS) and podocytopathy, marked by loss of podocin and synaptopodin, podocytopenia and extensive foot process effacement on electron microscopy. All these pathologic findings were prominently attenuated by NDP-MSH, a potent non-steroidogenic pan-MCR agonist. Surprisingly, MC1R deficiency in MC1R-null mice barely affected the severity of Adriamycin-elicited injury. Moreover, the beneficial effect of NDP-MSH was completely preserved in MC1R-null mice, suggesting that MC1R is likely non-essential for the protective action. A direct podocyte effect seems to contribute to the beneficial effect of NDP-MSH, because Adriamycin-inflicted cytopathic signs in primary podocytes prepared from WT mice were all mitigated by NDP-MSH, including apoptosis, loss of podocyte markers, de novo expression of the podocyte injury marker desmin, actin cytoskeleton derangement and podocyte hypermotility. Consistent with in vivo findings, the podoprotective activity of NDP-MSH was fully preserved in MC1R-null podocytes. Mechanistically, MC1R expression was predominantly distributed to glomerular endothelial cells in glomeruli but negligibly noted in podocytes in vivo and in vitro, suggesting that MC1R signaling is unlikely involved in direct podocyte protection. Ergo, melanocortin therapy protects against podocyte injury and ameliorates proteinuria and glomerulopathy in experimental FSGS, at least in part, via a podocyte-specific non-MC1R-mediated melanocortinergic signaling.

Lithium targeting of AMPK protects against cisplatin-induced acute kidney injury by enhancing autophagy in renal proximal tubular epithelial cells.

Autophagy has been demonstrated to be vital for kidney homeostasis and is centrally implicated in the pathogenesis of cisplatin-induced acute kidney injury (AKI). Lithium is a potent autophagy inducer in a number of cell types. However, it remains uncertain whether its autophagic activity is associated with a beneficial effect on renal tubular cells in AKI. This study aimed to examine the effect of lithium on renal autophagy in cisplatin-induced AKI. Mice or renal proximal tubular epithelial cells in culture were exposed to cisplatin-induced acute injury in the presence or absence of lithium treatment. AKI or tubular cell injury was evaluated, and cell signaling associated with autophagy was examined. Lithium pretreatment prominently ameliorated acute renal tubular damage in mice exposed to cisplatin insult, associated with enhanced autophagy in renal tubules, as assessed by measuring microtubule-associated protein 1A/1B-light chain 3 (LC3)BII/I expression and autophagosome formation. Consistently, in cisplatin-injured renal tubular cells in vitro, lithium enhanced autophagic activities, improved cell viability, and attenuated cell death. Mechanistically, lithium triggered AMPK-α phosphorylation and activation, which in turn positively correlated with the induced expression of autophagy-related molecules, like mammalian target of rapamycin and LC3BII/I. AMPK-α activation is likely required for lithium-induced tubular cell autophagy and protection in cisplatin-induced AKI because blockade of AMPK-α phosphorylation by compound C markedly abrogated lithium-induced autophagosome formation and mitigated the protective effect of lithium on AKI. Our findings suggest that lithium represents a promising therapeutic strategy for protecting renal tubular cells against cisplatin-induced AKI by enhancing autophagy via AMPK-α activation.-Bao, H., Zhang, Q., Liu, X., Song, Y., Li, X., Wang, Z., Li, C., Peng, A., Gong, R. Lithium targeting of AMPK protects against cisplatin-induced acute kidney injury by enhancing autophagy in renal proximal tubular epithelial cells.

Long noncoding RNA: an emerging player in diabetes and diabetic kidney disease.

Diabetic kidney disease (DKD) is among the most common complications of diabetes mellitus (DM), and remains the leading cause of end-stage renal diseases (ESRDs) in developed countries, with no definitive therapy yet available. It is imperative to decipher the exact mechanisms underlying DKD and identify novel therapeutic targets. Burgeoning evidence indicates that long non-coding RNAs (lncRNAs) are essential for diverse biological processes. However, their roles and the mechanisms of action remain to be defined in disease conditions like diabetes and DKD. The pathogenesis of DKD is twofold, so is the principle of treatments. As the underlying disease, diabetes per se is the root cause of DKD and thus a primary focus of therapy. Meanwhile, aberrant molecular signaling in kidney parenchymal cells and inflammatory cells may directly contribute to DKD. Evidence suggests that a number of lncRNAs are centrally involved in development and progression of DKD either via direct pathogenic roles or as indirect mediators of some nephropathic pathways, like TGF-ß1, NF-κB, STAT3 and GSK-3ß signaling. Some lncRNAs are thus likely to serve as biomarkers for early diagnosis or prognosis of DKD or as therapeutic targets for slowing progression or even inducing regression of established DKD. Here, we elaborated the latest evidence in support of lncRNAs as a key player in DKD. In an attempt to strengthen our understanding of the pathogenesis of DKD, and to envisage novel therapeutic strategies based on targeting lncRNAs, we also delineated the potential mechanisms of action as well as the efficacy of targeting lncRNA in preclinical models of DKD.

Blocking Macrophage Migration Inhibitory Factor Protects Against Cisplatin-Induced Acute Kidney Injury in Mice.

Macrophage migration inhibitory factor (MIF) is elevated in patients with acute kidney injury (AKI) and is suggested as a potential predictor for renal replacement therapy in AKI. In this study, we found that MIF also plays a pathogenic role and is a therapeutic target for AKI. In a cisplatin-induced AKI mouse model, elevated plasma MIF correlated with increased serum creatinine and the severity of renal inflammation and tubular necrosis, whereas deletion of MIF protected the kidney from cisplatin-induced AKI by largely improving renal functional and histological injury, and suppressing renal inflammation including upregulation of cytokines such as interleukin (IL)-1ß, tumor necrosis factor-alpha (TNF-α), IL-6, inducible nitric oxide synthase (iNOS), MCP-1, IL-8, and infiltration of macrophages, neutrophils, and T cells. We next developed a novel therapeutic strategy for AKI by blocking the endogenous MIF with an MIF inhibitor, ribosomal protein S19 (RPS19). Similar to the MIF-knockout mice, treatment with RPS19, but not the mutant RPS19, suppressed cisplatin-induced AKI. Mechanistically, we found that both genetic knockout and pharmacological inhibition of MIF protected against AKI by inactivating the CD74-nuclear factor κB (NF-κB) signaling. In conclusion, MIF is pathogenic in cisplatin-induced AKI. Targeting MIF with an MIF inhibitor RPS19 could be a promising therapeutic potential for AKI.

Ecdysone Elicits Chronic Renal Impairment via Mineralocorticoid-Like Pathogenic Activities.

BACKGROUND/AIMS: Ecdysteroids are steroidal insect molting hormones that also exist in herbs. Ecdysteroid-containing adaptogens have been popularly used to improve well-being and by bodybuilders for muscle growth. However, the use of ecdysone in mammals is also associated with kidney growth and enlargement, indications of disturbed kidney homeostasis. The underlying pathogenic mechanism remains to be clarified. METHODS: Virtual screening tools were employed to identify compounds that are homologous to ecdysone and to predict putative ecdysone-interacting proteins. The kidney effect of ecdysone was examined in vitro and in vivo and compared with that of aldosterone. Cellular apoptosis was estimated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Cell motility was assessed by scratch-wound cell migration assay. Blood urea nitrogen was measured to evaluate renal function. Western immunblot analysis was employed to determine the expression profile of interested proteins. RESULTS: Computational molecular structure analysis revealed that ecdysone is highly homologous to aldosterone. Moreover, virtual screening based on compound-protein interaction profiles identified the Mineralocorticoid Receptor (MR) to potentially interact with ecdysone. Accordingly, to assess potential biological functions of ecdysone in mammals, ecdysone was applied to mineralocorticoid-sensitive inner medullar collecting duct cells. Ecdysone induced mesenchymal accumulation of extracellular matrix and epithelial dedifferentiation characterized by de novo expression of α-smooth muscle actin. In addition, ecdysone elicited cellular apoptosis and retarded cell motility, akin to the effect of aldosterone. In vivo, daily treatment of mice with ecdysone increased cell apoptosis in the kidney, impaired renal function and elicited early signs of renal fibrogenesis, marked by deposition of collagen and fibronectin in tubulointerstitium, reminiscent of the action of aldosterone. The MR signaling pathway is likely responsible for the cellular and pathobiological effects of ecdysone, as evidenced by strong ecdysone-induced MR nuclear translocation in renal tubular cells both in vitro and in vivo, while blockade of MR by concomitant spironolactone treatment largely abolished the detrimental effects of ecdysone. CONCLUSION: Our findings suggest that ecdysone induces mineralocorticoid-dependent activities that impair renal function and elicit renal injury.

The ß isoform of GSK3 mediates podocyte autonomous injury in proteinuric glomerulopathy.

Converging evidence points to glycogen synthase kinase (GSK) 3 as a key player in the pathogenesis of podocytopathy and proteinuria. However, it remains unclear if GSK3 is involved in podocyte autonomous injury in glomerular disease. In normal kidneys, the ß isoform of GSK3 was found to be the major GSK3 expressed in glomeruli and intensely stained in podocytes. GSK3ß expression in podocytes was markedly elevated in experimental or human proteinuric glomerulopathy. Podocyte-specific somatic ablation of GSK3ß in adult mice attenuated proteinuria and ameliorated podocyte injury and glomerular damage in experimental adriamycin (ADR) nephropathy. Mechanistically, actin cytoskeleton integrity in podocytes was largely preserved in GSK3ß knockout mice following ADR insult, concomitant with a correction of podocyte hypermotility and lessened phosphorylation and activation of paxillin, a focal adhesion-associated adaptor protein. In addition, GSK3ß knockout diminished ADR-induced NFκB RelA/p65 phosphorylation selectively at serine 467; suppressed de novo expression by podocytes of NFκB-dependent podocytopathic mediators, including B7-1, cathepsin L, and MCP-1; but barely affected the induction of NFκB target pro-survival factors, such as Bcl-xL. Moreover, the ADR-elicited podocytopenia and podocyte death were significantly attenuated in GSK3ß knockout mice, associated with protection against podocyte mitochondrial damage and reduced phosphorylation and activation of cyclophilin F, a structural component of mitochondria permeability transition pores. Overall, our findings suggest that the ß isoform of GSK3 mediates autonomous podocyte injury in glomerulopathy by integrating multiple podocytopathic signalling pathways.

Genetic and Pharmacologic Targeting of Glycogen Synthase Kinase 3ß Reinforces the Nrf2 Antioxidant Defense against Podocytopathy.

Evidence suggests that the glycogen synthase kinase 3 (GSK3)-dictated nuclear exclusion and degradation of Nrf2 is pivotal in switching off the self-protective antioxidant stress response after injury. Here, we examined the mechanisms underlying this regulation in glomerular disease. In primary podocytes, doxorubicin elicited cell death and actin cytoskeleton disorganization, concomitant with overactivation of GSK3ß (the predominant GSK3 isoform expressed in glomerular podocytes) and minimal Nrf2 activation. SB216763, a highly selective small molecule inhibitor of GSK3, exerted a protective effect that depended on the potentiated Nrf2 antioxidant response, marked by increased Nrf2 expression and nuclear accumulation and augmented production of the Nrf2 target heme oxygenase-1. Ectopic expression of the kinase-dead mutant of GSK3ß in cultured podocytes reinforced the doxorubicin-induced Nrf2 activation and prevented podocyte injury. Conversely, a constitutively active GSK3ß mutant blunted the doxorubicin-induced Nrf2 response and exacerbated podocyte injury, which could be abolished by treatment with SB216763. In murine models of doxorubicin nephropathy or nephrotoxic serum nephritis, genetic targeting of GSK3ß by doxycycline-inducible podocyte-specific knockout or pharmacologic targeting by SB216763 significantly attenuated albuminuria and ameliorated histologic signs of podocyte injury, including podocytopenia, loss of podocyte markers, podocyte de novo expression of desmin, and ultrastructural lesions of podocytopathy (such as foot process effacement). This beneficial outcome was likely attributable to an enhanced Nrf2 antioxidant response in glomerular podocytes because the selective Nrf2 antagonist trigonelline abolished the proteinuria-reducing and podocyte-protective effect. Collectively, our results suggest the GSK3ß-regulated Nrf2 antioxidant response as a novel therapeutic target for protecting podocytes and treating proteinuric glomerulopathies.

Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3) and functions as an oncogenic factor in many cancer types. However, the role of EZH2 in renal fibrogenesis remains unexplored. In this study, we found high expression of EZH2 and H3K27me3 in cultured renal fibroblasts and fibrotic kidneys from mice with unilateral ureteral obstruction and humans with CKD. Pharmacologic inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) or GSK126 or siRNA-mediated silencing of EZH2 inhibited serum- and TGFß1-induced activation of renal interstitial fibroblasts in vitro, and 3-DZNeP administration abrogated deposition of extracellular matrix proteins and expression of α-smooth muscle actin in the obstructed kidney. Injury to the kidney enhanced Smad7 degradation, Smad3 phosphorylation, and TGFß receptor 1 expression, and 3-DZNeP administration prevented these effects. 3-DZNeP also suppressed phosphorylation of the renal EGF and PDGFß receptors and downstream signaling molecules signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 after injury. Moreover, EZH2 inhibition increased the expression of phosphatase and tensin homolog (PTEN), a protein previously associated with dephosphorylation of tyrosine kinase receptors in the injured kidney and serum-stimulated renal interstitial fibroblasts. Finally, blocking PTEN with SF1670 largely diminished the inhibitory effect of 3-DZNeP on renal myofibroblast activation. These results uncovered the important role of EZH2 in mediating the development of renal fibrosis by downregulating expression of Smad7 and PTEN, thus activating profibrotic signaling pathways. Targeted inhibition of EZH2, therefore, could be a novel therapy for treating CKD.

Glycogen synthase kinase 3ß orchestrates microtubule remodeling in compensatory glomerular adaptation to podocyte depletion.

Reminiscent of neural repair, following podocyte depletion, remnant-surviving podocytes exhibit a considerable adaptive capacity to expand and cover the denuded renal glomerular basement membrane. Microtubules, one of the principal cytoskeletal components of podocyte major processes, play a crucial role in podocyte morphogenesis and podocyte process outgrowth, branching, and elongation. Here, we demonstrated that the microtubule-associated proteins Tau and collapsin response mediator protein (CRMP) 2, key regulators of microtubule dynamics, were abundantly expressed by glomerular podocytes in vivo and in vitro, interacted with glycogen synthase kinase (GSK)3ß, and served as its putative substrates. GSK3ß overactivity induced by adriamycin injury or by a constitutively active mutant of GSK3ß augmented phosphorylation of Tau and CRMP2, concomitant with microtubule depolymerization, cell body shrinkage, and shortening of podocyte processes. Conversely, inhibition of GSK3ß by a dominant negative mutant or by lithium, a Food and Drug Administration-approved neuroprotective mood stabilizer, diminished Tau and CRMP2 phosphorylation, resulting in microtubule polymerization, podocyte expansion, and lengthening of podocyte processes. In a mouse model of adriamycin-induced podocyte depletion and nephropathy, delayed administration of a single low dose of lithium attenuated proteinuria and ameliorated progressive glomerulosclerosis despite no correction of podocytopenia. Mechanistically, lithium therapy obliterated GSK3ß overactivity, mitigated phosphorylation of Tau and CRMP2, and enhanced microtubule polymerization and stabilization in glomeruli in adriamycin-injured kidneys, associated with elongation of podocyte major processes. Collectively, our findings suggest that the GSK3ß-dictated podocyte microtubule dynamics might serve as a novel therapeutic target to reinforce the compensatory glomerular adaptation to podocyte loss.

What we need to know about the effect of lithium on the kidney.

Lithium has been a valuable treatment for bipolar affective disorders for decades. Clinical use of lithium, however, has been problematic due to its narrow therapeutic index and concerns for its toxicity in various organ systems. Renal side effects associated with lithium include polyuria, nephrogenic diabetes insipidus, proteinuria, distal renal tubular acidosis, and reduction in glomerular filtration rate. Histologically, chronic lithium nephrotoxicity is characterized by interstitial nephritis with microcyst formation and occasional focal segmental glomerulosclerosis. Nevertheless, this type of toxicity is uncommon, with the strongest risk factors being high serum levels of lithium and longer time on lithium therapy. In contrast, in experimental models of acute kidney injury and glomerular disease, lithium has antiproteinuric, kidney protective, and reparative effects. This paradox may be partially explained by lower lithium doses and short duration of therapy. While long-term exposure to higher psychiatric doses of lithium may be nephrotoxic, short-term low dose of lithium may be beneficial and ameliorate kidney and podocyte injury. Mechanistically, lithium targets glycogen synthase kinase-3ß, a ubiquitously expressed serine/threonine protein kinase implicated in the processes of tissue injury, repair, and regeneration in multiple organ systems, including the kidney. Future studies are warranted to discover the exact "kidney-protective dose" of lithium and test the effects of low-dose lithium on acute and chronic kidney disease in humans.

Therapeutic targeting of GSK3ß enhances the Nrf2 antioxidant response and confers hepatic cytoprotection in hepatitis C.

OBJECTIVE: Impaired adaptive response to oxidative injuries is a fundamental mechanism central to the pathogenesis of chronic hepatitis C (CHC). Glycogen synthase kinase (GSK) 3ß is an indispensable regulator of the oxidative stress response. However, the exact role of GSK3ß in CHC is uncertain and was examined. DESIGN: GSK3ß and Nrf2 signalling pathways were examined in JFH1 HCV infected Huh7.5.1 hepatocytes, and also in liver biopsy specimens from CHC patients. RESULTS: HCV infection elicited prominent Nrf2 antioxidant response in hepatocytes, marked by elevated expression of the Nrf2-dependent molecule haem oxygenase-1 and subsequent protection from apoptotic cell death. Inhibitory phosphorylation of GSK3ß seems to be essential and sufficient for HCV-induced Nrf2 response. Mechanistically, GSK3ß associated and physically interacted with Nrf2 in hepatocytes. In silico analysis revealed that Nrf2 encompasses multiple GSK3ß phosphorylation consensus motifs, denoting Nrf2 as a cognate substrate of GSK3ß. In the presence of TGFß1, the HCV-induced GSK3ß phosphorylation was blunted via a protein phosphatase 1-dependent mechanism and the cytoprotective Nrf2 response drastically impaired. This effect was counteracted by lithium, a selective inhibitor of GSK3ß. In liver biopsy specimens from CHC patients, the expression of phosphorylated GSK3ß positively correlated with Nrf2 expression and was inversely associated with the degree of liver injury. Moreover, CHC patients who received long-term lithium carbonate therapy primarily for concomitant psychiatric disorders exhibited much less liver injury, associated with enhanced hepatic expression of Nrf2. CONCLUSIONS: Inhibition of GSK3ß exerts hepatoprotection in CHC possibly through its direct regulation of Nrf2 antioxidant response.

P2X7 receptor inhibition protects against ischemic acute kidney injury in mice.

Activation of the purinergic P2X7 receptor (P2X7R) has been associated with the development of experimental nephritis and diabetic and hypertensive nephropathy. However, its role in acute kidney injury (AKI) remains unknown. In this study, we examined the effects of P2X7R inhibition in a murine model of ischemia-reperfusion (I/R)-induced AKI using A438079, a selective inhibitor of P2X7R. At 24 h after I/R, mice developed renal dysfunction and renal tubular damage, which was accompanied by elevated expression of P2X7R. Early administration of A438079 immediately or 6 h after the onset of reperfusion protected against renal dysfunction and attenuated kidney damage whereas delayed administration of A438079 at 24 h after restoration of perfusion had no protective effects. The protective actions of A438079 were associated with inhibition of renal tubule injury and cell death and suppression of renal expression of monocyte chemotactic protein-1 and regulated upon expression normal T cell expressed and secreted (RANTES). Moreover, I/R injury led to an increase in phosphorylation (activation) of extracellular signal-regulated kinases 1/2 in the kidney; treatment with A438079 diminished this response. Collectively, these results indicate that early P2X7R inhibition is effective against renal tubule injury and proinflammatory response after I/R injury and suggest that targeting P2X7R may be a promising therapeutic strategy for treatment of AKI.