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.

Therapeutic Targeting of SGLT2: A New Era in the Treatment of Diabetes and Diabetic Kidney Disease.

As the prevalence of diabetic kidney disease (DKD) continues to rise, so does the need for a novel therapeutic modality that can control and slow its progression to end-stage renal disease. The advent of sodium-glucose cotransporter-2 (SGLT2) inhibitors has provided a major advancement for the treatment of DKD. However, there still remains insufficient understanding of the mechanism of action and effectiveness of this drug, and as a result, its use has been very limited. Burgeoning evidence suggests that the SGLT2 inhibitors possess renal protective activities that are able to lower glycemic levels, improve blood pressure/hemodynamics, cause bodyweight loss, mitigate oxidative stress, exert anti-inflammatory and anti-fibrotic effects, reduce urinary albumin excretion, lower uric acid levels, diminish the activity of intrarenal renin-angiotensin-aldosterone system, and reduce natriuretic peptide levels. SGLT2 inhibitors have been shown to be safe and beneficial for use in patients with a GFR ≥30mL/min/1.73m2, associated with a constellation of signs of metabolic reprogramming, including enhanced ketogenesis, which may be responsible for the correction of metabolic reprogramming that underlies DKD. This article aims to provide a comprehensive overview and better understanding of the SGLT2 inhibitor and its benefits as it pertains to renal pathophysiology. It summarizes our recent understanding on the mechanisms of action of SGLT2 inhibitors, discusses the effects of SGLT2 inhibitors on diabetes and DKD, and presents future research directions and therapeutic potential.

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.

Microdose Lithium Protects against Pancreatic Islet Destruction and Renal Impairment in Streptozotocin-Elicited Diabetes.

Psychiatric use of lithium has been associated with hypoglycemic effects, but its effect on type 1 diabetes mellitus (T1D) is unknown. In streptozotocin (STZ) induced murine models of T1D, microdose lithium therapy improved hyperglycemia, attenuated body weight loss and prevented early signs of diabetic kidney injury. This beneficial effect was associated with preservation of pancreatic islet histology and ß-cell production of insulin as well as mitigated oxidative damage of islets. Mechanistically, lithium in islets cells induced inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß), the major molecular target of lithium that has been recently implicated in non-canonical regulation of Nrf2 activity. In turn, Nrf2 antioxidant response was potentiated in islets, marked by nuclear translocation of Nrf2 and augmented expression of its target antioxidant enzyme heme oxygenase 1 (HO-1). Conversely, cotreatment with trigonelline, a selective blockade of Nrf2, offset the lithium enhanced Nrf2 antioxidant response in islets, blunted the protective effect of lithium on pancreatic islets and ß-cells, and abolished the hypoglycemic activity of lithium in STZ-injured mice. Collectively, our findings suggest that microdose lithium confers a protective effect on islet ß-cells via targeting the GSK3ß-regulated Nrf2 antioxidant response and thereby ameliorates T1D and its related kidney impairment.

Triptolide potentiates the cytoskeleton-stabilizing activity of cyclosporine A in glomerular podocytes via a GSK3ß dependent mechanism.

Tripterygium wilfordii Hook F. (TwHF) is a traditional Chinese herb and has a broad spectrum of biological functions including immunosuppression and anti-inflammatory effects. When used in combination with other standard of care medications, such as glucocorticoids and calcineurin inhibitors like cyclosporine A, for treating glomerular diseases, TwHF demonstrates a remarkable dose-sparing effect, the molecular mechanism for which remains largely unknown. In an in vitro model of podocytopathy elicited by a diabetic milieu, triptolide, the major active component of TwHF, at low doses, potentiated the beneficial effect of cyclosporine A, and protected podocytes against diabetic milieu-elicited injury, mitigated cytoskeleton derangement, and preserved podocyte filtration barrier function, entailing a synergistic cytoskeleton-preserving and podocyte protective effect of triptolide and cyclosporine A. Mechanistically, inhibitory phosphorylation of GSK3ß, a key molecule recently implicated as a convergence point of podocytopathic pathways, is likely required for the synergistic effect of triptolide and cyclosporine A on podocyte protection, because the synergistic effect was largely blunted in cells expressing the constitutively active GSK3ß. Ergo, a synergistic podocyte cytoskeleton-stabilizing mechanism seems to underlie the cyclosporine A-sparing effect of triptolide in glomerulopathies. Combined triptolide and cyclosporine A therapy at reduced doses may be an invaluable regimen for treating diabetic nephropathy.

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.

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.

GSK3ß-mediated Keap1-independent regulation of Nrf2 antioxidant response: A molecular rheostat of acute kidney injury to chronic kidney disease transition.

Transition of acute kidney injury (AKI) to chronic kidney disease (CKD) represents an important cause of kidney failure. However, how AKI is transformed into CKD remains elusive. Following folic acid injury, mice developed AKI with ensuing CKD transition, featured by variable degrees of interstitial fibrosis and tubular cell atrophy and growth arrest. This lingering injury of renal tubules was associated with sustained oxidative stress that was concomitant with an impaired Nrf2 antioxidant defense, marked by mitigated Nrf2 nuclear accumulation and blunted induction of its target antioxidant enzymes, like heme oxygenase (HO)-1. Activation of the canonical Keap1/Nrf2 signaling, nevertheless, seems intact during CKD transition because Nrf2 in injured tubules remained activated and elevated in cytoplasm. Moreover, oxidative thiol modification and activation of Keap1, the cytoplasmic repressor of Nrf2, was barely associated with CKD transition. In contrast, glycogen synthase kinase (GSK)3ß, a key modulator of the Keap1-independent Nrf2 regulation, was persistently overexpressed and hyperactive in injured tubules. Likewise, in patients who developed CKD following AKI due to diverse etiologies, like volume depletion and exposure to radiocontrast agents or aristolochic acid, sustained GSK3ß overexpression was evident in renal tubules and coincided with oxidative damages, impaired Nrf2 nuclear accumulation and mitigated induction of antioxidant gene expression. Mechanistically, the Nrf2 response against oxidative insult was sabotaged in renal tubular cells expressing a constitutively active mutant of GSK3ß, but reinforced by ectopic expression of dominant negative GSK3ß in a Keap1-independent manner. In vivo in folic acid-injured mice, targeting GSK3ß in renal tubules via conditional knockout or by weekly microdose lithium treatment reinstated Nrf2 antioxidant response in the kidney and hindered AKI to CKD transition. Ergo, our findings suggest that GSK3ß-mediated Keap1-independent regulation of Nrf2 may serve as an actionable therapeutic target for modifying the long-term sequelae of AKI.

Brain natriuretic peptide mitigates TIMP2 induction and reinstates extracellular matrix catabolic activity via GSK3ß inhibition in glomerular podocytes exposed to a profibrogenic milieu.

Brain natriuretic peptide (BNP) has a demonstrable anti-fibrotic effect on diverse organ systems, including the kidney. To understand the molecular mechanism underlying this renoprotective effect, the efficacy of BNP was examined in an in vitro model of glomerular sclerosis by exposing glomerular podocytes to transforming growth factor (TGF)ß1-containing media that recapitulates the profibrogenic milieu in chronic glomerular disease. BNP mitigates extracellular matrix (ECM) accumulation in TGFß1-treated podocytes, as evidenced by Sirius red assay and staining, concomitant with a restoration of the ECM catabolizing activity, as assessed by pulse chase analysis. This effect was in parallel with a mitigating effect on TGFß1-elicited overexpression of tissue inhibitor of metalloproteinases (TIMP)2, a key inhibitor of a multitude of ECM-degrading metalloproteinases. Mechanistically, glycogen synthase kinase (GSK)3ß, a key player in pathogenesis of podocyte injury and glomerulopathies, seems to be involved. BNP treatment considerably induced GSK3ß inhibition, marked by inhibitory phosphorylation at the serine 9 residue, and this significantly correlated with the abrogated TIMP2 induction in TGFß1-injured podocytes. Moreover, genetic knockout of GSK3ß in podocytes is sufficient to attenuate the TGFß1 induced TIMP2 expression and ECM deposition, reminiscent of the effect of BNP. Conversely, ectopic expression of a nonphosphorylatable GSK3ß mutant abolished the inhibitory effect of BNP on TGFß1-elicited TIMP2 overexpression and ECM accumulation, signifying an essential role of GSK3ß inhibition in mediating the effect of BNP. Collectively, BNP possesses an anti-fibrotic activity in glomerular epithelial cells. This finding, if validated in vivo, may open a new avenue to the treatment of glomerulosclerosis.

Targeting Regulatory T Cells for Transplant Tolerance: New Insights and Future Perspectives.

BACKGROUND: Organ transplantation is considered the ultimate therapy for end-stage organ disease. While pharmacologic immunosuppression is the mainstay of therapeutic strategies to prolong the survival of the graft, long-term use of immunosuppressive medications carries the risk of organ toxicity, malignancies, serious opportunistic infections, and diabetes. Therapies that promote recipient tolerance in solid organ transplantation are able to improve patient outcomes by eliminating the need for long-term immunosuppression. SUMMARY: Establishing tolerance to an allograft has become an area of intense study and would be the ideal therapy in clinical practice. The discovery of a subset of T cells naturally committed to perform immunoregulation has led to further investigation into their role in the immunopathogenesis of transplantation. Evidence suggests that regulatory T cells (Tregs) are fundamentally involved in promoting allograft tolerance. Efforts to characterize specific markers for Tregs, while challenging, have identified Foxp3 gene expression as a crucial step in promoting the tolerance-inducing features of Tregs. A number of approaches, including those based on targeting the glycogen synthase kinase 3ß signaling pathway or activating the melanocortinergic pathway, have been tested as a way to promote Treg lineage commitment and maintenance as well as to facilitate immune tolerance. In order to be effective in clinical practice, Tregs must be allospecific and possess a specific phenotype to avoid suppression of other aspects of the immune system or increasing the risk of malignancy or infections. Multiple experimental and clinical studies have demonstrated the impact of currently used immunosuppressants on the immunoregulatory activities of Tregs and their Foxp3 expression status. Pharmacological induction of tolerogenic Tregs for inducing transplant tolerance, including epigenetic therapies, is in the ascendant. KEY MESSAGES: Therapies that promote Treg function and survival may represent a novel strategy for achieving immune tolerance in transplant patients.

Uremic cardiomyopathy: role of circulating digitalis like substances.

Patients with chronic renal failure develop a cardiomyopathy characterized by marked diastolic dysfunction and left ventricular hypertrophy. Interestingly, they also have substantial increases in the circulating concentrations of digitalis like substances. Digitalis like substances produce reactive oxygen species as part of the signal cascade induced by binding to the sodium pump and patients, and this signal cascade appears to induce hypertrophy of cardiac myocytes grown in culture. Also, patients with chronic renal failure develop an oxidant stress state without a known mechanism. From these data, we propose that it is these digitalis like substances which cause cardiomyopathy of renal failure as well as the systemic oxidant stress state.