Melanocortin therapy has demonstrated potential in improving membranous nephropathy in human patients. The underlying mechanisms remain unclear. Here, in a heterologous mouse model of THSD7A-associated membranous nephropathy, various melanocortin agents, including repository corticotropin injection, the nonsteroidogenic pan-melanocortin receptor agonist NDP-MSH, and the selective melanocortin 5 receptor (MC5R) agonist PG-901, attenuated proteinuria, and ameliorated glomerulopathy. In contrast, MC5R knockout exacerbated membranous nephropathy, while abolishing the beneficial effects of melanocortins. Mechanistically, MC5R expression was evident in podocytes in wild-type mice, and podocyte MC5R appears to protect against membranous nephropathy, as demonstrated by its necessity for melanocortin protection in cultured podocytes, and restoration of melanocortin therapeutic efficacy in MC5R knockout mouse after podocyte-specific reconstitution of MC5R. Furthermore, glomerular fixation of C5b-9 and C3 was diminished by MC5R agonism but enhanced by MC5R knockout, despite comparable glomerular deposition of the heterologous nephritogenic antibody and activation of C4. Comprehensive complement cascade analysis revealed that this anti-complement effect of MC5R signaling stems from an inhibition of podocyte expression of complement factors B and D, critical regulators of the complement amplification loop, involving a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent mechanism. Ergo, MC5R-mediated interception of the complement amplification loop in podocytes may serve as a novel therapeutic target for membranous nephropathy.

Rationale & Objective: Although renal artery stenosis (RAS) and heart failure (HF) have been linked, the incidence and predictors of HF among patients with RAS are not well described. Study Design: Post hoc analysis of the Cardiovascular Outcomes in Renal Atherosclerotic Lesions (CORAL) multicenter, open-label, randomized controlled trial (RCT). Settings and Participants: Patients with atherosclerotic RAS and elevated blood pressure, chronic kidney disease, or both, and without a history of HF at enrollment. Intervention: Medical therapy alone versus medical therapy plus renal artery stenting. Outcomes: Incident HF events. Results: This analysis included 808 participants enrolled in the CORAL trial without evidence of baseline HF. During a median follow-up of 4.8 years, 54 participants (6.7%) developed incident HF. HF incidence rates did not differ by randomized intervention (HR, 0.84; 95% confidence interval [CI], 0.49-1.43 for stent arm with medical arm as reference). Baseline diabetes (subdistribution hazard ratio (sHR), 2.07; 95% CI, 1.20-3.58), albuminuria (sHR, 1.12 per doubling of urinary albumin-creatinine ratio, 95% CI, 1.02-1.24), lower eGFR (sHR, 0.78 per 10 mL/min/1.73 m2 estimated glomerular fi ltration rate calculated with cystatin C and creatinine, 95% CI, 0.69-0.88), and peripheral vascular disease (PVD) (sHR, 2.18, 95% CI, 1.213.91) were independent predictors of incident HF. Participants who experienced incident HF had greater kidney function decline before HF events. Limitations: This is a post hoc analysis of a RCT. The number of HF events is small. Conclusions: In patients with RAS, rates of incident HF did not differ between participants randomized to optimal medical therapy alone versus optimal medical therapy plus renal artery stenting. The presence of diabetes, PVD, and worse kidney health at baseline were associated with future HF events.

Melanocortin therapeutics, exemplified by adrenocorticotropic hormone, have a proven steroidogenic-independent anti-proteinuric and glomerular protective effect. The biological functions of melanocortins are mediated by melanocortin receptors (MCR), including MC1R, which recent studies have shown to protect against glomerular disease. However, the role of other MCRs like MC5R is unknown. Here, Mc5r knockout exacerbated glomerulopathy in mice injured by adriamycin (ADR) or nephrotoxic serum (NTS), as demonstrated by increased albuminuria and podocyte injury. Conversely, selective MC5R agonism using a peptidomimetic agonist improved outcomes of glomerulopathies. Mechanistically, MC5R is expressed in glomerular podocytes. Reconstitution of MC5R in podocytes attenuated glomerular injury and proteinuria in Mc5r knockout mouse models of glomerulopathies, indicating a direct podocyte protective effect. In vitro, MC5R agonism in primary wild-type podocytes attenuated ADR-elicited cytoskeleton disruption, hypermotility and apoptosis, associated with restored inhibitory phosphorylation of glycogen synthases kinase 3β (GSK3β), a signaling transducer downstream of MC5R and at the nexus of multiple podocytopathic pathways. In parallel, ADR-induced phosphorylation and activation of GSK3β substrates, such as paxillin and NF-κB Rela/p65, were abrogated, leading to improved actin cytoskeleton integrity and diminished expression of mediators of podocyte injury, like MCP-1, B7-1 and Cathepsin L. This protective effect of MC5R agonism was blunted in wild-type podocytes expressing constitutively active GSK3β and was mimicked in Mc5r knockout podocytes by ectopic expression of dominant negative GSK3β. Consistently in ADR-injured Mc5r knockout mice, worsened podocytopathy was associated with enhanced GSK3β hyperactivity. These findings suggest that MC5R signaling protects against podocyte injury and may serve as a novel therapeutic target for glomerular diseases.

Microcystin-LR (MC-LR) is one of a large family of cyanotoxins which are naturally produced by cyanobacteria within harmful algal blooms occurring in bodies of water globally. Early findings of the toxicity of such blooms stemmed from fatalities of livestock drinking from affected water. Since then, various toxins have been identified such as the microcystins. Microcystin-LR has been studied as a representative congener due to its abundance and toxicity. While there have been extensive studies of microcystin-LR by oral route exposure, we have turned our attention to inhalation route exposure due to the recent findings of microcystin-containing lake and sea-spray aerosol. We have shown inflammatory outcomes in the airways of mice and in human cell culture models after microcystin aerosol exposure, and have found a consistent molecular patterns similar to those of Type 1/Type 17 driven neutrophilic asthma. Here we address the hypothesis that MC-LR will increase the inflammatory mediators of neutrophilic asthma leading to worsening symptoms. This is tested and characterized in both in vitro and in vivo models. We found that asthma symptoms and molecular signatures of inflammation are both worsened by MC-LR exposure in a mouse model of neutrophilic asthma. We found that 3D human airway cell culture models reconstructed from asthmatic donor cells are similarly affected, however healthy donor cells are nearly unaltered by comparison. Aggregating these findings with RNA sequencing data from all models, we developed a hypothetical molecular mechanism which relies on MC-LR mediated amplification of existing inflammatory signaling. We test this in a human reporter cell line of NF-κB activity and further demonstrate the mechanism by inhibitor testing. This study sheds light on the risk to asthmatic patients living near or recreating on affected bodies of water. Beyond asthma, we believe this study provides crucial insight into the findings over the last 40 years concerning disparate outcomes of MC-LR exposure as the result of exposure will be dependent on the signaling state of the tissue upon exposure.

BACKGROUND:Renal-artery stenosis can be associated with difficult to control hypertension, although renal-artery stenting has not been shown to improve clinical outcomes. Alternative antihypertensive medications could potentially result in quality of life benefits with renal-artery stenting. METHODS AND RESULTS:We performed a pre-specified quality of life sub-study of the CORAL trial-multicenter, randomized, open-label trial of renal-artery stenting vs. medical therapy in patients with atherosclerotic renal-artery stenosis. Longitudinal growth curve models were used to compare the Physical Symptoms Distress Index (PSDI), SF-36, and EQ-5D scores over time between treatment groups. We also sought to validate the approach of assessing quality of life in hypertension studies. Among 906 patients (mean age 69.2 ± 9.1 years, 49.7% men), symptom frequency and distress due to side effects from antihypertensive medications changed minimally over time, with no significant differences between treatment groups. There were also no clinically significant differences between treatment groups for the SF-36 and its subscales or the EQ-5D. In internal validation of the quality of life measures, the PSDI correlated well with number/type of antihypertensive medications, and generic health status measures correlated with late clinical events. CONCLUSIONS:In a large, multicenter, randomized clinical trial, we found no significant benefit of routine renal-artery stenting over medical management for the treatment of atherosclerotic renal-artery stenosis in terms of disease-specific or generic quality of life measures. As these quality of life measures are important to patients and are associated with medication compliance, future studies of antihypertensive treatments should consider including these quality of life measures as secondary outcomes. TRIAL REGISTRATION:ClinicalTrials.gov: NCT00081731.

Ethyl carbamate (EC) is a process contaminant found in fermented foods and alcoholic beverages. Metabolic conversion of ethyl carbamate generates vinyl carbamate (VC), a carcinogenic metabolite. EC, as a Group 2A probable human carcinogen, and the more potent VC, are known to cause tumors in rodents. However, their effects on the kidney are unknown and were explored here. Female A/J inbred mice received an intraperitoneal injection of vehicle or VC. Beginning 5 weeks after VC injection, mice showed signs of moribund state. Mouse necropsies revealed renal glomerular injury that histopathologically recapitulated human membranoproliferative glomerulonephritis (MPGN), as evidenced by light microscopy, immunostaining for immunoglobulins and complements, and electron microscopy. To determine the molecular pathomechanisms, a post-hoc analysis was performed on a publicly available RNA-Seq transcriptome of kidneys from control rats and rats treated with fermented wine containing high concentrations of EC. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the differentially expressed genes revealed that the complement and coagulation cascades were a top predicted biological process involved. Furthermore, pathway-based data integration and visualization revealed that key regulators of complement activation were altered by high EC treatment. Among these, complement factors (CF) D and H, critical positive and negative regulators of the alternative pathway, respectively, were most affected, with CFD induced by 3.49-fold and CFH repressed by 5.9-fold, underscoring a hyperactive alternative pathway. Consistently, exposure of primary glomerular endothelial cells to EC or VC resulted in induction of CFD and repression of CFH, accompanied by increased fixation of C3 and C5b9. This effect seems to be mediated by Ras, one of the top genes that interact with both EC and VC, as identified by analyzing the chemical-gene/protein interactions database. Indeed, EC or VC-elicited complement activation was associated with activation of Ras signaling, but was abolished by the Ras inhibitor farnesyl thiosalicylic acid. Collectively, our findings suggest that VC, a metabolite of EC, induces glomerular injury in mice akin to human MPGN, possibly via perturbing the expression of complement regulators, resulting in an effect that favors activation of the alternative complement pathway.

Membranous nephropathy (MN) continues to be a leading cause of nephrotic syndrome in non-diabetic adults. As a unique subtype in the serology-based classification of MN, thrombospondin type 1 domain containing 7A (THSD7A)-associated MN has attracted increasing interest, because, unlike other autoantigens, THSD7A is also expressed in preclinical species, facilitating the study of its role in MN. A heterologous mouse model of THSD7A-associated MN was previously established using a proprietary in-house antibody that was unfortunately not available to the research community. Here, we developed a mouse model of THSD7A-associated MN by administering a commercially available antibody targeting the most N-terminal part of THSD7A. Our model was characterized by heavy proteinuria and pathological features of human MN without sex differences. Complement depletion with cobra venom factor only partially attenuated proteinuria and glomerular injury in this model, entailing that complement-independent pathomechanisms also contribute. Consistently, in vitro in primary podocytes, exposure to the anti-THSD7A antibody caused evident podocytopathic changes, including disruption of actin cytoskeleton integrity, podocyte hypermobility, oxidative stress, and apoptotic cell death. These signs of podocytopathy were preserved, albeit to a lesser extent, after complement inactivation, indicating autonomous podocyte injury. Furthermore, as the first FDA-approved treatment for primary MN, adrenocorticotropic hormone therapy with repository corticotropin injection (Purified Cortrophin Gel®) appeared to be beneficial and significantly attenuated proteinuria and glomerular injury, suggesting that this model may be useful for developing novel treatments or understanding the pathogenesis of MN. Collectively, our model, based on the use of a commercially available anti-THSD7A antibody, will be an important tool for MN research.

Harmful algal blooms are increasing globally and pose serious health concerns releasing cyanotoxins. Microcystin-LR (MC-LR), one of the most frequently produced cyanotoxins, has recently been detected in aerosols generated by the normal motions of affected bodies of water. MC-LR aerosol exposure has been linked to a pro-inflammatory influence on the airways of mice; however, little is understood about the underlying mechanism or the potential consequences. This study aimed to investigate the pro-inflammatory effects of aerosolized MC-LR on murine airways. C57BL/6 and BALB/c mice were exposed to MC-LR aerosols, as these strains are predisposed to type 1/type 17 and type 2 immune responses, respectively. Exposure to MC-LR induced granulocytic inflammation in C57BL/6 but not BALB/c mice, as observed by increased expression of cytokines MIP-1α, CXCL1, CCL2, and GM-CSF compared with their respective vehicle controls. Furthermore, the upregulation of interleukins IL-17A and IL-12 is consistent with Th1- and Th17-driven type 1/type 17 inflammation. Histological analysis confirmed inflammation in the C57BL/6 lungs, with elevated neutrophils and macrophages in the bronchoalveolar lavage fluid and increased pro-inflammatory and pro-resolving oxidized lipids. In contrast, BALB/c mice showed no significant airway inflammation. These results highlight the ability of aerosolized MC-LR to trigger harmful airway inflammation, requiring further research, particularly into populations with predispositions to type 1/type 17 inflammation.

Emerging evidence suggests that GSK3β, a redox-sensitive transducer downstream of insulin signaling, acts as a convergent point for myriad pathways implicated in kidney injury, repair, and regeneration. However, its role in diabetic kidney disease remains controversial. In cultured glomerular podocytes, exposure to a milieu of type 2 diabetes elicited prominent signs of podocyte injury and degeneration, marked by loss of homeostatic marker proteins like synaptopodin, actin cytoskeleton disruption, oxidative stress and apoptosis, and stress-induced premature senescence, as shown by increased staining for senescence-associated β-galactosidase activity, amplified formation of γH2AX foci, and elevated expression of mediators of senescence signaling, like p21 and p16INK4A. These degenerative changes coincided with GSK3β hyperactivity, as evidenced by GSK3β overexpression and reduced inhibitory phosphorylation of GSK3β, and were averted by tideglusib, a highly-selective small molecule inhibitor of GSK3β. In agreement, post-hoc analysis of a publicly-available glomerular transcriptomics dataset from patients with type 2 diabetic nephropathy revealed that the curated diabetic nephropathy-related gene set was enriched in high GSK3β expression group. Mechanistically, GSK3β-modulated nuclear factor Nrf2 signaling is involved in diabetic podocytopathy, because GSK3β knockdown reinforced Nrf2 antioxidant response and suppressed oxidative stress, resulting in an improvement in podocyte injury and senescence. Conversely, ectopic expression of the constitutively active mutant of GSK3β impaired Nrf2 antioxidant response and augmented oxidative stress, culminating in an exacerbated diabetic podocyte injury and senescence. Moreover, IRS-1 was found to be a cognate substrate of GSK3β for phosphorylation at IRS-1S332, which negatively regulates IRS-1 activity. GSK3β hyperactivity promoted IRS-1 phosphorylation, denoting a desensitized insulin signaling. Consistently, in vivo in db/db mice with diabetic nephropathy, GSK3β was hyperactive in glomerular podocytes, associated with IRS-1 hyperphosphorylation, impaired Nrf2 response and premature senescence. Our finding suggests that GSK3β is likely a novel therapeutic target for treating type 2 diabetic glomerular injury.

Background: Epidemiological evidence suggests that hyperinsulinemia or insulin resistance is a significant risk factor for the development of diabetic complications such as DKD. However, whether hyperinsulinemia per se plays a causative role in the development of diabetic kidney injury is unknown and was explored here. Methods: Pre-diabetic db/db mice were examined for serum insulin levels, urinary albumin to creatinine ratios and renal histology. Conditionally immortalized mouse podocytes were cultured under non-permissive conditions and exposed to high ambient insulin conditions, following GSK3beta silencing, ectopic expression of a constitutively active GSK3beta mutant (S9A), or treatment with a small molecule GSK3beta inhibitor tideglusib. Podocyte injury was assessed and signaling pathways examined. Results: In pre-diabetic db/db mice, hyperinsulinemia was evident and associated with microalbuminuria and early signs of podocyte impairment, including diminished expression of homeostatic marker proteins like synaptopodin, as compared with db/m littermates. In vitro, prolonged exposure of differentiated podocytes to high ambient insulin induced podocytopathic changes, including cellular hypertrophy, loss of synaptopodin, and disruption of actin cytoskeleton integrity. This was associated with a desensitized insulin signaling and diminished inhibitory phosphorylation of GSK3beta, denoting GSK3beta hyperactivity. In pre-diabetic db/db mice, GSK3beta hyperactivity was confirmed in glomerular podocytes, correlating with the level of hyperinsulinemia or microalbuminuria. In cultured podocytes, ectopic expression of S9A caused podocyte hypertrophy and podocytopathic changes, reminiscent of the harmful effect of the hyperinsulinemic milieu. Conversely, GSK3beta knockdown mitigates podocyte injury elicited by hyperinsulinemic milieu. This protective effect was mimicked by the small molecule inhibitor tideglusib. Conclusions: GSK3beta hyperactivity is required and sufficient for Hyperinsulinemic milieu-elicited glomerular podocyte impairment and dysfunction.

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.

Objective: The Cardiovascular Outcomes in Renal Atherosclerotic Lesions (CORAL) trial, a multicenter randomized controlled trial with 947 patients, concluded that there was no benefit of renal artery stenting (RAS) over medical therapy. However, patients with chronic kidney disease (CKD) were not analyzed separately in the CORAL trial. CKD is a risk factor for cardiovascular and renal morbidity. We hypothesized that improved renal function after RAS would be associated with increased long-term survival and a lower risk of cardiovascular and renal events in patients with CKD. Methods: This post hoc analysis of the CORAL trial included 842 patients with CKD stages 2 to 4 at baseline who were randomized to optimal medical therapy alone (OMT; n = 432) or RAS plus OMT (RAS + OMT; n = 410). Patients were categorized as responders or nonresponders based on the change in the estimated glomerular filtration rate (eGFR) from baseline to last follow-up (median, 3.6 years; interquartile range, 2.6-4.6 years). Responders were defined by a 20% or greater increase in eGFR from baseline; all others were designated as nonresponders. Event-free survival was defined as freedom from death and multiple cardiovascular and renal complications. Event-free survival was analyzed using the Kaplan-Meier method and log-rank test. Multivariable Cox proportional hazards regression analysis was used to identify independent predictors of event-free survival. Results: The RAS + OMT group had a higher proportion of patients with improved renal function (>= 20% increase in eGFR over baseline), compared with the OMT group (25.6% vs 17.1%; P = .003). However, event-free survival was no different for the two cohorts (P = .18 by the log-rank test). Multivariable Cox proportional hazards regression analysis identified four variables that independently correlated with event-free survival for the stented cohort. Higher preoperative eGFR (hazard ratio, 0.98; 95% confidence interval [CI], 0.96-0.99; P = .002) and being a responder to stenting (hazard ratio, 0.49; 95% CI, 0.26-0.95; P = .033) increased event-free survival, whereas a history of congestive heart failure (hazard ratio, 2.52; 95% CI, 1.46-4.35; P < .001) and a higher preoperative systolic BP (hazard ratio, 1.02; 95% CI, 1.01-1.03; P = .002) decreased event-free survival. Within the stented group, 105 of 410 patients (25.6%) were responders. Event-free survival was superior for responders, compared with nonresponders (P = .009 by log-rank test). The only independent preoperative negative predictor of improved renal function after stenting was diabetes (odds ratio, 0.37; 95% CI, 0.16-0.84; P = .017), which decreased the probability of improved renal function after RAS + OMT. A subset of patients (23.4%) after RAS had worsened renal function, but OMT alone produced an equivalent incidence of worsened renal function. An increased urine albumin/creatinine ratio was an independent predictor of worsened renal function after RAS. Conclusions: CORAL participants who demonstrated improved kidney function after RAS + OMT demonstrated improved event-free survival. This finding reinforces the need for predictors of outcome to guide patient selection for RAS.