MASH is a leading cause of liver transplantation. Here, we investigated formoterol, a long-acting β2 adrenergic receptor agonist (LABA), in MASH. Mice treated with a high-fat diet (HFD) for sixteen weeks developed liver steatosis and were treated with formoterol or vehicle for four weeks. Steatosis largely resolved following formoterol treatment. To investigate mechanism, we evaluated mitochondrial biogenesis and found in HFD mice treated with formoterol versus vehicle that: PGC1α levels and electron transport chain components were significantly higher; mitochondrial number was increased; and lipids were decreased. Human HepaRG liver cells were then exposed to free fatty acids and/or formoterol. Formoterol attenuated lipid accumulation and increased ATP-linked basal and maximal respiration. Finally, a retrospective analysis of 59,644 patients with MASH showed that patients taking LABAs had fewer complications of advanced liver disease and lower mortality. Together, these data raise the possibility that LABAs, especially formoterol, could be a novel MASH treatment.

Podocytes have a remarkable ability to recover from injury; however, little is known about the recovery mechanisms involved in this process. We recently showed that formoterol, a long-acting β2-adrenergic receptor (β2-AR) agonist, induced mitochondrial biogenesis (MB) in podocytes and led to renoprotection in mice. However, it is not clear whether this effect was mediated by formoterol acting through the β2-AR or if it occurred through “off-target” effects. We genetically deleted the β2-AR specifically in murine podocytes and used these mice to determine whether formoterol acting through the podocyte β2-AR alone is sufficient for recovery of renal filtration function following injury. The podocyte-specific β2-AR knockout mice (β2-ARfl/fl/PodCre) were generated by crossing β2-AR floxed mice with podocin Cre (B6.Cg-Tg(NPHS2-cre)295Lbh/J) mice. These mice were then subjected to both acute and chronic glomerular injury using nephrotoxic serum (NTS) and adriamycin (ADR), respectively. The extent of injury was evaluated by measuring albuminuria and histological and immunostaining analysis of the murine kidney sections. A similar level of injury was observed in β2-AR knockout and control mice; however, the β2-ARfl/fl/PodCre mice failed to recover in response to formoterol. Functional evaluation of the β2-ARfl/fl/PodCre mice following injury plus formoterol showed similar albuminuria and glomerular injury to control mice that were not treated with formoterol. These results indicate that the podocyte β2-AR is a critical component of the recovery mechanism and may serve as a novel therapeutic target for treating podocytopathies.

We previously showed that the long-acting β2-AR agonist formoterol induced recovery from AKI in mice. To determine if formoterol protected against diabetic nephropathy, the most common cause of ESKD, we used a high fat diet (HFD), a murine type 2 diabetes model, and streptozotocin, a murine type 1 diabetes model. Following formoterol treatment there was a marked recovery from and reversal of diabetic nephropathy in HFD mice compared to those treated with vehicle alone at the ultrastructural, histological, and functional levels. Similar results were seen after formoterol treatment in mice receiving streptozotocin. To investigate effects in humans we performed a competing risk regression analysis with death as a competing risk to examine the association between patients with COPD, who use β2-AR agonists, and patients without COPD and progression to ESKD in a large national cohort of Veterans with stage 4 CKD between 2011-2013. Veterans were followed until 2016 or death. ESKD was defined as initiation of dialysis and/or receipt of kidney transplant. We found that COPD was associated with a 25.6% reduction in progression from stage 4 CKD to ESKD compared to no COPD after adjusting for age, diabetes, sex, race-ethnicity, comorbidities, and medication use. Sensitivity analysis showed a 33.2% reduction in ESKD in Veterans with COPD taking long-acting formoterol and a 20.8% reduction in ESKD in Veterans taking other β2-AR agonists when compared to those with no COPD. These data indicate that β2-AR agonists, especially formoterol, may be a treatment for diabetic nephropathy and perhaps other forms of CKD.

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-β1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-β1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-β1 signaling and fibrosis

Unconventional myosins, linked to deafness, are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice, specifically causes rhodopsin mislocalization, leading to impaired visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with MYO1C. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is important for rhodopsin localization to the photoreceptor OS, and for normal visual function.

Unconventional myosins linked to deafness are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice specifically affected opsin trafficking, leading to loss of visual function. Electroretinogram analysis of Myo1c knockout ( Myo1c -KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with the MYO1C cargo domain. In Myo1c -KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c -KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is critical for opsin trafficking to the photoreceptor OS and for normal visual function.

Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases.

ABSTRACTWhen activated, slit diaphragm proteins NEPHRIN and NEPH1 enable signaling pathways leading to podocyte actin cytoskeleton reorganization, which is critical for podocyte recovery from injury. However, the mechanisms through which these proteins are activated remain unknown. This study presents a novel concept showing ligand-induced activation of NEPHRIN and NEPH1. We first identified phosphatase SHP-2, which directly dephosphorylated these proteins. We next identified HGF, a known SHP-2 modulator, as a rapid inducer of NEPHRIN and NEPH1 phosphorylation. Using baculovirus expressed recombinant purified proteins, SPR (surface plasma resonance), molecular modeling and peptide binding approaches, we show that HGF directly binds NEPHRIN and NEPH1 extracellular domains. Further, using cultured podocytes and Drosophila nephrocytes, we demonstrate that while HGF treatment repaired injured podocytes, the addition of inhibitory NEPH1 or NEPHRIN peptides blocked HGF-induced recovery. Overall, this study shows novel activation and deactivation mechanisms for NEPHRIN and NEPH1 that are required for their function.

Podocytes have limited ability to recover from injury. Here, we demonstrate that increased mitochondrial biogenesis, to meet the metabolic and energy demand of a cell, accelerates podocyte recovery from injury. Analysis of events induced during podocyte injury and recovery showed marked upregulation of peroxisome proliferator-activated receptor-g coactivator-1 alpha (PGC-1 alpha), a transcriptional co-activator of mitochondrial biogenesis, and key components of the mitochondrial electron transport chain. To evaluate our hypothesis that increasing mitochondrial biogenesis enhanced podocyte recovery from injury, we treated injured podocytes with formoterol, a potent, specific, and long-acting beta 2-adrenergic receptor agonist that induces mitochondrial biogenesis in vitro and in vivo. Formoterol increased mitochondrial biogenesis and restored mitochondrial morphology and the injury-induced changes to the organization of the actin cytoskeleton in podocytes. Importantly, beta 2-adrenergic receptors were found to be present on podocyte membranes. Their knockdown attenuated formoterol-induced mitochondrial biogenesis. To determine the potential clinical relevance of these findings, mouse models of acute nephrotoxic serum nephritis and chronic (Adriamycin [doxorubicin]) glomerulopathy were used. Mice were treated with formoterol post-injury when glomerular dysfunction was established. Strikingly, formoterol accelerated the recovery of glomerular function by reducing proteinuria and ameliorating kidney pathology. Furthermore, formoterol treatment reduced cellular apoptosis and increased the expression of the mitochondrial biogenesis marker PGC-1 alpha and multiple electron transport chain proteins. Thus, our results support beta 2-adrenergic receptors as novel therapeutic targets and formoterol as a therapeutic compound for treating podocytopathies.

Fibroblast to myofibroblast differentiation is crucial for the initial healing response but excessive myofibroblast activation leads to pathological fibrosis. Therefore, it is imperative to understand the mechanisms underlying myofibroblast formation. Here we report that mitochondrial calcium ( m Ca 2+ ) signaling is a regulatory mechanism in myofibroblast differentiation and fibrosis. We demonstrate that fibrotic signaling alters gating of the mitochondrial calcium uniporter (mtCU) in a MICU1-dependent fashion to reduce m Ca 2+ uptake and induce coordinated changes in metabolism, i.e., increased glycolysis feeding anabolic pathways and glutaminolysis yielding increased α-ketoglutarate (αKG) bioavailability. m Ca 2+ -dependent metabolic reprogramming leads to the activation of αKG-dependent histone demethylases, enhancing chromatin accessibility in loci specific to the myofibroblast gene program, resulting in differentiation. Our results uncover an important role for the mtCU beyond metabolic regulation and cell death and demonstrate that m Ca 2+ signaling regulates the epigenome to influence cellular differentiation.

Beta2-adrenergic receptor (β2 -AR) is a G-protein-coupled adrenergic receptor family member, whose clinical significance has been extensively investigated in lung, cardiovascular and muscular diseases, but its role in kidney biology remains understudied. In this review, we discuss some of the recent studies, where the effect of agonist/antagonist-mediated activation/inhibition of β2 -AR on disease pathogenesis process was studied, and highlighted the role of β2 -AR in kidney biology. The expression of β2 -AR has been noted in many kidney subunits including proximal tubules, glomeruli and podocytes. In vivo studies have shown that in cultured proximal tubules β2 -AR is involved in Na-ATPase activity and transcellular Na-transport through protein kinase-C activation; whereas in cultured podocytes, it was associated with depolarization of the membrane. The animal studies further revealed that β2 -AR activation by short-acting β2 agonists attenuated monocyte activation, pro-inflammatory and pro-fibrotic responses through β-arrestin2 dependent NF-kB inactivation in diabetic kidney disease; in contrast, activation by long-acting β2 agonists restored mitochondrial and renal function in the acute kidney injury mice models through PGC-1α dependent mitochondrial biogenesis. In conclusion, the activation of β2 -AR may present a rapidly developing therapeutic target for renal diseases.

Definitive diagnosis of glomerular disease requires a kidney biopsy, an invasive procedure that may not be safe or feasible to perform in all patients. We developed a noninvasive, accurate, and economical diagnostic assay with easy commercial adaptability to detect recurrent focal segmental glomerulosclerosis (rFSGS) after kidney transplant. Since FSGS involves podocyte damage and death, our approach involved mRNA profiling of cultured podocytes treated with plasma from patients with rFSGS to identify upregulated genes involved in podocyte damage. For concept validation, three upregulated pro-apoptotic candidate genes (IL1 beta, BMF, and IGFBP3) were selected, and their promoter regions were cloned into a luciferase-based reporter vector and transfected into podocytes to generate stable podocyte cell lines. Strikingly, when exposed to rFSGS patient plasma, these cell lines showed increased reporter activity; in contrast, no reporter activity was noted with plasma from patients with non-recurrent FSGS or membranous nephropathy. Area under the receiver operating characteristics curves (AUCs) for models discriminating between rFSGS and other nephropathies (non-recurrent FSGS and membranous nephropathy) and between rFSGS and non-recurrent FSGS ranged from 0.81 to 0.86, respectively. Estimated sensitivities and specificities for the diagnosis of rFSGS were greater than 80% for the IL1 beta and BMF cell lines, and were slightly lower for the IGFBP3 cell line. Importantly, the novel approach outlined here for the diagnosis of rFSGS is widely applicable to the design of sensitive and specific diagnostic/prognostic assays for other glomerular diseases.

Transforming growth factor-β (TGF-β) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-β signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-β-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-β-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-β signaling through downregulation of canonical and non-canonical TGF-β pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-β signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-β responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-β-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.

Steroid-resistant nephrotic syndrome is a frequent cause of chronic kidney disease almost inevitably progressing to end-stage renal disease. More than 58 monogenic causes of SRNS have been discovered and majority of known steroid-resistant nephrotic syndrome causing genes are predominantly expressed in glomerular podocytes, placing them at the center of disease pathogenesis. Herein, we describe two unrelated families with steroid-resistant nephrotic syndrome with homozygous mutations in the KIRREL1 gene. One mutation showed high frequency in the European population (minor allele frequency 0.0011) and this patient achieved complete remission following treatment, but later progressed to chronic kidney disease. We found that mutant KIRREL1 proteins failed to localize to the podocyte cell membrane, indicating defective trafficking and impaired podocytes function. Thus, the KIRREL1 gene product has an important role in modulating the integrity of the slit diaphragm and maintaining glomerular filtration function.

Transforming growth factor-beta (TGF-beta) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-beta signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myolc is critical for TGF-beta-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myolc also protected from injury in the TGF-beta-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myolc significantly blunted TGF-beta signaling through downregulation of canonical and non-canonical TGF-beta pathways. Interestingly, nuclear rather than the cytoplasmic Myolc was found to play a central role in controlling TGF-beta signaling through transcriptional regulation. Differential expression analysis of nuclear Myolc-associated gene promoters showed that nuclear Myolc targeted the TGF-beta responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myolc-mediated regulation of TGF-beta-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.