Although oxidative stress plays central roles in postischemic renal injury, region-specific alterations in energy and redox metabolism caused by short-duration ischemia remain unknown. Imaging mass spectrometry enabled us to reveal spatial heterogeneity of energy and redox metabolites in the postischemic murine kidney. After 10-minute ischemia and 24-hour reperfusion (10mIR), in the cortex and outer stripes of the outer medulla, ATP substantially decreased, but not in the inner stripes of the outer medulla and inner medulla. 10mIR caused renal injury with elevation of fractional excretion of sodium, although histological damage by oxidative stress was limited. Ischemia-induced NADH elevation in the cortex indicated prolonged production of reactive oxygen species by xanthine oxidase (XOD). However, consumption of reduced glutathione after reperfusion suggested the amelioration of oxidative stress. An XOD inhibitor, febuxostat, which blocks the degradation pathway of adenine nucleotides, promoted ATP recovery and exerted renoprotective effects in the postischemic kidney. Because effects of febuxostat were canceled by silencing of the hypoxanthine phosphoribosyl transferase 1 gene in cultured tubular cells, mechanisms for the renoprotective effects appear to involve the purine salvage pathway, which uses hypoxanthine to resynthesize adenine nucleotides, including ATP. These findings suggest a novel therapeutic approach for acute ischemia/reperfusion renal injury with febuxostat through salvaging high-energy adenine nucleotides.
Kentaro Fujii, Akiko Kubo, Kazutoshi Miyashita, Masaaki Sato, Aika Hagiwara, Hiroyuki Inoue, Masaki Ryuzaki, Masanori Tamaki, Takako Hishiki, Noriyo Hayakawa, Yasuaki Kabe, Hiroshi Itoh, Makoto Suematsu
Mitophagy, by maintaining mitochondrial quality control, plays a key role in maintaining kidney function and is impaired in pathologic states. Macrophages are well-known for their pathogenic role in kidney fibrosis. Here, we report that PINK1/Parkin-mediated mitophagy in macrophages is compromised in experimental and human kidney fibrosis. We demonstrate downregulation of mitophagy regulators, mitofusin-2 (MFN2) and Parkin, downstream of PINK1 in kidney fibrosis. Loss of either Pink1 or Prkn promoted renal extracellular matrix accumulation and frequency of profibrotic/M2 macrophages. Pink1-/- or Prkn-/- bone-marrow-derived macrophages (BMDMs) showed enhanced expression of rictor. Mitochondria from TGF-β1-treated Pink1-/- BMDMs exhibited increased superoxide levels, and reduced respiration and ATP production. In addition, mitophagy in macrophages involves PINK1-mediated phosphorylation of downstream MFN2 and MFN2-facilitated recruitment of Parkin to damaged mitochondria, and macrophage-specific deletion of Mfn2 aggravates kidney fibrosis. Moreover, mitophagy regulators were downregulated in human CKD kidney and TGF-β1-treated human renal macrophages, whereas Mdivi1 treatment suppressed mitophagy mediators and promoted fibrotic response. Taken together, our study is the first to demonstrate that macrophage mitophagy plays a protective role against kidney fibrosis via regulating PINK1/MFN2/Parkin-mediated pathway.
Divya Bhatia, Kuei-Pin Chung, Kiichi Nakahira, Edwin Patino, Michelle C. Rice, Lisa K. Torres, Thangamani Muthukumar, Augustine M.K. Choi, Oleh M. Akchurin, Mary E. Choi
Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in two different mouse models of kidney disease. These effects were not related to changes in circulating leukocytes, as bone marrow transplant from miR-33 deficient animals did not have a similar impact on disease progression. Most importantly, targeted delivery of miR-33 peptide nucleic acid (PNA) inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides (pHLIP) as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease.
Nathan L. Price, Verónica Miguel, Wen Ding, Abhishek K. Singh, Shipra Malik, Noemi Rotllan, Anna Moshnikova, Jakub Toczek, Caroline Zeiss, Mehran M. Sadeghi, Noemi Arias, Ángel Baldán, Oleg A. Andreev, Diego Rodríguez-Puyol, Raman Bahal, Yana K. Reshetnyak, Yajaira Suárez, Carlos Fernández-Hernando, Santiago Lamas
Glomerular disease is characterized by proteinuria and glomerulosclerosis, two pathologic features caused by podocyte injury and mesangial cell activation, respectively. However, whether these two events are linked remains elusive. Here, we report that sonic hedgehog (Shh) is the mediator that connects podocyte damage to mesangial activation and glomerulosclerosis. Shh was induced in glomerular podocytes in various models of proteinuric chronic kidney diseases (CKD). However, mesangial cells in the glomeruli, but not podocytes, responded to hedgehog ligand. In vitro, Shh was induced in podocytes after injury and selectively promoted mesangial cell activation and proliferation. In a mini-organ culture of isolated glomeruli, Shh promoted mesangial activation but did not affect the integrity of podocytes. Podocyte-specific ablation of Shh in vivo exhibited no effect on proteinuria after adriamycin injection but hampered mesangial activation and glomerulosclerosis. Consistently, pharmacologic blockade of Shh signaling decoupled proteinuria from glomerulosclerosis. In humans, Shh was upregulated in glomerular podocytes in patients with CKD and its circulating level was associated with glomerulosclerosis but not proteinuria. These studies demonstrate that Shh mechanistically links podocyte injury to mesangial activation in the pathogenesis of glomerular diseases. Our findings also illustrate a crucial role for podocyte-mesangial communication in connecting proteinuria to glomerulosclerosis.
Dong Zhou, Haiyan Fu, Yang Han, Lu Zhang, Shijia Liu, Lin Lin, Donna B. Stolz, Youhua Liu
Background: In this study, we aimed to identify the lipidomic predictors of early type-2 diabetic kidney disease (DKD) progression which are currently undefined DKD progression. Methods: This longitudinal study included 92 American Indians with type-2 diabetes. Serum lipids (406 from 18 classes) were quantified using mass spectrometry from baseline samples when iothalamate glomerular filtration rate (GFR) was ≥90 mL/min. Affymetrix GeneChip Array was used to measure renal transcript expression. DKD Progression was defined as ≥40% decline in GFR during follow up. Results: Participants had a mean age of 45±9 years and median urine albumin-creatinine ratio of 43 (interquartile range 11 to 144). The 32 progressors had significantly higher relative abundance of polyunsaturated triacylglycerols (TAG)s and a lower abundance of C16-20 acylcarnitines (AC)s (p<0.001). In a Cox regression model the main effect terms of unsaturated free fatty acids and phosphatidylethanolamines and the interaction terms of C16-20 ACs and short, low-double-bond TAGs by categories of albuminuria independently predicted progression of DKD. Renal expression of acetyl-CoA carboxylase encoding gene (ACACA) correlated with serum diacylglycerols in the glomerular compartment (r=0.36, p=0.006), and with low-double-bond TAGs in the tubulointerstitial compartment (r=0.52, p<0.001). Conclusion: Collectively, the findings reveal a previously unrecognized link between lipid markers of impaired mitochondrial β-oxidation and enhanced lipogenesis, with DKD progression, in individuals with preserved GFR. Renal acetyl-CoA carboxylase activation accompanies these lipidomic changes and suggests that it may be the underlying mechanism linking lipid abnormalities to DKD progression. Funding: R24DK082841, K08DK106523, R03DK121941, P30DK089503, P30DK081943, P30DK020572
Farsad Afshinnia, Viji Nair, Jiahe Lin, Thekkelnaycke M. Rajendiran, Tanu Soni, Jaeman Byun, Kumar Sharma, Patrice E. Fort, Thomas W. Gardner, Helen C. Looker, Robert G. Nelson, Frank C. Brosius, Eva L. Feldman, George Michailidis, Matthias Kretzler, Subramaniam Pennathur
Hereditary renal cystic diseases are characterized by defects in primary cilia of renal tubular epithelial cells and abnormality of tubular epithelium, which ultimately result in the development of renal cysts. However, the mechanism leading from abnormality of the tubular epithelium to cystogenesis is not well understood. In this report, we demonstrate a critical role for Robo2 in regulating epithelial development, including ciliogenesis, polarization, and differentiation. We found that Robo2 deficiency results in cystic kidneys, and the cyst cells showed defective cilia and polarity defects in tubular epithelium. The cyst cells, less than terminally differentiated, continue to proliferate. We further established that Robo2 works with p53 as well as polarity and ciliary proteins (Par3, PKCς, ZO-2, and Claudin-2) to regulate these processes. Robo2 binds to Baiap2 (also known as IRSp53) through the IRSp53/MIM homology domain in renal epithelial cells. This binding allows Robo2 to phosphorylate MDM2 at Ser166 via Baiap2 and maintain p53 homeostasis. Disruption of the Robo2-Baiap2 complex causes MDM2 to be subjected to dephosphorylation, leading to a high level of active p53, and initiated p53-mediated cellular senescence via p21 and decreased the expression of ZO-1, ZO-2, PKCς, Par3, and Claudin-2 proteins, resulting in defects in epithelial development, including ciliogenesis, polarization, and differentiation. Importantly, double knockout of Robo2 and p53 rescued all the epithelial defects in kidneys compared with those in Robo2-knockout kidneys. Taken together, the present results demonstrate that Robo2 deficiency causes renal cystic disease, which is largely dependent on defective Robo2-Baiap2 integrated signaling in kidneys.
Qinggang Li, Shaoyuan Cui, Qian Ma, Ying Liu, Hongyu Yu, GuangRui Geng, Ewud Agborbesong, Chongyu Ren, Kai Wei, Yingjie Zhang, Jurong Yang, Xueyuan Bai, Guangyan Cai, Yuansheng Xie, Xiaogang Li, Xiangmei Chen
The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin–mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation–related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis — all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.
Victor G. Puelles, James W. van der Wolde, Nicola Wanner, Markus W. Scheppach, Luise A. Cullen-McEwen, Tillmann Bork, Maja T. Lindenmeyer, Lukas Gernhold, Milagros N. Wong, Fabian Braun, Clemens D. Cohen, Michelle M. Kett, Christoph Kuppe, Rafael Kramann, Turgay Saritas, Claudia R. van Roeyen, Marcus J. Moeller, Leon Tribolet, Richard Rebello, Yu B.Y. Sun, Jinhua Li, Gerard Müller-Newen, Michael D. Hughson, Wendy E. Hoy, Fermin Person, Thorsten Wiech, Sharon D. Ricardo, Peter G. Kerr, Kate M. Denton, Luc Furic, Tobias B. Huber, David J. Nikolic-Paterson, John F. Bertram
Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions, rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teff) to restore tolerance, by exploiting dendritic cell (DC) antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide, to elucidate mechanisms of tolerance employed by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLN of immunized relative to naïve mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339-calcitriol liposomes suppressed expansion, differentiation and function of Teff and induced Foxp3+ and IL-10+ peripheral (p)Treg in an antigen-specific manner, which was dependent on PD-L1. Peptide-calcitriol liposomes modulated CD40 expression by human DCs, and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture’s vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide-calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.
Ryan Galea, Hendrik Nel, Meghna Talekar, Xiao Liu, Joshua D. Ooi, Megan Huynh, Sara Hadjigol, Kate J. Robson, Yi Tian Ting, Suzanne Cole, Karyn Cochlin, Shannon Hitchcock, Bijun Zeng, Suman Yekollu, Martine Boks, Natalie Goh, Helen Roberts, Jamie Rossjohn, Hugh H. Reid, Ben J. Boyd, Ravi Malaviya, David J. Shealy, Daniel G. Baker, Loui Madakamutil, A. Richard Kitching, Brendan J. O’Sullivan, Ranjeny Thomas
Dysregulated actions of bone-derived phosphaturic hormone fibroblast growth factor 23 (FGF23) result in several inherited diseases, such as X-linked hypophosphatemia (XLH), and contribute substantially to the mortality in kidney failure. Mechanisms governing FGF23 production are poorly defined. We herein found that ablation of the Gq/11α–like, extralarge Gα subunit (XLαs), a product of GNAS, exhibits FGF23 deficiency and hyperphosphatemia in early postnatal mice (XLKO). FGF23 elevation in response to parathyroid hormone, a stimulator of FGF23 production via cAMP, was intact in XLKO mice, while skeletal levels of protein kinase C isoforms α and δ (PKCα and PKCδ) were diminished. XLαs ablation in osteocyte-like Ocy454 cells suppressed the levels of FGF23 mRNA, inositol 1,4,5-trisphosphate (IP3), and PKCα/PKCδ proteins. PKC activation in vivo via injecting phorbol myristate acetate (PMA) or by constitutively active Gqα-Q209L in osteocytes and osteoblasts promoted FGF23 production. Molecular studies showed that the PKC activation–induced FGF23 elevation was dependent on MAPK signaling. The baseline PKC activity was elevated in bones of Hyp mice, a model of XLH. XLαs ablation significantly, but modestly, reduced serum FGF23 and elevated serum phosphate in Hyp mice. These findings reveal a potentially hitherto-unknown mechanism of FGF23 synthesis involving a G protein–coupled IP3/PKC pathway, which may be targeted to fine-tune FGF23 levels.
Qing He, Lauren T. Shumate, Julia Matthias, Cumhur Aydin, Marc N. Wein, Jordan M. Spatz, Regina Goetz, Moosa Mohammadi, Antonius Plagge, Paola Divieti Pajevic, Murat Bastepe
We induced chronic kidney disease (CKD) with adenine in WT mice, mice with osteocyte-specific deletion of Cyp27b1, encoding the 25-hydroxyvitamin D 1(OH)ase [Oct-1(OH)ase–/–], and mice with global deletion of Cyp27b1 [global-1α(OH)ase–/–]; we then compared extraskeletal calcification. After adenine treatment, mice displayed increased blood urea nitrogen, decreased serum 1,25(OH)2D, and severe hyperparathyroidism. Skeletal expression of Cyp27b1 and of sclerostin and serum sclerostin all increased in WT mice but not in Oct-1α(OH)ase–/– mice or global-1α(OH)ase–/– mice. In contrast, skeletal expression of BMP2 and serum BMP2 rose in the Oct-1α(OH)ase–/– mice and in the global-1α(OH)ase–/– mice. Extraskeletal calcification occurred in muscle and blood vessels of mice with CKD and was highest in Oct-1α(OH)ase–/–mice. In vitro, recombinant sclerostin (100 ng/mL) significantly suppressed BMP2-induced osteoblastic transdifferentiation of vascular smooth muscle A7r5 cells and diminished BMP2-induced mineralization. Our study provides evidence that local osteocytic production of 1,25(OH)2D stimulates sclerostin and inhibits BMP2 production in murine CKD, thus mitigating osteoblastic transdifferentiation and mineralization of soft tissues. Increased osteocytic 1,25(OH)2D production, triggered by renal malfunction, may represent a “primary defensive response” to protect the organism from ectopic calcification by increasing sclerostin and suppressing BMP2 production.
Loan Nguyen-Yamamoto, Ken-Ichiro Tanaka, Rene St–Arnaud, David Goltzman
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