Podocyte injury and loss are key drivers of primary and secondary glomerular diseases, such as focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). We previously demonstrated the renoprotective role of protein S (PS) and its cognate tyrosine-protein kinase receptor, TYRO3, in models of FSGS and DKD and that their signaling exerts anti-apoptotic and anti-inflammatory effects to confer protection against podocyte loss. Among the three TAM receptors (TYRO3, AXL, and MER), only TYRO3 expression is largely restricted to podocytes, and glomerular TYRO3 mRNA expression negatively correlates with human glomerular disease progression. We, therefore, posited that the agonism PS-TYRO3 signaling could serve as a potential therapeutic approach to attenuate glomerular disease progression. As PS function is not limited to TYRO3-mediated signal transduction but includes its anticoagulant activity, we focused on the development of TYRO3 agonist as an optimal therapeutic approach to glomerular disease. Among the small molecule TYRO3 agonist compounds screened, compound-10 (C-10) showed a select activation of TYRO3 without any effects on AXL or MER. We also confirmed that C-10 directly binds to TYRO3, but not the other receptors. In vivo, C-10 attenuated proteinuria, glomerular injury, and podocyte loss in mouse models of adriamycin-induced nephropathy and db/db model of type 2 diabetes. Moreover, these renoprotective effects of C-10 are lost in Tyro3 knockout mice, indicating that C-10 is a select agonist of TYRO3 activity that mitigates podocyte injury and glomerular disease. Therefore, C-10, a novel TYRO3 agonist, could be potentially developed as a new therapy for glomerular disease.
Fang Zhong, Hong Cai, Jia Fu, Zeguo Sun, Zhengzhe Li, David Bauman, Lois Wang, Bhaskar Das, Kyung Lee, John He
FOXD1+ derived stromal cells give rise to pericytes and fibroblasts that support the kidney vasculature and interstitium but are also major precursors of myofibroblasts. ZEB2 is a SMAD-interacting transcription factor that is expressed in developing kidney stromal progenitors. Here we show that Zeb2 is essential for normal FOXD1+ stromal progenitor development. Specific deletion of mouse Zeb2 in FOXD1+ stromal progenitors (Zeb2 cKO) leads to abnormal interstitial stromal cell development, differentiation, and kidney fibrosis. Immunofluorescent staining analyses revealed abnormal expression of interstitial stromal cell markers MEIS1/2/3, CDKN1C, and CSPG4 (NG2) in newborn and 3-week-old Zeb2 cKO mouse kidneys. Zeb2 deficient FOXD1+ stromal progenitors also took on a myofibroblast fate that led to kidney fibrosis and kidney failure. Cell marker studies further confirmed that these myofibroblasts expressed pericyte and resident fibroblast markers including PDGFRβ, CSPG4, Desmin, GLI1, and NT5E. Notably, increased interstitial collagen deposition associated with loss of Zeb2 in FOXD1+ stromal progenitors was accompanied by increased expression of activated SMAD1/5/8, SMAD2/3, SMAD4, and AXIN2. Thus, our study identifies a key role of ZEB2 in maintaining the cell fate of FOXD1+ stromal progenitors during kidney development whereas loss of ZEB2 leads to differentiation of FOXD1+ stromal progenitors into myofibroblasts and kidney fibrosis.
Sudhir Kumar, Xueping Fan, Hila Milo Rasouly, Richa Sharma, David J. Salant, Weining Lu
Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic nephropathy, is characterized by phenotypic variability exceeding genic effects. Dysregulated metabolism and immune cell function are key disease modifiers. The tryptophan metabolites, kynurenines, produced through IDO1, are known immunomodulators. Here, we study the role of tryptophan metabolism in PKD using an orthologous disease model (C57Bl/6J Pkd1RC/RC). We found elevated kynurenine and IDO1 levels in Pkd1RC/RC kidneys versus wildtype. Further, IDO1 levels were increased in ADPKD cell lines. Genetic Ido1 loss in Pkd1RC/RC animals resulted in reduced PKD severity as measured by %kidney weight/body weight and cystic index. Consistent with an immunomodulatory role of kynurenines, Pkd1RC/RC;Ido1-/- mice presented with significant changes in the cystic immune microenvironment (CME) versus controls. Kidney macrophage numbers decreased and CD8+ T cell numbers increased, both known PKD modulators. Also, pharmacological IDO1 inhibition in Pkd1RC/RC mice and kidney specific Pkd2 knockout mice with rapidly progressive PKD resulted in less severe PKD versus controls with similar changes in the CME as in the genetic model. Our data suggest that tryptophan metabolism is dysregulated in ADPKD and that its inhibition results in changes to the CME and slows disease progression, making IDO1 a novel therapeutic target for ADPKD.
Dustin T. Nguyen, Emily K. Kleczko, Nidhi Dwivedi, Marie-Louise T. Monaghan, Berenice Y. Gitomer, Michel B. Chonchol, Eric T. Clambey, Raphael A. Nemenoff, Jelena Klawitter, Katharina Hopp
Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with aging and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using 2 murine models of injury and repair, we show that obstructive injury generated senescent epithelia, which persisted after resolution of the original injury, promoted ongoing fibrosis, and impeded adaptive repair. Depletion of senescent cells with ABT-263 reduced fibrosis in reversed ureteric obstruction and after renal ischemia/reperfusion injury. We validated these findings in humans, showing that senescence and fibrosis persisted after relieved renal obstruction. We next characterized senescent epithelia in murine renal injury using single-cell RNA-Seq. We extended our classification to human kidney and liver disease and identified conserved profibrotic proteins, which we validated in vitro and in human disease. We demonstrated that increased levels of protein disulfide isomerase family A member 3 (PDIA3) augmented TGF-β–mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduced kidney fibrosis during ongoing renal injury and as such represented a new potential therapeutic pathway. Analysis of the signaling pathways of senescent epithelia connected senescence to organ fibrosis, permitting rational design of antifibrotic therapies.
Eoin D. O’Sullivan, Katie J. Mylonas, Rachel Bell, Cyril Carvalho, David P. Baird, Carolynn Cairns, Kevin M. Gallagher, Ross Campbell, Marie Docherty, Alexander Laird, Neil C. Henderson, Tamir Chandra, Kristina Kirschner, Bryan Conway, Gry H. Dihazi, Michael Zeisberg, Jeremy Hughes, Laura Denby, Hassan Dihazi, David A. Ferenbach
Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and pro-inflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of pro-inflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron significantly decreased TGF-β expression and suppressed TGF-β-driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, confirming the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.
Edwin Patino, Divya Bhatia, Steven Z. Vance, Ada Antypiuk, Rie Uni, Chantalle Campbell, Carlo G. Castillo, Shahd Jaouni, Francesca Vinchi, Mary E. Choi, Oleh Akchurin
Glucocorticoids remain a cornerstone of therapeutic regimes for autoimmune and chronic inflammatory diseases, for example, in different forms of crescentic glomerulonephritis because of their rapid anti-inflammatory effects, low cost, and wide availability. Despite their routine use for decades, the underlying cellular mechanisms by which steroids exert their therapeutic effects need to be fully elucidated. Here, we demonstrate that high-dose steroid treatment rapidly reduced the number of proinflammatory CXCR3+ CD4+ T cells in the kidney by combining high-dimensional single-cell and morphological analyses of kidney biopsies from patients with antineutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. Using an experimental model of crescentic glomerulonephritis, we show that the steroid-induced decrease in renal CD4+ T cells is a consequence of reduced T-cell recruitment, which is associated with an ameliorated disease course. Mechanistic in vivo and in vitro studies revealed that steroids act directly on renal tissue cells, such as tubular epithelial cells, but not on T cells, which resulted in an abolished renal expression of CXCL9 and CXCL10, as well as in the prevention of CXCR3+ CD4+ T-cell recruitment to the inflamed kidneys. Thus, we identified the CXCL9/10-CXCR3 axis as a previously unrecognized cellular and molecular target of glucocorticoids providing protection from immune-mediated pathology.
Jan-Hendrik Riedel, Lennart Robben, Hans-Joachim Paust, Yu Zhao, Nariaki Asada, Ning Song, Anett Peters, Anna Kaffke, Alina C. Borchers, Gisa Tiegs, Larissa Seifert, Nicola M. Tomas, Elion Hoxha, Ulrich O. Wenzel, Tobias B. Huber, Thorsten Wiech, Jan-Eric Turner, Christian F. Krebs, Ulf Panzer
Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The syndrome of inappropriate secretion of anti-diuretic hormone (vasopressin) raised the question if vasopressin could be produced outside of the brain and whether the kidney could be a source of vasopressin. We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects the pre-pro-vasopressin, we found that immunoreactive pre-pro-vasopressin protein is found in mouse and human kidneys. Moreover, we found that murine collecting duct cells make biologically active vasopressin which increases in response to NaCl mediated hypertonicity, and that water restriction increases the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.
Juan P. Arroyo, Andrew S. Terker, Yvonne Zuchowski, Jason A. Watts, Fabian Bock, Cameron Meyer, Wentian Luo, Meghan E. Kapp, Edward R. Gould, Adam X Miranda, Joshua Carty, Ming Jiang, Roberto M. Vanacore, Elizabeth Hammock, Matthew H. Wilson, Roy Zent, Mingzhi Zhang, Gautam Bhave, Raymond C. Harris
The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause systemic developmental syndrome, including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic disease have remained unclear. We performed whole-exome sequence analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis; we identified a probable pathogenic novel variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 LG4-5 domain. Heterozygous and homozygous knock-in mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels; this suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Re-examinations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.
Jun-Ya Kaimori, Yamato Kikkawa, Daisuke Motooka, Tomoko Namba-Hamano, Ayako Takuwa, Atsuko Imai-Okazaki, Kaori Kobayashi, Arisa Tanigawa, Yuko Kotani, Yoshihiro Uno, Kazuto Yoshimi, Koki Hattori, Yuta Asahina, Sachio Kajimoto, Yohei Doi, Tatsufumi Oka, Yusuke Sakaguchi, Tomoji Mashimo, Kiyotoshi Sekiguchi, Akihiro Nakaya, Motoyoshi Nomizu, Yoshitaka Isaka
Lipoprotein modification by reactive dicarbonyls, including isolevuglandin (IsoLG), produces dysfunctional particles. Kidneys participate in lipoprotein metabolism, including tubular uptake. However, the process beyond the proximal tubule is unclear, as is the effect of kidney injury on this pathway. We found that patients and animals with proteinuric injury have increased urinary apolipoprotein AI (apoAI), IsoLG, and IsoLG adduct enrichment of the urinary apoAI fraction compared with other proteins. Proteinuric mice, induced by podocyte specific injury, showed more tubular absorption of IsoLG-apoAI and increased expression of lipoprotein transporters in proximal tubular cells compared with uninjured animals. Renal lymph reflects composition of the interstitial compartment, and showed increased apoAI and IsoLG in proteinuric animals, supporting a tubular cell-interstitium-lymph pathway for renal handling of lipoproteins. IsoLG-modified apoAI was not only a marker of renal injury, but also directly damaged renal cells. IsoLG-apoAI increased inflammatory cytokines in cultured tubular epithelial cells, activated lymphatic endothelial cells and caused greater contractility of renal lymphatic vessels than unmodified apoAI. In vivo, inhibition of IsoLG by a dicarbonyl scavenger reduced both albuminuria and urinary apoAI and decreased tubular epithelial cell and lymphatic endothelial cell injury, lymphangiogenesis, and interstitial fibrosis. Our results indicate that IsoLG-modified apolipoprotein AI is a novel pathogenic mediator and therapeutic target in kidney disease.
Jianyong Zhong, Hai-Chun Yang, Elaine L. Shelton, Taiji Matsusaka, Amanda J. Clark, Valery Yermalitsky, Zahra Mashhadi, Linda S. May-Zhang, MacRae F. Linton, Agnes B. Fogo, Annet Kirabo, Sean S. Davies, Valentina Kon
Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia-reperfusion-injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, neutrophil recruitment and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation result in decreased JNK activation leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.
Katharina Thomas, Lisa Zondler, Nadine Ludwig, Marina Kardell, Corinna Lüneburg, Katharina Henke, Sina Mersmann, Andreas Margraf, Tilmann Spieker, Tobias Tekath, Ana Velic, Richard Holtmeier, Juliane Hermann, Vera Jankowski, Melanie Meersch, Dietmar Vestweber, Martin Westphal, Johannes Roth, Michael A. Schaefers, John A. Kellum, Clifford A. Lowell, Jan Rossaint, Alexander Zarbock
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