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American Society for Microbiology Journals: Antimicrobial Agents and Chemotherapy 2022. 66. https://doi.org/10.1128/aac.00017-22
Repurposing the Kinase Inhibitor Mavelertinib for Giardiasis Therapy
Samantha A. Michaels, Matthew A. Hulverson, Grant R. Whitman, Linh T. Tran, Ryan Choi, Erkang Fan, Case W. McNamara, Melissa S. Love, Kayode K. Ojo
Abstract
A phenotypic screen of the ReFRAME compound library was performed to identify cell-active inhibitors that could be developed as therapeutics for giardiasis. A primary screen against Giardia lamblia GS clone H7 identified 85 cell-active compounds at a hit rate of 0.72%. A cytotoxicity counterscreen against HEK293T cells was carried out to assess hit compound selectivity for further prioritization. Mavelertinib (PF-06747775), a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), was identified as a potential new therapeutic based on indication, activity, and availability after reconfirmation. Mavelertinib has in vitro efficacy against metronidazole-resistant 713-M3 strains. Other EGFR-TKIs screened in follow-up assays exhibited insignificant inhibition of G. lamblia at 5 μM, suggesting that the primary molecular target of mavelertinib may have a different mechanistic binding mode from human EGFR-tyrosine kinase. Mavelertinib, dosed as low as 5 mg/kg of body weight or as high as 50 mg/kg, was efficacious in the acute murine Giardia infection model. These results suggest that mavelertinib merits consideration for repurposing and advancement to giardiasis clinical trials while its analogues are further developed.
ACS Infectious Diseases 2021 7 (5), 943-947; DOI: 10.1021/acsinfecdis.0c00919
A Curious Case for Development of Kinase Inhibitors as Antigiardiasis Treatments Using Advanced Drug Techniques
Samantha A. Michaels, Kelly M. Hennessey, Neal Paragas, Alexander R. Paredez, Kayode K. Ojo
Abstract
Giardiasis is a neglected parasitic diarrheal disease that is particularly associated with poverty. Current treatment options are limited in the face of growing resistance, but the reduced kinome of Giardia lamblia increases the likelihood of identifying nonredundant essential kinases as potential drug targets. Repurposing known and newly identified kinase inhibitors in drug development programs for novel giardiasis therapeutics could therefore be a cost-effective and time saving approach. Innovative improvements to physiologically-based pharmacokinetic modeling coupled with emerging imaging technologies and a CRISPR-interference method could accelerate progress toward the goal of more effective giardiasis therapeutics based on kinase inhibition.
Journal of Antimicrobial Chemotherapy, Volume 75, Issue 5, May 2020, Pages 1218–1227, https://doi.org/10.1093/jac/dkz567
Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model
Samantha A Michaels, Han-Wei Shih, Bailin Zhang, Edelmar D Navaluna, Zhongsheng Zhang, Ranae M Ranade, J Robert Gillespie, Ethan A Merritt, Erkang Fan, Frederick S Buckner, Alexander R Paredez, Kayode K Ojo
Abstract
Background
Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia.
Objectives
To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites.
Methods
Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model.
Results
Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays.
Conclusions
The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.
Commun Biol 2, 326 (2019). https://doi.org/10.1038/s42003-019-0571-7
Cell-specific image-guided transcriptomics identifies complex injuries caused by ischemic acute kidney injury in mice
Miyazaki, T., Gharib, S.A., Hsu, Y.A. et al. Cell-specific image-guided transcriptomics identifies complex injuries caused by ischemic acute kidney injury in mice.
Abstract
The kidney’s inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.
Nat Commun 9, 4262 (2018). https://doi.org/10.1038/s41467-018-06288-w
Klose, A.D., Paragas, N.
Automated quantification of bioluminescence images
Abstract
We developed a computer-aided analysis tool for quantitatively determining bioluminescent reporter distributions inside small animals. The core innovations are a body-fitting animal shuttle and a statistical mouse atlas, both of which are spatially aligned and scaled according to the animal’s weight, and hence provide data congruency across animals of varying size and pose. In conjunction with a multispectral bioluminescence tomography technique capitalizing on the spatial framework of the shuttle, the in vivo biodistribution of luminescent reporters can rapidly be calculated and, thus, enables operator-independent and computer-driven data analysis. We demonstrate its functionality by quantitatively monitoring a bacterial infection, where the bacterial organ burden was determined and validated with the established serial-plating method. In addition, the statistical mouse atlas was validated and compared to existing techniques providing an anatomical reference. The proposed data analysis tool promises to increase data throughput and data reproducibility and accelerate human disease modeling in mice.
J Clin Invest. 2014 Jul;124(7):2963-76. doi: 10.1172/JCI71630. Epub 2014 Jun 17.
α-Intercalated cells defend the urinary system from bacterial infection.
Paragas N, Kulkarni R, Werth M, Schmidt-Ott KM, Forster C, Deng R, Zhang Q, Singer E, Klose AD, Shen TH, Francis KP, Ray S, Vijayakumar S, Seward S, Bovino ME, Xu K, Takabe Y, Amaral FE, Mohan S, Wax R, Corbin K, Sanna-Cherchi S, Mori K, Johnson L, Nickolas T, D'Agati V, Lin CS, Qiu A, Al-Awqati Q, Ratner AJ, Barasch J.
Abstract
α-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.
Comment in
Kidney α-intercalated cells and lipocalin 2: defending the urinary tract. [J Clin Invest. 2014]
PMID: 24937428 PMCID: PMC4071397
Tissue Antigens. 2014 Apr;83(4):273-85. doi: 10.1111/tan.12330.
Expression of the innate defense receptor S5D-SRCRB in the urogenital tract.
Miró-Julià C1, Escoda-Ferran C, Carrasco E, Moeller JB, Vadekaer DF, Gao X, Paragas N, Oliver J, Holmskov U, Al-Awqati Q, Lozano F.
Abstract
S5D-SRCRB is a novel mouse secretory glycoprotein belonging to the ancient and highly conserved scavenger receptor cysteine-rich superfamily of protein receptors. Available evidence indicates that S5D-SRCRB interacts with conserved microbial cell wall components, as well as with some endogenous proteins, and presents a restricted tissue expression pattern. This study further analyzes the expression of S5D-SRCRB along the mouse urogenital tract. Immunohistochemical staining for S5D-SRCRB was observed in spermatocytes from seminiferous tubules and in the epithelial surface from urethra and bladder, as well as in kidney tubules, mainly from medulla and papilla. Double stainings showed that S5D-SRCRB is expressed in both principal (P) and intercalated (IC) cells from renal collecting ducts (CD). By using an in vitro cell model of IC cell differentiation, preferential expression of S5D-SRCRB was observed in the apical border of terminally differentiated IC. Colocalization of S5D-SRCRB with galectin-3 (Gal-3) was also observed in kidney and bladder, but not in testis, supporting concurrent biochemical studies demonstrating the carbohydrate-dependent interaction of Gal-3 and S5D-SRCRB. Furthermore, upregulation of S5D-SRCRB expression was observed in in vitro and in vivo models of bacterial aggression, reinforcing the emerging view that CD, and specially IC, are important players in innate defense of the urinary tract against infection. Taken together, the results indicate that S5D-SRCRB is an integral component of the urogenital tract involved in innate immune functions.
PMID: 24641504
N Engl J Med. 2013 Aug 15;369(7):621-9. doi: 10.1056/NEJMoa1214479. Epub 2013 Jul 17.
Mutations in DSTYK and dominant urinary tract malformations.
Sanna-Cherchi S1, Sampogna RV, Papeta N, Burgess KE, Nees SN, Perry BJ, Choi M, Bodria M, Liu Y, Weng PL, Lozanovski VJ, Verbitsky M, Lugani F, Sterken R, Paragas N, Caridi G, Carrea A, Dagnino M, Materna-Kiryluk A, Santamaria G, Murtas C, Ristoska-Bojkovska N, Izzi C, Kacak N, Bianco B, Giberti S, Gigante M, Piaggio G, Gesualdo L, Kosuljandic Vukic D, Vukojevic K, Saraga-Babic M, Saraga M, Gucev Z, Allegri L, Latos-Bielenska A, Casu D, State M, Scolari F, Ravazzolo R, Kiryluk K, Al-Awqati Q, D'Agati VD, Drummond IA, Tasic V, Lifton RP, Ghiggeri GM, Gharavi AG.
Abstract
BACKGROUND: Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood. METHODS: We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies. RESULTS: Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine-threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases. CONCLUSIONS: We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.).
Comment in
Genetics and urinary tract malformations. [Am J Kidney Dis. 2014]
Genetics: DSTYK gene linked to urinary tract defects. [Nat Rev Urol. 2013]
PMID: 23862974 PMCID: PMC3846391
Biochim Biophys Acta. 2012 Sep;1823(9):1451-8. doi: 10.1016/j.bbamcr.2012.06.014. Epub 2012 Jun 19.
NGAL-Siderocalin in kidney disease.
Paragas N1, Qiu A, Hollmen M, Nickolas TL, Devarajan P, Barasch J.
Abstract
Kidney damage induces the expression of a myriad of proteins in the serum and in the urine. The function of these proteins in the sequence of damage and repair is now being studied in genetic models and by novel imaging techniques. One of the most intensely expressed proteins is lipocalin2, also called NGAL or Siderocalin. While this protein has been best studied by clinical scientists, only a few labs study its underlying metabolism and function in tissue damage. Structure-function studies, imaging studies and clinical studies have revealed that NGAL-Siderocalin is an endogenous antimicrobial with iron scavenging activity. This review discusses the "iron problem" of kidney damage, the tight linkage between kidney damage and NGAL-Siderocalin expression and the potential roles that NGAL-Siderocalin may serve in the defense of the urogenital system. This article is part of a Special Issue entitled: Cell Biology of Metals.
PMID: 22728330
PMCID: PMC3664277
Nephrol Dial Transplant. 2011 Jul;26(7):2387-90. doi: 10.1093/ndt/gfr258. Epub 2011 May 9.
Urinary NGAL is a useful clinical biomarker of HIV-associated nephropathy.
Sola-Del Valle DA1, Mohan S, Cheng JT, Paragas NA, Sise ME, D'Agati VD, Barasch J.
Abstract
BACKGROUND: Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is expressed by kidney tubules that are acutely damaged, but few studies have investigated the association of neutrophil gelatinase-associated lipocalin (NGAL) with different forms of chronic kidney disease (CKD). HIV-associated nephropathy (HIVAN) is a progressive form of CKD characterized by collapsing focal segmental glomerulosclerosis and microcytic tubular dilatation that typically leads to end-stage renal disease (ESRD). METHODS: Previously, we reported that microcystic tubular dilatations specifically expressed NGAL RNA, implying that the detection of uNGAL protein could mark advanced HIVAN. To test this idea, we performed a comparative study of diverse proteinuric glomerulopathies in 25 patients who were HIV positive. RESULTS: Eighteen patients had HIVAN and seven had other glomerulopathies (four membranoproliferative glomerulonephritis, one membranous glomerulonephritis, one amyloid and one malarial GN). HIVAN and non-HIVAN patients did not differ with respect to age, ethnicity, serum creatinine, estimated GFR, proteinuria or the prevalence of hypocomplementemia (6 versus 29%, P = 0.18), but HIVAN patients were less likely to have HCV infections. HIVAN patients expressed 4-fold higher levels of uNGAL than the patients with other glomerulopathies [387 ± 338 versus 94 ± 101 μg/g urine creatinine (uCr), P = 0.02]. A cutpoint of 121.5 μg uNGAL/g uCr demonstrated 94% sensitivity and 71% specificity for the diagnosis of HIVAN, with an area under the receiver operator characteristic curve of 0.88. CONCLUSION: In summary, while HIVAN disease is currently diagnosed only by kidney biopsy, uNGAL can distinguish HIVAN from other proteinuric glomerulopathies in the HIV-infected patient, likely because of its specific expression from characteristic microcysts.
PMID: 21555394 PMCID: PMC3164447
Nat Med. 2011 Feb;17(2):216-22. doi: 10.1038/nm.2290. Epub 2011 Jan 16.
The Ngal reporter mouse detects the response of the kidney to injury in real time.
Paragas N1, Qiu A, Zhang Q, Samstein B, Deng SX, Schmidt-Ott KM, Viltard M, Yu W, Forster CS, Gong G, Liu Y, Kulkarni R, Mori K, Kalandadze A, Ratner AJ, Devarajan P, Landry DW, D'Agati V, Lin CS, Barasch J.
Abstract
Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.
PMID:21240264 PMCID: PMC3059503
J Am Soc Nephrol. 2011 Jan;22(1):113-23. doi: 10.1681/ASN.2010080888.
The Sweet Pee model for Sglt2 mutation.
Ly JP1, Onay T, Sison K, Sivaskandarajah G, Sabbisetti V, Li L, Bonventre JV, Flenniken A, Paragas N, Barasch JM, Adamson SL, Osborne L, Rossant J, Schnermann J, Quaggin SE.
Author information
Abstract
Inhibiting renal glucose transport is a potential pharmacologic approach to treat diabetes. The renal tubular sodium-glucose transporter 2 (SGLT2) reabsorbs approximately 90% of the filtered glucose load. An animal model with sglt2 dysfunction could provide information regarding the potential long-term safety and efficacy of SGLT2 inhibitors, which are currently under clinical investigation. Here, we describe Sweet Pee, a mouse model that carries a nonsense mutation in the Slc5a2 gene, which results in the loss of sglt2 protein function. The phenotype of Sweet Pee mutants was remarkably similar to patients with mutations in the Scl5a2 gene. The Sweet Pee mutants had improved glucose tolerance, higher urinary excretion of calcium and magnesium, and growth retardation. Renal physiologic studies demonstrated a prominent distal osmotic diuresis without enhanced natriuresis. Sweet Pee mutants did not exhibit increased KIM-1 or NGAL, markers of acute tubular injury. After induction of diabetes, Sweet Pee mice had better overall glycemic control than wild-type control mice, but had a higher risk for infection and an increased mortality rate (70% in homozygous mutants versus 10% in controls at 20 weeks). In summary, the Sweet Pee model allows study of the long-term benefits and risks associated with inhibition of SGLT2 for the management of diabetes. Our model suggests that inhibiting SGLT2 may improve glucose control but may confer increased risks for infection, malnutrition, volume contraction, and mortality.
Comment in
Risks and benefits of Sweet Pee. [J Am Soc Nephrol. 2011]
PMID: 2120925 PMCID: PMC3014040
Nat Chem Biol. 2010 Aug;6(8):602-9. doi: 10.1038/nchembio.402. Epub 2010 Jun 27.
Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
Bao G1, Clifton M, Hoette TM, Mori K, Deng SX, Qiu A, Viltard M, Williams D, Paragas N, Leete T, Kulkarni R, Li X, Lee B, Kalandadze A, Ratner AJ, Pizarro JC, Schmidt-Ott KM, Landry DW, Raymond KN, Strong RK, Barasch J.
Abstract
The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.
Comment in
Bioinorganic chemistry: Getting a grip on iron. [Nat Chem Biol. 2010]
PMID: 20581821 PMCID: PMC2907470
J Am Soc Nephrol. 2009 Aug;20(8):1687-92. doi: 10.1681/ASN.2009010065. Epub 2009 Jul 23.
Urinary NGAL marks cystic disease in HIV-associated nephropathy.
Paragas N1, Nickolas TL, Wyatt C, Forster CS, Sise M, Morgello S, Jagla B, Buchen C, Stella P, Sanna-Cherchi S, Carnevali ML, Mattei S, Bovino A, Argentiero L, Magnano A, Devarajan P, Schmidt-Ott KM, Allegri L, Klotman P, D'Agati V, Gharavi AG, Barasch J.
Abstract
Nephrosis and a rapid decline in kidney function characterize HIV-associated nephropathy (HIVAN). Histologically, HIVAN is a collapsing focal segmental glomerulosclerosis with prominent tubular damage. We explored the expression of neutrophil gelatinase-associated lipocalin (NGAL), a marker of tubular injury, to determine whether this protein has the potential to aid in the noninvasive diagnosis of HIVAN. We found that expression of urinary NGAL was much higher in patients with biopsy-proven HIVAN than in HIV-positive and HIV-negative patients with other forms of chronic kidney disease. In the HIV-transgenic mouse model of HIVAN, NGAL mRNA was abundant in dilated, microcystic segments of the nephron. In contrast, urinary NGAL did not correlate with proteinuria in human or in mouse models. These data show that marked upregulation of NGAL accompanies HIVAN and support further study of uNGAL levels in large cohorts to aid in the noninvasive diagnosis of HIVAN and screen for HIVAN-related tubular damage.
Comment in
The development of urinary biomarkers for kidney disease is the search for our renal troponin. [J Am Soc Nephrol. 2009]
PMID:19628667 PMCID: PMC272398
Dev Cell. 2009 Jan;16(1):35-46. doi: 10.1016/j.devcel.2008.12.002.
Scara5 is a ferritin receptor mediating non-transferrin iron delivery.
Li JY1, Paragas N, Ned RM, Qiu A, Viltard M, Leete T, Drexler IR, Chen X, Sanna-Cherchi S, Mohammed F, Williams D, Lin CS, Schmidt-Ott KM, Andrews NC, Barasch J.
Abstract
Developing organs require iron for a myriad of functions, but embryos deleted of the major adult transport proteins, transferrin or its receptor transferrin receptor1 (TfR1(-/-)), still initiate organogenesis, suggesting that non-transferrin pathways are important. To examine these pathways, we developed chimeras composed of fluorescence-tagged TfR1(-/-) cells and untagged wild-type cells. In the kidney, TfR1(-/-) cells populated capsule and stroma, mesenchyme and nephron, but were underrepresented in ureteric bud tips. Consistently, TfR1 provided transferrin to the ureteric bud, but not to the capsule or the stroma. Instead of transferrin, we found that the capsule internalized ferritin. Since the capsule expressed a novel receptor called Scara5, we tested its role in ferritin uptake and found that Scara5 bound serum ferritin and then stimulated its endocytosis from the cell surface with consequent iron delivery. These data implicate cell type-specific mechanisms of iron traffic in organogenesis, which alternatively utilize transferrin or non-transferrin iron delivery pathways.
Comment in
Identification of ferritin receptors: their role in iron homeostasis, hepatic injury, and inflammation.[Gastroenterology. 2009]
A Tf-independent iron transport system required for organogenesis. [Dev Cell. 2009]
PMID: 19154717 PMCID: PMC2652503
Development. 2007 Sep;134(17):3177-90.
beta-catenin/TCF/Lef controls a differentiation-associated transcriptional program in renal epithelial progenitors.
Schmidt-Ott KM1, Masckauchan TN, Chen X, Hirsh BJ, Sarkar A, Yang J, Paragas N, Wallace VA, Dufort D, Pavlidis P, Jagla B, Kitajewski J, Barasch J.
Abstract
In the embryonic kidney, progenitors in the metanephric mesenchyme differentiate into specialized renal epithelia in a defined sequence characterized by the formation of cellular aggregates, conversion into polarized epithelia and segmentation along a proximal-distal axis. This sequence is reiterated throughout renal development to generate nephrons. Here, we identify global transcriptional programs associated with epithelial differentiation utilizing an organ culture model of rat metanephric mesenchymal differentiation, which recapitulates the hallmarks of epithelialization in vivo in a synchronized rather than reiterative fashion. We observe activation of multiple putative targets of beta-catenin/TCF/Lef-dependent transcription coinciding with epithelial differentiation. We show in cultured explants that isolated activation of beta-catenin signaling in epithelial progenitors induces, in a TCF/Lef-dependent manner, a subset of the transcripts associated with epithelialization, including Pax8, cyclin D1 (Ccnd1) and Emx2. This is associated with anti-apoptotic and proliferative effects in epithelial progenitors, whereas cells with impaired TCF/Lef-dependent transcription are progressively depleted from the epithelial lineage. In vivo, TCF/Lef-responsive genes comprise a conserved transcriptional program in differentiating renal epithelial progenitors and beta-catenin-containing transcriptional complexes directly bind to their promoter regions. Thus, beta-catenin/TCF/Lef-mediated transcriptional events control a subset of the differentiation-associated transcriptional program and thereby participate in maintenance, expansion and stage progression of the epithelial lineage.
PMID: 17693601
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Dev Biol. 2006 Nov 1;299(1):238-49. Epub 2006 Jul 31.
c-kit delineates a distinct domain of progenitors in the developing kidney.
Schmidt-Ott KM1, Chen X, Paragas N, Levinson RS, Mendelsohn CL, Barasch J.
Abstract
Early inductive events in mammalian nephrogenesis depend on an interaction between the ureteric bud and the metanephric mesenchyme. However, mounting evidence points towards an involvement of additional cell types--such as stromal cells and angioblasts--in growth and patterning of the nephron. In this study, through analysis of the stem cell factor (SCF)/c-kit ligand receptor pair, we describe an additional distinct cell population in the early developing kidney. While SCF is restricted to the ureteric bud, c-kit-positive cells are located within the renal interstitium, but are negative for Foxd1, an established marker of stromal cells. In fact, the c-kit-positive domain is continuous with a central mesodermal cell mass ventral and lateral to the dorsal aorta, while Foxd1-expressing stromal cells are continuous with a dorsal perisomitic cell population suggesting distinct intraembryonic origins for these cell types. A subset of c-kit-positive cells expresses Flk-1 and podocalyxin, suggesting that this cell population includes angioblasts and their progenitors. c-kit activation is not required for the survival of these cells in vivo, because white spotting (c-kit(W/W)) mice, carrying a natural inactivating mutation of c-kit, display normal intrarenal distribution of the c-kit-positive cells at E13.5. In addition, early kidney development in these mutants is preserved up to the stage when anemia compromises global embryonic development. In contrast, under defined conditions in organ cultures of metanephric kidneys, c-kit-positive cells, including the Flk-1-positive subset, undergo apoptosis after treatment with STI-571, an inhibitor of c-kit tyrosine phosphorylation. This is associated with reductions in ureteric bud branching and nephron number. Conversely, exogenous SCF expands the c-kit-positive population, including Flk-1-positive angioblasts, and accelerates kidney development in vitro. These data suggest that ureteric bud-derived SCF elicits growth-promoting effects in the metanephric kidney by expanding one or more components of the interstitial c-kit-positive progenitor pool.
PMID: 16942767
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Curr Opin Nephrol Hypertens. 2006 Jul;15(4):442-9.
Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia.
Schmidt-Ott KM1, Mori K, Kalandadze A, Li JY, Paragas N, Nicholas T, Devarajan P, Barasch J.
Abstract
PURPOSE OF REVIEW: Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily of carrier proteins. NGAL is the first known mammalian protein which specifically binds organic molecules called siderophores, which are high-affinity iron chelators. Here, we review the expression, siderophore-dependent biological activities and clinical significance of NGAL in epithelial development and in kidney disease. RECENT FINDINGS: NGAL expression is rapidly induced in the nephron in response to renal epithelial injury. This has led to the establishment of NGAL assays that detect renal damage in the human. Additionally, only when complexed with siderophore and iron as a trimer, NGAL induces mesenchymal-epithelial transition (or nephron formation) in embryonic kidney in vitro and protects adult kidney from ischemia-reperfusion injury in vivo. While the structure of the NGAL: siderophore: iron complex has thus far only been solved for bacterially synthesized siderophores, new evidence suggests the presence of mammalian siderophore-like molecules. SUMMARY: NGAL is rapidly and massively induced in renal epithelial injury and NGAL: siderophore: iron complexes may comprise a physiological renoprotective mechanism. The data have implications for the diagnosis and treatment of acute renal injury.
PMID: 16775460
J Am Soc Nephrol. 2005 Jul;16(7):1993-2002. Epub 2005 May 25.
Novel regulators of kidney development from the tips of the ureteric bud.
Schmidt-Ott KM1, Yang J, Chen X, Wang H, Paragas N, Mori K, Li JY, Lu B, Costantini F, Schiffer M, Bottinger E, Barasch J.
Abstract
Mammalian nephrogenesis depends on the interaction between the ureteric bud and the metanephric mesenchyme. As the ureteric bud undergoes branching and segmentation, the stalks differentiate into the collecting system of the mature kidney, while the tip cells interact with the adjacent cells of the metanephric mesenchyme, inducing their conversion into nephrons. This induction is mediated by secreted factors. For identifying novel mediators, the tips of the ureteric tree were isolated and microarray analyses were performed using manually refined, multistep gene ontology annotations. For identifying conserved factors, two databases were developed, one from mouse E12.5 and one from rat E13.5 ureteric buds. The overlap of mouse and rat data sets yielded 20 different transcripts that were enriched in the ureteric bud compared with metanephric mesenchyme and predicted to code for secreted proteins. Real-time reverse transcriptase-PCR and in situ hybridization confirmed these identifications. One of the genes that was highly specific to the ureteric bud tip was cytokine-like factor 1 (CLF-1). Recombinant CLF-1 in complex with its physiologic ligand, cardiotrophin-like cytokine (CLC), triggered phosphorylation of signal transducer and activator of transcription 3 in mesenchyme, a pathway characteristic of mesenchymal-to-epithelial conversion. Indeed, when applied to isolated rat metanephric mesenchyme, CLF-1/CLC (3 nM) induced mature nephron structures expressing glomerular and tubular markers. These results underline the power of this first comprehensive gene expression analysis of the ureteric bud tip to identify bioactive molecules.
PMID: 15917337
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Nat Genet. 2002 Sep;32(1):109-15. Epub 2002 Aug 26.
Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.
Batourina E1, Choi C, Paragas N, Bello N, Hensle T, Costantini FD, Schuchardt A, Bacallao RL, Mendelsohn CL.
Erratum in
Nat Genet 2002 Oct;32(2):331.
Abstract
Almost 1% of human infants are born with urogenital abnormalities, many of which are linked to irregular connections between the distal ureters and the bladder. During development, ureters migrate by an unknown mechanism from their initial integration site in the Wolffian ducts up to the base of the bladder in a process that we call ureter maturation. Rara(-/-) Rarb2(-/-) mice display impaired vitamin A signaling and develop syndromic urogenital malformations similar to those that occur in humans, including renal hypoplasia, hydronephrosis and mega-ureter, abnormalities also seen in mice with mutations in the proto-oncogene Ret. Here we show that ureter maturation depends on formation of the 'trigonal wedge', a newly identified epithelial outgrowth from the base of the Wolffian ducts, and that the distal ureter abnormalities seen in Rara(-/-) Rarb2(-/-) and Ret(-/-) mutant mice are probably caused by a failure of this process. Our studies indicate that formation of the trigonal wedge may be essential for correct insertion of the distal ureters into the bladder, and that these events are mediated by the vitamin A and Ret signaling pathways.
PMID: 12195422