A novel nanoluciferase transgenic reporter measures proteinuria in zebrafish

Abstract

The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies. The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies. Translational StatementThe zebrafish has become a useful tool for modeling kidney disease. However, proteinuria, an important clinical indicator of kidney dysfunction, is not easily detected in the zebrafish system. Here, we describe a novel transgenic zebrafish line that measures proteinuria associated with both glomerular and tubular dysfunction. This new resource will impact future treatments for patients by permitting rapid and economical preclinical screening of drugs to treat kidney disease associated with glomerular or tubular dysfunction. The zebrafish has become a useful tool for modeling kidney disease. However, proteinuria, an important clinical indicator of kidney dysfunction, is not easily detected in the zebrafish system. Here, we describe a novel transgenic zebrafish line that measures proteinuria associated with both glomerular and tubular dysfunction. This new resource will impact future treatments for patients by permitting rapid and economical preclinical screening of drugs to treat kidney disease associated with glomerular or tubular dysfunction. Proteinuria is a key clinical indicator of kidney disease.1National Kidney FoundationK/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification.Am J Kidney Dis. 2002; 39: S1-266PubMed Google Scholar Using urine dipsticks, the presence of protein in the urine can be determined in seconds. Further evaluation of the molecular size of proteins in the urine indicates the site of functional deficit along the nephron; the presence of large proteins in the urine suggests dysfunction in the glomerular filter, whereas the presence of low-molecular-weight proteins indicates proximal tubule dysfunction.2Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (59) Google Scholar,3Lawrence M.G. Altenburg M.K. Sanford R. et al.Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.Proc Natl Acad Sci U S A. 2017; 114: 2958-2963Crossref PubMed Scopus (78) Google Scholar The zebrafish is a popular model organism for the study of kidney development and disease,4Outtandy P. Russell C. Kleta R. Bockenhauer D. Zebrafish as a model for kidney function and disease.Pediatr Nephrol. 2019; 34: 751-762Crossref PubMed Scopus (50) Google Scholar, 5Poureetezadi S.J. Wingert R.A. Little fish, big catch: zebrafish as a model for kidney disease.Kidney Int. 2016; 89: 1204-1210Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Morales E.E. Wingert R.A. Zebrafish as a model of kidney disease.Results Probl Cell Differ. 2017; 60: 55-75Crossref PubMed Scopus (43) Google Scholar but a quantitative reporter of proteinuria is currently lacking. Embryonic zebrafish develop a functioning pronephric kidney that has molecular and structural analogy to a human nephron.7Drummond I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar The pronephros consists of a single midline fused glomerulus attached to bilateral tubules that are segmented along their anterior-posterior axis into distinct proximal and distal domains.7Drummond I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar,8Wingert R.A. Selleck R. Yu J. et al.The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.PLoS Genet. 2007; 3: 1922-1938Crossref PubMed Scopus (247) Google Scholar Until recently, the best method of determining kidney function in zebrafish embryos was the use of fluorescent dextrans of varying molecular weights that distinguish between glomerular and proximal tubule dysfunction.9Christou-Savina S. Beales P.L. Osborn D.P.S. Evaluation of zebrafish kidney function using a fluorescent clearance assay.J Vis Exp. 2015; 96e52540Google Scholar However, fluorescent dextran is not specific to the cellular machinery of protein endocytosis; it is passively internalized in the fluid bulk. As such, these methods are useful, but they are not quantitative, do not specifically identify megalin-mediated endocytosis defects in the proximal tubule, and cannot be used in a high-throughput manner. Recently, a zebrafish transgenic reporter (fabp10a:½vdbp-mCherry) has enabled the embryo medium to be collected and assayed for low-molecular-weight proteins.10Chen Z. Luciani A. Mateos J.M. et al.Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases.Kidney Int. 2020; 97: 1150-1163Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar This powerful system permits the quantification of proteinuria as a readout of dysfunction of the lrp2/megalin endocytosis pathway in the proximal tubule. It reports proximal tubular dysfunction after downstream processing with immunofluorescence imaging or enzyme-linked immunosorbent assays to quantify protein uptake in the proximal tubules and proteinuria. This genetic tool highlights the potential of transgenics to develop the reporters of proteinuria in the zebrafish. Here, we describe a novel nanoluciferase (NL)-based reporter of proteinuria in zebrafish. Excreted NL protein in the embryo medium is detected by luminescence after simple addition of its substrate luciferin. With this new system, perturbations in both proximal tubular and glomerular function can be assayed, and its simple and easy application makes it amenable to use in a high-throughput manner. For detailed methods see Supplementary Methods. Zebrafish were maintained and staged according to established protocols11Kimmel C.B. Ballard W.W. Kimmel S.R. et al.Stages of embryonic development of the zebrafish.Dev Dyn. 1995; 203: 253-310Crossref PubMed Scopus (8997) Google Scholar and in accordance with the project licenses of Martin Lowe (70/9091) and Rachel Lennon (P1AE9A736) under the current guidelines of the UK Animals Act 1986. Embryos were injected at the stage indicated in the text and were treated with drugs/fluorescent tracers by either incubation in the embryo medium (after dechorionation) or microinjection into the common cardinal vein. For morpholino treatments, final concentrations of 0.15 mM (nphs1MOex25) and 0.25 mM (nphs2MOex3) were used. For lrp2a and ocrl knockdowns, a p53 morpholino was also coinjected at the same concentration (0.24 mM). CRISPR-Cas9 depletion is described in the paper by Wu et al.12Wu R.S. Lam I.I. Clay H. et al.A rapid method for directed gene knockout for screening in G0 zebrafish.Dev Cell. 2018; 46: 112-125.e4Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar gRNAs were resuspended in nuclease-free water to a concentration of 20 μM. For the injection mix, 4 μM of each gRNA was combined with Cas9 (NEB #M0646) and Cas9 buffer. Embryos were grown to 4 days post fertilization (dpf); then 3 embryos per well were placed in 1 well of a 96-well dish. The E3 embryo medium was removed and replaced with 200 μl of fresh E3. μl of E3 medium was removed each well and placed in the well of a fresh 96-well of substrate the was then to each were then at for 1 and then assayed for luminescence a 3 in zebrafish embryos was as C. in to zebrafish 3: PubMed Scopus Google Scholar were using for zebrafish col4a3, and were by 4 zebrafish embryos were a and were an zebrafish embryos were in or at 4 embryos were then in after in were then a and for The used were and were collected a using a The were as 1 and were collected using the with were according to described S. et the of glomerular 2016; PubMed Scopus Google Scholar were a were in for were and to the of the glomerular basement membrane The of for each control col4a4 and For per were used and the was The of along this was and then this was by the The was and a was using for to develop a transgenic reporter to study endocytosis in the zebrafish pronephric proximal tubule. important of in the proximal tubule is the which has H. and Cell 2002; 3: PubMed Scopus Google Scholar function and processing is H. H. et protein is important for processing of in kidney proximal Nephrol. PubMed Google Scholar which that with varying to M. et of the Full Text Full Text PDF PubMed Scopus Google Scholar For our reporter we fused protein to A a protein that is in size that the size of the reporter protein its the glomerular and we fused the D3 domain of protein to the of NL to which has a size of to that the NL-D3 protein For this we NL-D3 in and used to the protein The NL-D3 was then used in uptake a of the and the of 4 large was by the and measuring and this that the with NL-D3 of uptake with control with NL-D3 control luminescence after incubation with NL in that NL-D3 is by and To develop a in reporter of proximal tubule we to the zebrafish used the Tol2 gene and methods in zebrafish.Dev Dyn. PubMed Scopus Google Scholar to generate transgenic zebrafish that NL-D3 under the control of the liver-specific fabp10a promoter To the under the control of the promoter embryo transgenic zebrafish in luminescence embryos A in luminescence was in NL-D3 reporter fish also the of NL-D3 in with the luminescence in the that liver-specific of NL-D3 in transgenic zebrafish in the robust of this reporter into the of NL-D3 transgenic zebrafish and not impact fish or analysis of NL-D3 zebrafish and To NL-D3 the glomerular and is by the pathway in the pronephric proximal tubule, we lrp2a gene that for using a described morpholino S. et of in the zebrafish Cell PubMed Scopus Google Scholar of a fluorescent dextran in proximal tubules was as or of lrp2a was to uptake of a fluorescent dextran in proximal tubules when with that these embryos proximal tubule For the NL-D3 proteinuria lrp2a at 4 were placed in 1 well of a 96-well the embryo medium was and embryos were further NL-D3 in the embryo medium was a that 3 embryos per well in the luminescence with 1 embryo per well also luminescence of the embryo medium lrp2a after 1 4 and for each This that the NL-D3 reporter is to proteinuria after as as 1 but the between and was at we used this in our luminescence the were to NL-D3 using a NL-D3 of lrp2a an in NL-D3 in the embryo medium gene in proximal tubule endocytosis is and Lowe which is associated with low-molecular-weight D. A. et in Lowe a proximal tubular J Nephrol. 3: PubMed Scopus Google L. The in Lowe and disease Nephrol. 2017; PubMed Scopus Google Scholar of the zebrafish ocrl using a et development in a zebrafish model for Lowe Genet. PubMed Scopus Google et al.The Lowe protein is for endocytosis in the zebrafish pronephric Genet. 2015; PubMed Scopus Google Scholar a in fluorescent dextran uptake in the proximal tubule and induced NL-D3 proteinuria also the of that proximal tubules and proteinuria. and are drugs that are that kidney by proximal tubule and they used to the zebrafish proximal tubule L. et renal in a novel system to study a J PubMed Scopus Google Scholar The analysis of NL-D3 into the embryo medium that of proteinuria by with Treating embryos with the of further the proteinuria to that a is in the NL-D3 reporter. A was with a an in proteinuria, and a an these the NL-D3 transgenic fish as a tool for proteinuria to proximal tubular dysfunction after genetic or further the of the of glomerular specific genes to the NL-D3 reporter also be used to proteinuria associated with glomerular dysfunction. and for proteins and that the in these genes which is by proteinuria. depletion of and in zebrafish has in human patients with in and et and are between the zebrafish pronephros and PubMed Scopus Google S. I.A. of the pronephric filtration in zebrafish and the domain protein PubMed Scopus Google Scholar Using morpholino to used by et et and are between the zebrafish pronephros and PubMed Scopus Google Scholar we clearance of a dextran the and that these glomerular dysfunction. also NL-D3 in the embryo medium zebrafish depleted of either or with control that the NL-D3 reporter as a reporter of both glomerular and proximal tubular dysfunction analysis of glomerular development in the zebrafish pronephros suggests that a filtration is established at 4 Z. C. et of the pronephric glomerulus development in 2016; PubMed Scopus Google Scholar tubule this, with a and segmentation by post fertilization I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar,8Wingert R.A. Selleck R. Yu J. et al.The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.PLoS Genet. 2007; 3: 1922-1938Crossref PubMed Scopus (247) Google I.A. Zebrafish kidney Cell PubMed Scopus Google Scholar the NL-D3 reporter be used at of development to proteinuria associated with proximal tubule dysfunction and ocrl or glomerular dysfunction that proteinuria is in the reporter between and in lrp2a or ocrl of the same embryos between and a in proteinuria in lrp2a but embryos depleted of ocrl or not proteinuria that the NL-D3 line can proteinuria associated with tubular dysfunction but glomerular dysfunction and tubular dysfunction are not The potential for the NL-D3 reporter line to glomerular dysfunction is of given the of kidney associated with glomerular is Alport which is by in or in genes that to the which is to the The is to to the it to the by J. Alport its the glomerular filtration and for future Scopus Google Scholar, et renal and renal in with Alport Int. Full Text Full Text PDF PubMed Scopus Google Scholar, D. L. et gene to Alport glomerular disease.Kidney Int. Full Text Full Text PDF PubMed Scopus (54) Google Scholar in or to reduced or in the affecting its with Alport with by proteinuria and a in kidney function to kidney The of type in the zebrafish glomerulus has not Zebrafish type which are of the type genes in in to the of for in 4 zebrafish a when the glomerulus is Z. C. et of the pronephric glomerulus development in 2016; PubMed Scopus Google Scholar of and were which is given that type gene is for and R. M. et al.The structural genes for 1 and of human type are and an promoter Full Text PDF PubMed Google R. The genes for the and of human basement membrane type are and by a promoter of J. 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Clay H. et al.A rapid method for directed gene knockout for screening in G0 zebrafish.Dev Cell. 2018; 46: 112-125.e4Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar of in 4 gRNAs were not with this 1 gRNA in of the 3 in the site for of and gRNAs in it is that a of our and are of in Alport C. et of and of Alport 2020; PubMed Scopus Google Scholar analysis that for and col4a4 were reduced when each gene was using gRNAs analysis of the glomerular filtration by defects in the glomerular of the of and see Supplementary of that and col4a4 and was glomerular in human patients with Alport and knockout of Alport D. et a model for Alport PubMed Scopus Google and functional defects in of for Alport Cell PubMed Scopus Google Scholar to the and zebrafish model of proteinuria, which is a of Alport of or col4a4 by CRISPR-Cas9 in clearance of a dextran glomerular dysfunction in these determined or col4a4 proteinuria in our transgenic NL-D3 reporter This analysis that of either or col4a4 in zebrafish induced proteinuria for for col4a4 when with embryos In of either or col4a4 defects in the glomerular filtration and induced proteinuria, the of in Alport genes in both knockout and human that the zebrafish NL-D3 transgenic reporter line can be used for glomerular dysfunction proteinuria is a key clinical as in Alport we to glomerular filtration using to the potential for this system for col4a4 were used as a model of Alport for these In col4a4 captopril was used 3 to to and we assayed for proteinuria 4 to is an and has used in zebrafish to et in zebrafish is associated with and the pathway and J 2015; PubMed Scopus Google we that also that captopril zebrafish embryos not but proteinuria in col4a4 also used to the in zebrafish embryos 3 to that a to reduced glomerular thereby glomerular In zebrafish a analysis that at a concentration of μM was to of col4a4 with this concentration of proteinuria in these to a that was but col4a4 Using the NL-D3 reporter we also detected the of zebrafish is a that by that μM not proteinuria in NL-D3 μM in an in proteinuria Similarly, injection of or a in proteinuria in the NL-D3 reporter is a that injection water into the and has to glomerular S. et membrane defects in glomerular disease.Pediatr Nephrol. 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In of this, we clearance of a dextran In NL-D3 we detected an in luminescence is given in the clinical to the of a concentration of into zebrafish embryos was to glomerular an in clearance of both a dextran and NL-D3 proteinuria which was these that the NL-D3 reporter line can proteinuria associated with in and glomerular also the potential of this new tool in kidney function Here, we describe a new high-throughput tool for the quantification of proteinuria in the zebrafish that the NL-D3 transgenic reporter can in both glomerular and proximal tubular into the impact of and that in proteinuria can be the of our NL-D3 it is to in future and the NL-D3 reporter to with the that the reporter is a transgenic system, and in of the be To the impact of we used 3 embryos per well of 1 embryo per these measures the in embryos was of the The of the NL system also that embryos or are the embryo medium these not be To the impact of we also used 96-well to luminescence in also that the NL-D3 reporter is at tubular dysfunction by lrp2a depletion to NL-D3 proteinuria between and However, glomerular dysfunction by depletion of not in NL-D3 proteinuria at this This is to the glomerulus not 4 and we that the use of the NL-D3 reporter be to for glomerular studies. we not NL-D3 proteinuria in ocrl between and between 4 and This highlights the to at a of to that are when using the NL-D3 proteinuria reporter system. that the NL-D3 protein to the glomerular and be to be in the proximal tubule megalin-mediated However, the glomerulus also as a to NL-D3 given that we genetic or of glomerular filtration an in the presence of NL-D3 in the embryo medium. filtration of NL-D3 the glomerular function to which was to be to the filter, but has a A. H. of proteins in using J Google Scholar suggests that the and the of are important for the size of the glomerular L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (59) Google Scholar,3Lawrence M.G. Altenburg M.K. Sanford R. et al.Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.Proc Natl Acad Sci U S A. 2017; 114: 2958-2963Crossref PubMed Scopus (78) Google Scholar Thus, we that these of NL-D3 simple the glomerular in the healthy However, when we see in our of Alport the and are and NL-D3 is to the filter, the reabsorption in the tubules and to the proteinuria we In our we a new model of Alport that of the disease in human The in the glomerulus of Alport zebrafish were not as as in human this is to the embryos days depletion of or col4a4 in zebrafish is a model of the of Alport that and col4a4 are in the zebrafish glomerulus by in were to in the glomerulus by in which be to the as However, we cannot that zebrafish are distinct in their of this is as specific human and zebrafish when their are H. in the zebrafish the type 2007; PubMed Scopus Google Scholar their and of the human suggests that and are to with 1 A. et of the glomerular basement that the of is by the Full Text Full Text PDF PubMed Scopus Google Scholar will be to is in the pronephric glomerulus in zebrafish, and it is then an of which type in the pronephric at this stage into the of type and their in For this we used a gRNA to in R.S. Lam I.I. Clay H. et al.A rapid method for directed gene knockout for screening in G0 zebrafish.Dev Cell. 2018; 46: 112-125.e4Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar which is not to to each gene to 4 and the of However, the can to H. in the zebrafish the type 2007; PubMed Scopus Google Scholar and the of and col4a4 suggests that a of Alport zebrafish will be to and these be used to the in A line also be used for a to chemicals that ameliorate proteinuria associated with Alport In our the development of a reporter for proteinuria in the zebrafish system. the of this system in of and the feasibility of high-throughput we that this new proteinuria reporter will be a addition to the of kidney the The the in the for with zebrafish The used in this study were with and the of to and for their with the The also the in the in the of and for their and the for to the for the were in under the of Rachel with Supplementary

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