The zebrafish paralog six2b is required for early proximal pronephros morphogenesis.

The transcription factor Six2 plays a crucial role in maintaining self-renewing nephron progenitor cap mesenchyme (CM) during metanephric kidney development. In mouse and human, expression at single-cell resolution has detected Six2 in cells as they leave the CM pool and differentiate. The role Six2 may play in these cells as they differentiate remains unknown. Here, we took advantage of the zebrafish pronephric kidney which forms directly from intermediate mesoderm to test six2b function during pronephric tubule development and differentiation. Expression of six2b during early zebrafish development was consistent with a role in pronephros formation. Using morpholino knock-down and CRISPR/Cas9 mutagenesis, we show a functional role for six2b in the development of proximal elements of the pronephros. By 48 h post-fertilization, six2b morphants and mutants showed disrupted pronephric tubule morphogenesis. We observed a lower-than-expected frequency of phenotypes in six2b stable genetic mutants suggesting compensation. Supporting this, we detected increased expression of six2a in six2b stable mutant embryos. To further confirm six2b function, F0 crispant embryos were analyzed and displayed similar phenotypes as morphants and stable mutants. Together our data suggests a conserved role for Six2 during nephrogenesis and a role in the morphogenesis of the proximal tubule.

Xenopus Ssbp2 is required for embryonic pronephros morphogenesis and terminal differentiation.

The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for kidney organogenesis. By tandem- affinity purification from kidney-induced Xenopus animal caps, we identified single-stranded DNA binding protein 2 (Ssbp2) interacts with the Ldb1-Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1-Lhx1-Ssbp transcriptional complexes in embryonic development.

Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3.

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 â€‹kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 â€‹kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

Histone Deacetylases Cooperate with NF-κB to Support the Immediate Migratory Response after Zebrafish Pronephros Injury.

Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability to recover spontaneously from AKI; however, little progress has been made in the development of supportive treatments. Increasing evidence suggest that histone deacetylases (HDAC) and NF-κB promote the pathogenesis of AKI, and inhibition of Hdac activity has a protective effect in murine models of AKI. However, the role of HDAC at the early stages of recovery is unknown. We used the zebrafish pronephros model to study the role of epigenetic modifiers in the immediate repair response after injury to the tubular epithelium. Using specific inhibitors, we found that the histone deacetylase Hdac2, Hdac6, and Hdac8 activities are required for the repair via collective cell migration. We found that hdac6, hdac8, and nfkbiaa expression levels were upregulated in the repairing epithelial cells shortly after injury. Depletion of hdac6, hdac8, or nfkbiaa with morpholino oligonucleotides impaired the repair process, whereas the combined depletion of all three genes synergistically suppressed the recovery process. Furthermore, time-lapse video microscopy revealed that the lamellipodia and filopodia formation in the flanking cells was strongly reduced in hdac6-depleted embryos. Our findings suggest that Hdac activity and NF-κB are synergistically required for the immediate repair response in the zebrafish pronephros model of AKI, and the timing of HDAC inhibition might be important in developing supportive protocols in the human disease.

Thiosulfate sulfurtransferase prevents hyperglycemic damage to the zebrafish pronephros in an experimental model for diabetes.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate ß-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.

Blood Flow Regulates Glomerular Capillary Formation in Zebrafish Pronephros.

Background: The renal glomerulus is a tuft of capillaries in Bowman's capsule and functions as a blood-filtration unit in the kidney. The unique glomerular capillary tuft structure is relatively conserved through vertebrate species. However, the morphogenetic mechanism governing glomerular capillary tuft formation remains elusive. Methods: To clarify how glomerular capillaries develop, we analyzed glomerular capillary formation in the zebrafish pronephros by exploiting fluorescence-based bio-imaging technology. Results: During glomerular capillary formation in the zebrafish pronephros, endothelial cells initially sprouted from the dorsal aorta and formed the capillaries surrounding the bilateral glomerular primordia in response to podocyte progenitor-derived vascular endothelial growth factor-A. After formation, blood flow immediately occurred in the glomerular primordia-associated capillaries, while in the absence of blood flow, they were transformed into sheet-like structures enveloping the glomerular primordia. Subsequently, blood flow induced formation of Bowman's space at the lateral sides of the bilateral glomerular primordia. Concomitantly, podocyte progenitors enveloped their surrounding capillaries while moving toward and coalescing at the midline. These capillaries then underwent extensive expansion and remodeling to establish a functional glomerular capillary tuft. However, stopping blood flow inhibited the remodeling of bilateral glomerular primordia, which therefore remained unvascularized but covered by the vascular sheets. Conclusions: We delineated the morphogenetic processes governing glomerular capillary tuft formation in the zebrafish pronephros and demonstrated crucial roles of blood flow in its formation. Blood flow maintains tubular structures of the capillaries surrounding the glomerular primordia and promotes glomerular incorporation of these vessels by inducing the remodeling of glomerular primordia.

Single-cell mRNA profiling reveals changes in solute carrier expression and suggests a metabolic switch during zebrafish pronephros development.

Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues and organs. Little is known about the adjustments that kidneys undergo in response to these challenges. We performed single-cell RNA sequencing of zebrafish pronephric duct cells to understand how the developing kidney responds to changes in filtered substrates and intrinsic energy requirements. We found high levels of glucose transporters early in development and increased expression of monocarboxylate transporters at later times. This indicates that the zebrafish embryonic kidney displays a high glucose transporting capacity during early development, which is replaced by the ability to absorb monocarboxylates and amino acids at later stages. This change in transport capacity was accompanied by the upregulation of mitochondrial carriers, indicating a switch to increased oxidative phosphorylation to meet the increasing energy demand of a developing kidney.NEW & NOTEWORTHY The zebrafish embryonic kidney has high levels of glucose transporters during early development, which are replaced by monocarboxylate and amino acid transporters later on. Inhibition of Na+-glucose cotransporter-dependent glucose transport by sotagliflozin also increased slc2a1a expression, supporting the idea that the glucose transport capacity is dynamically adjusted during zebrafish pronephros development. Concurrent upregulation of mitochondrial SCL25 transporters at later stages supports the idea that the pronephros adjusts to changing substrate supplies and/or energy demands during embryonic development.

Mutations in PRDM15 Are a Novel Cause of Galloway-Mowat Syndrome.

BACKGROUND: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. METHODS: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. RESULTS: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. CONCLUSIONS: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.

Cadmium exposure induces pyroptosis of lymphocytes in carp pronephros and spleens by activating NLRP3.

Cadmium (Cd) is a type of toxic metal, in most cases, coming from fuel burning and aquatic plants. The cells of organisms can be caused serious damage, including pyroptosis, exposure to low concentrations of Cd in long-term. Pyroptosis is a recently discovered Caspase-1-mediated cell death. In this study, lymphocytes were extracted from the pronephros and spleens in carps, respectively. After treating cells with low concentration of Cd, the mRNA and protein expression levels of pyroptosis-related genes, NLRP3, Caspase-1, and pro-inflammatory cytokines, increased obviously. And the content of reactive oxygen species (ROS) and mitochondria reactive oxygen species (mtROS) increased significantly, we also found the activities of CAT, GSH-px and T-SOD reduce significantly, and the content of MDA have a clear upward trend. We then added NLRP3 inhibitor, Glyburide, to the Cd-treated group, further confirming that NLRP3 is a key gene in pyroptosis pathways by detecting the mRNA and protein expression levels. Besides, the rupture of the cell membrane was also confirmed by Hoechst/PI double staining, red fluorescence increased obviously in the Cd treatment group. The experiment revealed that Cd exposure induces pyroptosis of lymphocytes in carp pronephros and spleens by activating NLRP3. Inhibition of NLRP3 activity can slow down the degree of lymphocytes pyroptosis. Thus, the above information provides a new avenue toward understanding the partial mechanism of Cd exposure-induced pyroptosis.

Claudin-7b and Claudin-h are required for controlling cilia morphogenesis in the zebrafish kidney.

Claudins are a family of proteins which are the most important components of the tight junctions. The location of Claudins on the renal tubule epithelial determines its paracellular transport characteristics, but whether Claudins have other functions in kidneys remains still unclear. Here, we showed that the transcripts encoding two Claudin family proteins, claudin-7b (cldn-7b) and claudin-h (cldn-h), were expressed in the transporting cells in the zebrafish pronephros. By knocking down of cldn-7b and cldn-h in zebrafish, we showed that these claudins morphants exhibited cystic kidneys accompanied with body curvature. Further analysis showed that down regulation of cldn-7b or cldn-h led to multiple defects in apico-basolateral polarity, cilia morphology and ciliary function in kidney. Moreover, the ciliary defect was confirmed by depletion of Cldn-7b or Cldn-h using CRISPR/Cas9 system. We also showed that both cldn-7b and cldn-h were genetically interacted with a well-known ciliary gene, arl13b. Deletion of arl13b led to curly cilia in the pronephros that phenocopied with cldn-7b and cldn-h morphants. Taken together, our data suggested that the tight junction protein, Cldn-7b and Cldn-h, regulate kidney development and function by affecting cilia morphology.

A Technique for Studying Glomerular Filtration Integrity in the Zebrafish Pronephros.

With the advances in next-generation sequencing and rapid filtering of candidate variants in diseased patients, it has been increasingly important to develop translatable in vivo models to study genetic changes. This allows for functional validation of pathogenic mutations and establishes a system to understand the etiology of disease. Due to the ease of genetic manipulation and rapid ex utero development, the zebrafish has become a valuable resource to study important biological processes, including nephrogenesis. The development and function of the zebrafish pronephros are akin to that of mammals. As such, they offer a tractable model to study kidney disease, especially diabetic nephropathy. However, in order to study kidney dysfunction in zebrafish it is imperative that an appropriate readout is available. The appearance of macro-proteins in patient's urine is indicative of defective kidney function. In this technical chapter, we describe the in vivo use of fluorescently tagged dextrans of different molecular weights to reveal the integrity of the zebrafish glomerular filtration barrier.

Visualizing gene expression during zebrafish pronephros development and regeneration.

The vertebrate kidney is comprised of functional units known as nephrons. Defects in nephron development or activity are a common feature of kidney disease. Current medical treatments are unable to ameliorate the dire consequences of nephron deficit or injury. Although there have been tremendous advancements in our understanding of nephron ontogeny and the response to damage, many significant knowledge gaps still remain. The zebrafish embryo kidney, or pronephros, is an ideal model for many renal development and regeneration studies because it is comprised of nephrons that share conserved features with the nephron units that comprise the mammalian metanephric kidney. In this chapter, we provide an overview about the benefits of using the zebrafish pronephros to study the mechanisms underlying nephrogenesis as well as epithelial repair and regeneration. We subsequently detail methods for the spatiotemporal assessment of gene and protein expression in zebrafish embryos that can be used to extend the understanding of nephron development and disease, and thereby create new opportunities to identify therapeutic strategies for regenerative medicine.

Genetic compensation by epob in pronephros development in epoa mutant zebrafish.

Zebrafish erythropoietin a (epoa) is a well characterized regulator of red blood cell formation. Recent morpholino mediated knockdown data have also identified epoa being essential for physiological pronephros development in zebrafish, which is driven by blocking apoptosis in developing kidneys. Yet, zebrafish mutants for epoa have not been described so far. In order to compare a transient knockdown vs. permanent knockout for epoa in zebrafish on pronephros development, we used CRISPR/Cas9 technology to generate epoa knockout zebrafish mutants and we performed structural and functional studies on pronephros development. In contrast to epoa morphants, epoa-/- zebrafish mutants showed normal pronephros structure; however, a previously uncharacterized gene in zebrafish, named epob, was identified and upregulated in epoa-/- mutants. epob knockdown altered pronephros development, which was further aggravated in epoa-/- mutants. Likewise, epoa and epob morphants regulated similar and differential gene signatures related to kidney development in zebrafish. In conclusion, stable loss of epoa during embryonic development can be compensated by epob leading to phenotypical discrepancies in epoa knockdown and knockout zebrafish embryos.

Tfap2a is a novel gatekeeper of nephron differentiation during kidney development.

Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular compartments with discrete physiological identities. Here, from a forward genetic screen using zebrafish, we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that activates the terminal differentiation program of distal segments in the pronephros. We found that tfap2a acts downstream of Iroquois homeobox 3b (irx3b), a distal lineage transcription factor, to operate a circuit consisting of tfap2b, irx1a and genes encoding solute transporters that dictate the specialized metabolic functions of distal nephron segments. Interestingly, this regulatory node is distinct from other checkpoints of differentiation, such as polarity establishment and ciliogenesis. Thus, our studies reveal insights into the genetic control of differentiation, where tfap2a is essential for regulating a suite of segment transporter traits at the final tier of zebrafish pronephros ontogeny. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to facilitate drug discovery and regenerative therapies.

Investigating the RAS can be a fishy business: interdisciplinary opportunities using Zebrafish.

The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since the discovery of renin, its biological role continues to broaden with the identification and characterization of new peptides. The evolutionarily distant zebrafish is a remarkable model for studying the kidney due to its genetic tractability and accessibility for in vivo imaging. The zebrafish pronephros is an especially useful kidney model due to its structural simplicity yet complex functionality, including capacity for glomerular and tubular filtration. Both the pronephros and mesonephros contain renin-expressing perivascular cells, which respond to RAS inhibition, making the zebrafish an excellent model for studying the RAS. This review summarizes the physiological and genetic tools currently available for studying the zebrafish kidney with regards to functionality of the RAS, using novel imaging techniques such as SPIM microscopy coupled with targeted single cell ablation and synthesis of vasoactive RAS peptides.

Homeogene emx1 is required for nephron distal segment development in zebrafish.

Vertebrate kidneys contain nephron functional units where specialized epithelial cell types are organized into segments with discrete physiological roles. Many gaps remain in our understanding of how segment regions develop. Here, we report that the transcription factor empty spiracles homeobox gene 1 (emx1) is a novel nephron segment regulator during embryonic kidney development in zebrafish. emx1 loss of function altered the domains of distal segments without changes in cell turnover or traits like size and morphology, indicating that emx1 directs distal segment fates during nephrogenesis. In exploring how emx1 influences nephron patterning, we found that retinoic acid (RA), a morphogen that induces proximal and represses distal segments, negatively regulates emx1 expression. Next, through a series of genetic studies, we found that emx1 acts downstream of a cascade involving mecom and tbx2b, which encode essential distal segment transcription factors. Finally, we determined that emx1 regulates the expression domains of irx3b and irx1a to control distal segmentation, and sim1a to control corpuscle of Stannius formation. Taken together, our work reveals for the first time that emx1 is a key component of the pronephros segmentation network, which has implications for understanding the genetic regulatory cascades that orchestrate vertebrate nephron patterning.

ppargc1a controls nephron segmentation during zebrafish embryonic kidney ontogeny.

Nephron segmentation involves a concert of genetic and molecular signals that are not fully understood. Through a chemical screen, we discovered that alteration of peroxisome proliferator-activated receptor (PPAR) signaling disrupts nephron segmentation in the zebrafish embryonic kidney (Poureetezadi et al., 2016). Here, we show that the PPAR co-activator ppargc1a directs renal progenitor fate. ppargc1a mutants form a small distal late (DL) segment and an expanded proximal straight tubule (PST) segment. ppargc1a promotes DL fate by regulating the transcription factor tbx2b, and restricts expression of the transcription factor sim1a to inhibit PST fate. Interestingly, sim1a restricts ppargc1a expression to promote the PST, and PST development is fully restored in ppargc1a/sim1a-deficient embryos, suggesting Ppargc1a and Sim1a counterbalance each other in an antagonistic fashion to delineate the PST segment boundary during nephrogenesis. Taken together, our data reveal new roles for Ppargc1a during development, which have implications for understanding renal birth defects.

EphA7 regulates claudin6 and pronephros development in Xenopus.

Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we studied the roles of the Eph receptor EphA7 and its soluble form in Xenopus pronephros development. EphA7 is specifically expressed in pronephric tubules at tadpole stages and knockdown of EphA7 by a translation blocking morpholino led to defects in tubule cell differentiation and morphogenesis. A soluble form of EphA7 (sEphA7) was also identified. Interestingly, the membrane level of claudin6 (CLDN6), a tetraspan transmembrane tight junction protein, was dramatically reduced in the translation blocking morpholino injected embryos, but not when a splicing morpholino was used, which blocks only the full length EphA7. In cultured cells, EphA7 binds and phosphorylates CLDN6, and reduces its distribution at the cell surface. Our work suggests a role of EphA7 in the regulation of cell adhesion during pronephros development, whereas sEphA7 works as an antagonist.

Peroxiredoxin1, a novel regulator of pronephros development, influences retinoic acid and Wnt signaling by controlling ROS levels.

Peroxiredoxin1 (Prdx1) is an antioxidant enzyme belonging to the peroxiredoxin family of proteins. Prdx1 catalyzes the reduction of H2O2 and alkyl hydroperoxide and plays an important role in different biological processes. Prdx1 also participates in various age-related diseases and cancers. In this study, we investigated the role of Prdx1 in pronephros development during embryogenesis. Prdx1 knockdown markedly inhibited proximal tubule formation in the pronephros and significantly increased the cellular levels of reactive oxygen species (ROS), which impaired primary cilia formation. Additionally, treatment with ROS (H2O2) severely disrupted proximal tubule formation, whereas Prdx1 overexpression reversed the ROS-mediated inhibition in proximal tubule formation. Epistatic analysis revealed that Prdx1 has a crucial role in retinoic acid and Wnt signaling pathways during pronephrogenesis. In conclusion, Prdx1 facilitates proximal tubule formation during pronephrogenesis by regulating ROS levels.

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to "dial" the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.