Modeling oxidative injury response in human kidney organoids.

BACKGROUND: Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. METHODS: To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. RESULTS: We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. CONCLUSION: This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

The Utility of Human Kidney Organoids in Modeling Kidney Disease.

The formation of three-dimensional kidney tissue (organoids) from human pluripotent stem cell lines provides a valuable tool to examine kidney function in an in vitro model and could be used for regenerative medicine approaches. Kidney organoids have the potential to model kidney diseases and congenital defects, be used for drug development, and to further our understanding of acute kidney injury, fibrosis, and chronic kidney disease. In this review, we examine the current stage of pluripotent stem cell-derived kidney organoid technology, challenges, shortcomings, and regenerative potential of kidney organoids in the future.

Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model.

BACKGROUND: Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn't clear. METHODS: We examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish. RESULTS: OCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment. CONCLUSIONS: Our data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

The human nephrin Y1139RSL motif is essential for podocyte foot process organization and slit diaphragm formation during glomerular development.

Nephrin is an immunoglobulin-type cell-adhesion molecule with a key role in the glomerular interpodocyte slit diaphragm. Mutations in the nephrin gene are associated with defects in the slit diaphragm, leading to early-onset nephrotic syndrome, typically resistant to treatment. Although the endocytic trafficking of nephrin is essential for the assembly of the slit diaphragm, nephrin's specific endocytic motifs remain unknown. To search for endocytic motifs, here we performed a multisequence alignment of nephrin and identified a canonical YXXØ-type motif, Y1139RSL, in the nephrin cytoplasmic tail, expressed only in primates. Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, a human podocyte line, and a human nephrin-expressing zebrafish model, we found that Y1139RSL is a novel endocytic motif and a structural element for clathrin-mediated nephrin endocytosis that functions as a phosphorylation-sensitive signal. We observed that Y1139RSL motif-mediated endocytosis helps to localize nephrin to specialized plasma membrane domains in podocytes and is essential for normal foot process organization into a functional slit diaphragm between neighboring foot processes in zebrafish. The importance of nephrin Y1139RSL for healthy podocyte development was supported by population-level analyses of genetic variations at this motif, revealing that such variations are very rare, suggesting that mutations in this motif have autosomal-recessive negative effects on kidney health. These findings expand our understanding of the mechanism underlying nephrin endocytosis and may lead to improved diagnostic tools or therapeutic strategies for managing early-onset, treatment-resistant nephrotic syndrome.

Enhancing regeneration after acute kidney injury by promoting cellular dedifferentiation in zebrafish.

Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified 4-(phenylthio)butanoic acid (PTBA), a histone deacetylase inhibitor (HDI), as an enhancer of renal recovery, and showed that PTBA treatment increased RTEC proliferation and reduced renal fibrosis. Here, we investigated the regenerative mechanisms of PTBA in zebrafish models of larval renal injury and adult cardiac injury. With respect to renal injury, we showed that delivery of PTBA using an esterified prodrug (UPHD25) increases the reactivation of the renal progenitor gene Pax2a, enhances dedifferentiation of RTECs, reduces Kidney injury molecule-1 (Kim-1) expression, and lowers the number of infiltrating macrophages. Further, we found that the effects of PTBA on RTEC proliferation depend upon retinoic acid signaling and demonstrate that the therapeutic properties of PTBA are not restricted to the kidney but also increase cardiomyocyte proliferation and decrease fibrosis following cardiac injury in adult zebrafish. These studies provide key mechanistic insights into how PTBA enhances tissue repair in models of acute injury and lay the groundwork for translating this novel HDI into the clinic.This article has an associated First Person interview with the joint first authors of the paper.

The role of macrophages during acute kidney injury: destruction and repair.

Acute kidney injury (AKI) is defined by a rapid decline in renal function. Regardless of the initial cause of injury, the influx of immune cells is a common theme during AKI. While an inflammatory response is critical for the initial control of injury, a prolonged response can negatively affect tissue repair. In this review, we focus on the role of macrophages, from early inflammation to resolution, during AKI. These cells serve as the innate defense system by phagocytosing cellular debris and pathogenic molecules and bridge communication with the adaptive immune system by acting as antigen-presenting cells and secreting cytokines. While many immune cells function to initiate inflammation, macrophages play a complex role throughout AKI. This complexity is driven by their functional plasticity: the ability to polarize from a "pro-inflammatory" phenotype to a "pro-reparative" phenotype. Importantly, experimental and translational studies indicate that macrophage polarization opens the possibility to generate novel therapeutics to promote repair during AKI. A thorough understanding of the biological roles these phagocytes play during both injury and repair is necessary to understand the limitations while furthering the therapeutic application.

The Lhx1-Ldb1 complex interacts with Furry to regulate microRNA expression during pronephric kidney development.

The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specification of the kidney, but few Lhx1 interacting proteins or downstream targets have been identified. By tandem-affinity purification, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the pair, and confirmed these microRNA clusters influence specification of the kidney. Therefore, our data shows that a constitutively-active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney field.

The 14-3-3 protein PAR-5 regulates the asymmetric localization of the LET-99 spindle positioning protein.

PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in Caenorhabditis elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization.

CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo.

Microtubule dynamics are thought to play an important role in regulating microtubule interactions with cortical force generating motor proteins that position the spindle during asymmetric cell division. CLASPs are microtubule-associated proteins that have a conserved role in regulating microtubule dynamics in diverse cell types. Caenorhabditis elegans has three CLASP homologs in its genome. CLS-2 is known to localize to kinetochores and is needed for chromosome segregation at meiosis and mitosis; however CLS-1 and CLS-3 have not been reported to have any role in embryonic development. Here, we show that depletion of CLS-2 in combination with either CLS-1 or CLS-3 results in defects in nuclear rotation, maintenance of spindle length, and spindle displacement in the one-cell embryo. Polarity is normal in these embryos, but reduced numbers of astral microtubules reach all regions of the cortex at the time of spindle positioning. Analysis of the microtubule plus-end tracker EB1 also revealed a reduced number of growing microtubules reaching the cortex in CLASP depleted embryos, but the polymerization rate of astral microtubules was not slower than in wild type. These results indicate that C. elegans CLASPs act partially redundantly to regulate astral microtubules and position the spindle during asymmetric cell division. Further, we show that these spindle pole-positioning roles are independent of the CLS-2 binding proteins HCP-1 and HCP-2.