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Journal of the American Society of... Apr 2023G protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with...
SIGNIFICANCE STATEMENT
G protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with salt-sensitive or essential hypertension, this association has been inconsistent among different study populations. In addition, studies elucidating how GRK4 may modulate cellular signaling are sparse. In an analysis of how GRK4 affects the developing kidney, the authors found that GRK4 modulates mammalian target of rapamycin (mTOR) signaling. Loss of GRK4 in embryonic zebrafish causes kidney dysfunction and glomerular cysts. Moreover, GRK4 depletion in zebrafish and cellular mammalian models results in elongated cilia. Rescue experiments suggest that hypertension in carriers of GRK4 variants may not be explained solely by kinase hyperactivity; instead, elevated mTOR signaling may be the underlying cause.
BACKGROUND
G protein-coupled receptor kinase 4 (GRK4) is considered a central regulator of blood pressure through phosphorylation of renal dopaminergic receptors and subsequent modulation of sodium excretion. Several nonsynonymous genetic variants of GRK4 have been only partially linked to hypertension, although these variants demonstrate elevated kinase activity. However, some evidence suggests that function of GRK4 variants may involve more than regulation of dopaminergic receptors alone. Little is known about the effects of GRK4 on cellular signaling, and it is also unclear whether or how altered GRK4 function might affect kidney development.
METHODS
To better understand the effect of GRK4 variants on the functionality of GRK4 and GRK4's actions in cellular signaling during kidney development, we studied zebrafish, human cells, and a murine kidney spheroid model.
RESULTS
Zebrafish depleted of Grk4 develop impaired glomerular filtration, generalized edema, glomerular cysts, pronephric dilatation, and expansion of kidney cilia. In human fibroblasts and in a kidney spheroid model, GRK4 knockdown produced elongated primary cilia. Reconstitution with human wild-type GRK4 partially rescues these phenotypes. We found that kinase activity is dispensable because kinase-dead GRK4 (altered GRK4 that cannot result in phosphorylation of the targeted protein) prevented cyst formation and restored normal ciliogenesis in all tested models. Hypertension-associated genetic variants of GRK4 fail to rescue any of the observed phenotypes, suggesting a receptor-independent mechanism. Instead, we discovered unrestrained mammalian target of rapamycin signaling as an underlying cause.
CONCLUSIONS
These findings identify GRK4 as novel regulator of cilia and of kidney development independent of GRK4's kinase function and provide evidence that the GRK4 variants believed to act as hyperactive kinases are dysfunctional for normal ciliogenesis.
Topics: Humans; Animals; Mice; Phosphorylation; Cilia; Zebrafish; Hypertension; Kidney; Sodium; TOR Serine-Threonine Kinases; Receptors, G-Protein-Coupled; Cysts; Mammals
PubMed: 36810260
DOI: 10.1681/ASN.0000000000000082 -
Computational and Structural... 2023Diabetic nephropathy (DN) is one of the most established microvascular complications of diabetes and a key cause of end-stage renal disease. It is well established that...
Diabetic nephropathy (DN) is one of the most established microvascular complications of diabetes and a key cause of end-stage renal disease. It is well established that gene susceptibility to DN plays a critical role in disease pathophysiology. Therefore, many genetic studies have been performed to categorize candidate genes in prominent diabetic cohorts, aiming to investigate DN pathogenesis and etiology. In this study, we performed a meta-analysis on the expression profiles of GSE1009, GSE30122, GSE96804, GSE99340, GSE104948, GSE104954, and GSE111154 to identify critical transcriptional factors associated with DN progression. The analysis was conducted for all individual datasets for each kidney tissue (glomerulus, tubules, and kidney cortex). We identified distinct clusters of susceptibility genes that were dysregulated in a renal compartment-specific pattern. Further, we recognized a small but a closely connected set of these susceptibility genes enriched for podocyte differentiation, several of which were characterized as genes encoding critical transcriptional factors (TFs) involved in DN development and podocyte function. To validate the role of identified TFs in DN progression, we functionally validated the three main TFs (DACH1, LMX1B, and WT1) identified through differential gene expression and network analysis using the hyperglycemic zebrafish model. We report that hyperglycemia-induced altered gene expression of the key TF genes leads to morphological abnormalities in zebrafish glomeruli, pronephric tubules, proximal and distal ducts. This study demonstrated that altered expression of these TF genes could be associated with hyperglycemia-induced nephropathy and, thus, aids in understanding the molecular drivers, essential genes, and pathways that trigger DN initiation and development.
PubMed: 36659918
DOI: 10.1016/j.csbj.2022.12.054 -
Kidney International Jan 2023Pronephric kidneys have a single large nephron that provides essential osmoregulation in amphibians and fish until the adult kidney forms. As mammalian kidneys evolved...
Pronephric kidneys have a single large nephron that provides essential osmoregulation in amphibians and fish until the adult kidney forms. As mammalian kidneys evolved from the simple pronephric kidneys of the early vertebrates, understanding the structure and function of pronephroi gives insight into the blueprints underlying all nephrons. The article in this issue by Corkins et al. uses single-cell sequencing to demonstrate an extraordinary segmental complexity and the organizational roadmap that mammalian nephrons are based upon.
Topics: Animals; Nephrons; Kidney; Pronephros; Mammals
PubMed: 36603975
DOI: 10.1016/j.kint.2022.09.013 -
Disease Models & Mechanisms Dec 2022Meckel syndrome, nephronophthisis, Joubert syndrome and Bardet-Biedl syndrome are caused by mutations in proteins that localize to the ciliary transition zone (TZ). The...
Meckel syndrome, nephronophthisis, Joubert syndrome and Bardet-Biedl syndrome are caused by mutations in proteins that localize to the ciliary transition zone (TZ). The phenotypically distinct syndromes suggest that these TZ proteins have differing functions. However, mutations in a single TZ gene can result in multiple syndromes, suggesting that the phenotype is influenced by modifier genes. We performed a comprehensive analysis of ten zebrafish TZ mutants, including mks1, tmem216, tmem67, rpgrip1l, cc2d2a, b9d2, cep290, tctn1, nphp1 and nphp4, as well as mutants in ift88 and ift172. Our data indicate that variations in phenotypes exist between different TZ mutants, supporting different tissue-specific functions of these TZ genes. Further, we observed phenotypic variations within progeny of a single TZ mutant, reminiscent of multiple disease syndromes being associated with mutations in one gene. In some mutants, the dynamics of the phenotype became complex with transitory phenotypes that are corrected over time. We also demonstrated that multiple-guide-derived CRISPR/Cas9 F0 'crispant' embryos recapitulate zygotic null phenotypes, and rapidly identified ciliary phenotypes in 11 cilia-associated gene candidates (ankfn1, ccdc65, cfap57, fhad1, nme7, pacrg, saxo2, c1orf194, ttc26, zmynd12 and cfap52).
Topics: Animals; Cilia; Zebrafish; Penetrance; Syndrome; Polycystic Kidney Diseases; Biological Variation, Population; Zebrafish Proteins; Vesicular Transport Proteins
PubMed: 36533556
DOI: 10.1242/dmm.049568 -
Journal of the American Society of... Mar 2023Mutations in hepatocyte nuclear factor-1 β ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram...
SIGNIFICANCE STATEMENT
Mutations in hepatocyte nuclear factor-1 β ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram fibroblasts to induced renal tubule epithelial cells (iRECs) and, as we demonstrate, can induce ectopic pronephric tissue in Xenopus ectodermal organoids. Using these two systems, we analyzed the effect of HNF1B mutations found in patients with cystic dysplastic kidney disease. We found cross-species conserved targets of HNF1B, identified transcripts that are differentially regulated by the patient-specific mutant protein, and functionally validated novel HNF1B targets in vivo . These results highlight evolutionarily conserved transcriptional mechanisms and provide insights into the genetic circuitry of nephrogenesis.
BACKGROUND
Hepatocyte nuclear factor-1 β (HNF1B) is an essential transcription factor during embryogenesis. Mutations in HNF1B are the most common monogenic causes of congenital cystic dysplastic renal malformations. The direct functional consequences of mutations in HNF1B on its transcriptional activity are unknown.
METHODS
Direct reprogramming of mouse fibroblasts to induced renal tubular epithelial cells was conducted both with wild-type HNF1B and with patient mutations. HNF1B was expressed in Xenopus ectodermal explants. Transcriptomic analysis by bulk RNA-Seq identified conserved targets with differentially regulated expression by the wild-type or R295C mutant. CRISPR/Cas9 genome editing in Xenopus embryos evaluated transcriptional targets in vivo .
RESULTS
HNF1B is essential for reprogramming mouse fibroblasts to induced renal tubular epithelial cells and induces development of ectopic renal organoids from pluripotent Xenopus cells. The mutation R295C retains reprogramming and inductive capacity but alters the expression of specific sets of downstream target genes instead of diminishing overall transcriptional activity of HNF1B. Surprisingly, targets associated with polycystic kidney disease were less affected than genes affected in congenital renal anomalies. Cross-species-conserved transcriptional targets were dysregulated in hnf1b CRISPR-depleted Xenopus embryos, confirming their dependence on hnf1b .
CONCLUSIONS
HNF1B activates an evolutionarily conserved program of target genes that disease-causing mutations selectively disrupt. These findings provide insights into the renal transcriptional network that controls nephrogenesis.
Topics: Animals; Mice; Hepatocyte Nuclear Factor 1-beta; Kidney; Kidney Diseases, Cystic; Mutation; Xenopus laevis
PubMed: 36522156
DOI: 10.1681/ASN.2022010076 -
The Journal of Physiology Jan 2023Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently...
Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently reported as an ion channel in neurons for mechanosensing and pain sensing, is also distributed in diverse non-neuronal tissues, such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in a complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes, we found that TACAN inhibits the channel activity of PKD2 gain-of-function mutant F604P. TACAN fragments containing the first and last transmembrane domains interacted with the PKD2 C- and N-terminal fragments, respectively. The TACAN N-terminus acted as a blocking peptide, and TACAN inhibited the function of PKD2 by the binding of PKD2 with TACAN. By patch clamping in mammalian cells, we found that TACAN inhibits both the single-channel conductance and the open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechanosensitivity of the PKD2-TACAN channel complex. KEY POINTS: TACAN inhibits the function of PKD2 in vitro and in vivo. TACAN N-terminal S1-containing fragment T160X interacts with the PKD2 C-terminal fragment N580-L700, and its C-terminal S6-containing fragment L296-D343 interacts with the PKD2 N-terminal A594X. TACAN inhibits the function of the PKD2 channel by physical interaction. The complex of PKD2 with TACAN, but not PKD2 alone, confers mechanosensitivity.
Topics: Animals; Zebrafish; TRPP Cation Channels; Ion Channels; Polycystic Kidney, Autosomal Dominant; Kidney; Mammals
PubMed: 36420836
DOI: 10.1113/JP283895 -
PloS One 2022E26 transformation specific (ETS) family transcription factors are expressed during embryogenesis and are involved in various cellular processes such as proliferation,...
E26 transformation specific (ETS) family transcription factors are expressed during embryogenesis and are involved in various cellular processes such as proliferation, migration, differentiation, angiogenesis, apoptosis, and survival of cellular lineages to ensure appropriate development. Dysregulated expression of many of the ETS family members is detected in different cancers. The human ELF3, a member of the ETS family of transcription factors, plays a role in the induction and progression of human cancers is well studied. However, little is known about the role of ELF3 in early development. Here, the zebrafish elf3 was cloned, and its expression was analyzed during zebrafish development. Zebrafish elf3 is maternally deposited. At different developmental stages, elf3 expression was detected in different tissue, mainly neural tissues, endoderm-derived tissues, cartilage, heart, pronephric duct, blood vessels, and notochord. The expression levels were high at the tissue boundaries. Elf3 loss-of-function consequences were examined by using translation blocking antisense morpholino oligonucleotides, and effects were validated using CRISPR/Cas9 knockdown. Elf3-knockdown produced short and bent larvae with notochord, craniofacial cartilage, and fin defects. The extracellular matrix (ECM) in the fin and notochord was disorganized. Neural defects were also observed. Optic nerve fasciculation (bundling) and arborization in the optic tectum were defective in Elf3-morphants, and fragmentation of spinal motor neurons were evident. Dysregulation of genes encoding ECM proteins and matrix metalloprotease (MMP) and disorganization of ECM may play a role in the observed defects in Elf3 morphants. We conclude that zebrafish Elf3 is required for epidermal, mesenchymal, and neural tissue development.
Topics: Animals; Humans; DNA-Binding Proteins; Extracellular Matrix; Gene Expression Regulation, Developmental; Morphogenesis; Proto-Oncogene Proteins c-ets; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 36383615
DOI: 10.1371/journal.pone.0276255 -
Journal of Medical Genetics Jun 2023is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy...
BACKGROUND
is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy number variations (CNVs) in have been associated with the neurodevelopmental disorder Stocco dos Santos syndrome, but not with congenital anomalies of the urinary tract and the visceral or the cardiovascular system.
METHODS
Here, exome sequencing and CNV analyses besides expression studies in zebrafish and mouse and (KD) experiments using a splice blocking morpholino in zebrafish were performed to study the role of during embryonic development.
RESULTS
In this study, we identified putative disease-causing SNVs and CNVs in in six individuals from four families with congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems (CNS). Embryonic mouse and zebrafish expression studies showed expression in the upper and lower urinary tract, the developing cloaca, the heart and the cerebral CNS. KD studies in zebrafish larvae revealed pronephric cysts, anomalies of the cloaca and the heart, decreased eye-to-head ratio and higher mortality compared with controls. These phenotypes could be rescued by co-injection of human wild-type mRNA and morpholino.
CONCLUSION
The identified SNVs and CNVs in affected individuals with congenital anomalies of the urinary tract, the anorectal, the cardiovascular and the central nervous systems, and subsequent embryonic mouse and zebrafish studies suggest as a developmental gene for different organ systems.
Topics: Pregnancy; Female; Humans; Animals; Mice; Zebrafish; DNA Copy Number Variations; Morpholinos; Urinary Tract; Central Nervous System; Cardiovascular System
PubMed: 36379543
DOI: 10.1136/jmg-2022-108738 -
Environmental Science and Pollution... Feb 2023The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any...
The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any defects in kidney development and function are likely associated with a decreased ability to osmoregulate. Previous work has shown that early-life stage (ELS) zebrafish (Danio rerio) acutely exposed to Deepwater Horizon (DWH) crude oil exhibit transcriptional changes in key genes involved in pronephros development and function, as well as pronephric morphological defects and whole-animal osmoregulatory impairment. The objective of this study was to examine the acute effects of crude oil exposure during zebrafish ELS on pronephros function by assessing its fluid clearance capacity and glomerular filtration integrity. Following a 72-h exposure to control conditions, 20% or 40% dilutions of high-energy water-accommodated fractions (HEWAF) of DWH crude oil, zebrafish were injected into the common cardinal vein either with fluorescein-labeled (FITC) 70-kDa dextran to assess glomerular filtration integrity or with FITC-inulin to assess pronephric clearance capacity. Fluorescence was quantified after the injections at predetermined time intervals by fluorescence microscopy. The results demonstrated a diminished pronephric fluid clearance capacity and failed glomerular perfusion when larvae were exposed to 40% HEWAF dilutions, whereas only a reduced glomerular filtration selectivity was observed in zebrafish previously exposed to the 20% HEWAF dilution.
Topics: Animals; Zebrafish; Petroleum; Kidney; Larva; Petroleum Pollution; Water Pollutants, Chemical
PubMed: 36280635
DOI: 10.1007/s11356-022-23805-z -
Developmental Biology Jan 2023Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field....
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 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 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.
Topics: Animals; Xenopus laevis; Tretinoin; Xenopus Proteins; Paired Box Transcription Factors; Gene Expression Regulation, Developmental; Pronephros; Kidney; Aldehyde Dehydrogenase 1 Family; Retinal Dehydrogenase
PubMed: 36279927
DOI: 10.1016/j.ydbio.2022.10.009