The endogenous capacity of the kidney to repair is limited, and generation of new nephrons after injury for adequate function recovery remains a need. Discovery of factors that promote the endogenous ...regenerative capacity of the injured kidney or generation of transplantable kidney tissue represent promising therapeutic strategies. While several encouraging results are obtained after administration of stem or progenitor cells, stem cell secretome, or extracellular vesicles in experimental kidney injury models, very little data exist in the clinical setting to make conclusions about their efficacy. In this review, we provide an overview of the cutting-edge knowledge on kidney regeneration, including pre-clinical methodologies used to elucidate regenerative pathways and describe the perspectives of regenerative medicine for kidney patients.
Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, ...nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.
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•NEK8 acts with ATR at the replication fork to control fork dynamics and origin firing•NEK8 suppresses DNA damage by regulating S phase CDK activity•A ciliopathy-causing NEK8 mutant is defective in the replication stress response•Replication stress or loss of NEK8 perturbs ciliation and 3D renal cell architecture
We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the ...functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pluripotent stem cell‐derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off‐target cells and phenotypic alterations, preventing ...maturity. Here, various dynamic hydrogel architectures are created, conferring a controlled and biomimetic environment for organoid encapsulation. How hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers are investigated. The authors observe that stiff hydrogel encapsulation leads to an absence of certain renal cell types and signs of an epithelial–mesenchymal transition (EMT), whereas encapsulation in soft, stress‐relaxing hydrogels leads to all major renal segments, fewer fibrosis or EMT associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. The findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure–property–function relationships can enable the rational design of materials, bringing us closer to functional engraftments and disease‐modeling applications.
Current kidney organoid protocols often lead to off‐target cells and phenotypic alterations. Various dynamic hydrogel architectures are investigated for organoid encapsulation. Encapsulation in soft, fast stress‐relaxing hydrogels leads to all major renal segments, fewer fibrosis or epithelial‐mesenchymal transition (EMT) associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids.
Nephronophthisis-related ciliopathies (NPHP-RCs) are developmental and degenerative kidney diseases that are frequently associated with extrarenal pathologies such as retinal degeneration, obesity, ...and intellectual disability. We recently identified mutations in a gene encoding the centrosomal protein SDCCAG8 as causing NPHP type 10 in humans. To study the role of Sdccag8 in disease pathogenesis, we generated a Sdccag8 gene-trap mouse line. Homozygous Sdccag8(gt/gt) mice lacked the wild-type Sdccag8 transcript and protein, and recapitulated the human phenotypes of NPHP and retinal degeneration. These mice exhibited early onset retinal degeneration that was associated with rhodopsin mislocalization in the photoreceptors and reduced cone cell numbers, and led to progressive loss of vision. By contrast, renal histologic changes occurred later, and no global ciliary defects were observed in the kidneys. Instead, renal pathology was associated with elevated levels of DNA damage response signaling activity. Cell culture studies confirmed the aberrant activation of DNA damage response in Sdccag8(gt/gt)-derived cells, characterized by elevated levels of γH2AX and phosphorylated ATM and cell cycle profile abnormalities. Our analysis of Sdccag8(gt/gt) mice indicates that the pleiotropic phenotypes in these mice may arise through multiple tissue-specific disease mechanisms.
Ciliopathy nephronophthisis (NPHP), a common cause of end-stage renal disease (ESRD) in children and young adults, is characterized by disintegration of the tubular basement membrane accompanied by ...irregular thickening and attenuation, interstitial fibrosis and tubular atrophy, and occasionally cortico-medullary cyst formation. Pharmacological approaches that delay the development of ESRD could potentially extend the window of therapeutic opportunity for this group of patients, generating time to find an appropriate donor or even for new treatments to mature. In this review we provide an overview of compounds that have been tested to ameliorate kidney cysts and/or fibrosis. We also revisit paclitaxel as a potential strategy to target fibrosis in NPHP. At low dosage this chemotherapy drug shows promising results in rodent models of renal fibrosis. Possible adverse events and safety of paclitaxel treatment in pediatric patients would need to be investigated, as would the efficacy, optimum dose, and administration schedule for the treatment of renal fibrosis in NPHP patients. Paclitaxel is an approved drug for human use with known pharmacokinetics, which could potentially be used in other ciliopathies through targeting the microtubule skeleton.
Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the disease remains poorly understood, partly due to the lack of appropriate animal models. Joubert syndrome (JBTS) is an ...inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS that displays the kidney, eye, and brain abnormalities that define the syndrome. Mutant mice present with cystic kidney disease as neonates. Newborn kidneys contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt signaling pathway; however, an increase in the protein Gli3 repressor reveals abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct cells from mutant mice have abnormal primary cilia and are unable to form spheroid structures in vitro. Treatment of mutant cells with the Hh agonist purmorphamine restored normal spheroid formation. Renal epithelial cells from a JBTS patient with CEP290 mutations showed similar impairments to spheroid formation that could also be partially rescued by exogenous stimulation of Hh signaling. These data implicate abnormal Hh signaling as the cause of NPHP and suggest that Hh agonists may be exploited therapeutically.
Recent human genetic studies have suggested an intriguing link between ciliary signaling defects and altered DNA damage responses in nephronophthisis (NPH) and related ciliopathies. However, the ...molecular mechanism and the role of altered DNA damage response in kidney degeneration and fibrosis have remained elusive. We recently identified the kinase-regulated DNA damage response target Apoptosis Antagonizing Transcription Factor (AATF) as a master regulator of the p53 response. Here, we characterized the phenotype of mice with genetic deletion of Aatf in tubular epithelial cells. Mice were born without an overt phenotype, but gradually developed progressive kidney disease. Histology was notable for severe tubular atrophy and interstitial fibrosis as well as cysts at the corticomedullary junction, hallmarks of human nephronophthisis. Aatf deficiency caused ciliary defects as well as an accumulation of DNA double strand breaks. In addition to its role as a p53 effector, we found that AATF suppressed RNA:DNA hybrid (R loop) formation, a known cause of DNA double strand breaks, and enabled DNA double strand break repair in vitro. Genome-wide transcriptomic analysis of Aatf deficient tubular epithelial cells revealed several deregulated pathways that could contribute to the nephronophthisis phenotype, including alterations in the inflammatory response and anion transport. These results suggest that AATF is a regulator of primary cilia and a modulator of the DNA damage response, connecting two pathogenetic mechanisms in nephronophthisis and related ciliopathies.
Background
Epigenetic regulation has been suggested to be a link between environmental intrauterine exposures and development of asthma and allergy. The placenta is an essential part of the ...intrauterine environment. We have previously found the innate immune receptor CD14 to be differentially expressed on the mRNA level in placentas in relation to lifestyle and parental allergen sensitization. We here hypothesized that the promoter region of CD14 may be subject to differential DNA methylation and therefore a link between intrauterine exposure and mRNA expression.
Methods
Ninety‐four placentas from the ALADDIN (Assessment of Lifestyle and Allergic Disease During Infancy) study were investigated. We used methylation‐sensitive high‐resolution melting (MS‐HRM) analysis to semi‐quantitatively analyze the DNA methylation of the promoter region of CD14 in 36 placentas known to have different CD14 mRNA expression. EpiTYPER was used to validate the MS‐HRM data and to analyze an additional 58 placentas selected on mothers living on a farm or not.
Results
MS‐HRM analysis on 36 placenta samples revealed a relation between methylation of the CD14 promoter region with the level of CD14 mRNA expression. The MS‐HRM and EpiTYPER data correlated highly significantly. EpiTYPER analysis of the additional 58 placentas demonstrated that DNA methylation in the CD14 promoter was significantly lower in placentas of mothers living on a farm compared with mothers not living on a farm.
Conclusion
Our data suggest that epigenetic regulation of CD14 in placenta might be involved in the protective effect of ‘living on a farm’, with regard to allergy development.
Cilia are sensory organelles that project from the surface of almost all cells. Nephronophthisis (NPH) and NPH-related ciliopathies are degenerative genetic diseases caused by mutation of ...cilia-associated genes. These kidney disorders are characterized by progressive loss of functional tubular epithelial cells which is associated with inflammation, progressive fibrosis, and cyst formation, ultimately leading to end-stage renal disease. However, disease mechanisms remain poorly understood. Here, we show that targeted deletion of cilia in renal epithelial cells enhanced susceptibility to necroptotic cell death under inflammatory conditions. Treatment of non-ciliated cells with tumor necrosis factor (TNF) α and the SMAC mimetic birinapant resulted in Ripk1-dependent cell death, while viability of ciliated cells was almost not affected. Cell death could be enhanced and shifted toward necroptosis by the caspase inhibitor emricasan, which could be blocked by inhibitors of Ripk1 and Ripk3. Moreover, combined treatment of ciliated and non-ciliated cells with TNFα and cycloheximide induced a cell death response that could be partially rescued with emricasan in ciliated cells. In contrast, non-ciliated cells responded with pronounced cell death that was blocked by necroptosis inhibitors. Consistently, combined treatment with interferon-γ and emricasan induced cell death only in non-ciliated cells. Mechanistically, enhanced necroptosis induced by loss of cilia could be explained by induction of Ripk3 and increased abundance of autophagy components, including p62 and LC3 associated with the Ripk1/Ripk3 necrosome. Genetic ablation of cilia in renal tubular epithelial cells in mice resulted in TUNEL positivity and increased expression of Ripk3 in kidney tissue. Moreover, loss of Nphp1, the most frequent cause of NPH, further increased susceptibility to necroptosis in non-ciliated epithelial cells, suggesting that necroptosis might contribute to the pathogenesis of the disease. Together, these data provide a link between cilia-related signaling and cell death responses and shed new light on the disease pathogenesis of NPH-related ciliopathies.
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