Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease representing the most frequent genetic cause of end-stage kidney failure in children and adolescents. NPH is caused by genetic variants in >20 NPHP genes. Although nearly all NPHP genes encode ciliary proteins, classifying NPH as a renal ciliopathy, there is evidence for a pathogenic role of a compromised DNA damage response (DDR). Here, we present a novel Nphp7/Glis2-deficient mouse model with an early stop codon using CRISPR/Cas9-mediated genome editing (Glis2Y122X). Homozygous mice displayed dilated kidney tubules progressing to cystic kidney disease with significant fibrosis at a higher age. Interestingly, the kidneys of these animals exhibited an accumulation of DNA damage (DD) early on, even before any functional impairment of the kidneys became apparent. Interactome analysis for GLIS2 revealed an array of DDR-related proteins within the GLIS2 protein complex. Consistent with the in vivo data, the knockdown of Glis2 in kidney epithelial cells led to increased DNA damage. Moreover, supporting the role of GLIS2 in the DDR, we demonstrate that a substantial proportion of GLIS2 is present within the chromatin fraction of cells, which is further increased upon UV-induced DD. Live-cell imaging revealed the rapid recruitment of green fluorescent protein (GFP)-tagged GLIS2 to sites of laser-induced DD, a response diminished in Glis2Y122X and a variant of Glis2 resembling a known patient mutation. Overall, our data provide compelling evidence for the direct involvement of GLIS2 in the DDR, highlighting the loss of genome stability as an important factor contributing to the pathogenesis of renal ciliopathies.NEW & NOTEWORTHY Nephronophthisis (NPH) is a pediatric cystic kidney disease and ciliopathy. We present a novel Glis2/Nphp7-deficient mouse model that shows early accumulation of DNA damage before detectable kidney dysfunction. The GLIS2 protein complex includes DNA damage response factors. GLIS2 localizes to chromatin and rapidly relocates to sites of DNA damage. These findings position GLIS2 as a direct player in genome stability, highlighting impaired DDR as a key contributor to NPH pathogenesis.

Bardet-Biedl Syndrome (BBS) is a genetic disorder marked by considerable genetic and phenotypic diversity. BBS often presents as a combination of retinitis pigmentosa, obesity, polydactyly, and cystic kidney disease and is considered a model ciliopathy. The syndrome is caused by pathogenic variants in BBS genes, some of which encode components of a ciliary multi-protein complex, known as the BBSome, as well as a chaperonin-like complex, which is required for BBSome assembly. In this study, we describe the occurrence of kidney cysts in a BBS mouse model. Specifically, loss of BBS8 led to the development of cystic kidney disease by the end of the first year of life. In addition to transcriptional changes of key genes involved in regulated cell death and inflammation, proteomic approaches revealed increased expression and altered phosphorylation of histone deacetylase HDAC2 in knockout kidneys. Consistently, loss of Bbs8 resulted in a reduction of acetylated alpha-tubulin in primary cilia. This leads to diminished stability and altered dynamics of primary cilia, potentially contributing to the formation of cystic kidneys and other BBS manifestations previously described in Bbs8 deficient mice. ### Competing Interest Statement The authors have declared no competing interest.

Mycophenolate Mofetil (MMF) has an established role as a therapeutic agent in childhood nephrotic syndrome. While other immunosuppressants have been shown to positively affect podocytes, direct effects of MMF on podocytes remain largely unknown. The present study examines the effects of MMF’s active component Mycophenolic Acid (MPA) on the transcriptome of podocytes and investigates its biological significance. We performed transcriptomics in cultured murine podocytes exposed to MPA to generate hypotheses on podocyte-specific effects of MPA. Accordingly, we further analyzed biological MPA effects on actin cytoskeleton morphology after treatment with bovine serum albumin (BSA) by immunofluorescence staining, as well as on cell survival following exposure to TNF-α and cycloheximide by neutral red assay. MPA treatment significantly (adjusted p < 0.05) affected expression of 351 genes in podocytes. Gene Ontology term enrichment analysis particularly clustered terms related to actin and inflammation-related cell death. Indeed, quantification of the actin cytoskeleton of BSA treated podocytes revealed a significant increase of thickness and number of actin filaments after treatment with MPA. Further, MPA significantly reduced TNFα and cycloheximide induced cell death. MPA has a substantial effect on the transcriptome of podocytes in vitro, particularly including functional clusters related to non-immune cell dependent mechanisms. This may provide a molecular basis for direct beneficial effects of MPA on the structural integrity and survival of podocytes under pro-inflammatory conditions.

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM), and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function (LOF) is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 LOF resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Taken together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.

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.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome. Several studies have shown roles for PCDH19 in neuronal proliferation, migration, and synapse function, yet most of them have focused on cortical and hippocampal neurons. As epilepsy can also be caused by impaired interneuron migration, we studied the role of PCDH19 in cortical interneurons during embryogenesis. We show that cortical interneuron migration is affected by altering PCDH19 dosage by means of overexpression in brain slices and medial ganglionic eminence (MGE) explants. We also detect subtle defects when PCDH19 expression was reduced in MGE explants, suggesting that the dosage of PCDH19 is important for proper interneuron migration. We confirm this finding in vivo by showing a mild reduction in interneuron migration in heterozygote, but not in homozygote PCDH19 knockout animals. In addition, we provide evidence that subdomains of PCDH19 have a different impact on cell survival and interneuron migration. Intriguingly, we also observed domain-dependent differences in migration of the non-targeted cell population in explants, demonstrating a non-cell-autonomous effect of PCDH19 dosage changes. Overall, our findings suggest new roles for the extracellular and cytoplasmic domains of PCDH19 and support that cortical interneuron migration is dependent on balanced PCDH19 dosage.

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, cancer, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFAT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to pinpoint new HBP regulators. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFAT2 instead of the more common paralog GFAT1. Compared with GFAT1, GFAT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFAT2 activity was also observed in other mESCs and, consistently, the GFAT2/GFAT1 ratio decreased with differentiation of mouse and human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFAT2 for metabolite entry into the HBP.

Mammalian kidneys constantly filter large amounts of liquid, with almost complete retention of albumin and other macromolecules in the plasma. Breakdown of the three-layered renal filtration barrier results in loss of albumin into urine (albuminuria) across the wall of small renal capillaries, and is a leading cause of chronic kidney disease. However, exactly how the renal filter works and why its permeability is altered in kidney diseases is poorly understood. Here we show that the permeability of the renal filter is modulated through compression of the capillary wall. We collect morphometric data prior to and after onset of albuminuria in a mouse model equivalent to a human genetic disease affecting the renal filtration barrier. Combining quantitative analyses with mathematical modelling, we demonstrate that morphological alterations of the glomerular filtration barrier lead to reduced compressive forces that counteract filtration pressure, thereby resulting in capillary dilatation, and ultimately albuminuria. Our results reveal distinct functions of the different layers of the filtration barrier and expand the molecular understanding of defective renal filtration in chronic kidney disease.

Background: Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolarization and regeneration. Krüppel-like factor 4 (Klf4), a transcription factor regulating cell homeostasis and pluripotency, is highly expressed in human and mice during lung development and linked to lung growth arrest. Aim: To study (1) if hyperoxia-induced lung injury reduces Klf4 in ATII cells in mice; (2) if ATII-specific loss of Klf4 depletes number of ATII cells; (3) the function of Klf4 in ATII cells in vitro. Methods: (1) Newborn mice were exposed to 85% O2 (HYX) or room air (NOX). (2) Cell-specific deletion of Klf4 was induced in mice during late lung development. (3) Overexpression (Klf4OE) and ablation of Klf4 (Klf4del; CRISPR/Cas9) in murine lung epithelial cells (MLE-12). Results: (1) Dual immunofluorescent staining determined Klf4 in ATII cells during late murine lung development and in epithelial cells in the human fetal lung. Reduced ATII markers after HYX were linked to reduced Klf4 expression and lung growth. (2) ATII-specific Klf4 ablation during late lung development reduced ATII cells in mice. (3) In MLE-12, HYX decreased Klf4 expression and cell survival. Klf4OE and Klf4del in MLE-12 induced apoptosis and decreased epithelial cell markers. Klf4OE aggravated HYX-related decreased proliferation, activated Smad2 and blocked Stat3 signaling. Inhibition of Smad2, using an inhibitor, activated Stat3. Klf4del activated Stat3 and inhibited Smad2. Conclusion: We identified a novel Klf4-Smad2-Stat3 axis cell homeostasis that HYX disrupts in ATII cells and in lungs of newborn mice, suggesting Klf4 as potential target regulating lung regeneration.

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.

Lithium is the mainstay treatment for bipolar disorder. However, it leads to nephrogenic diabetes insipidus (NDI) and hypercalcemia in 10-20% of patients. Lithium-induced NDI is caused by the inability of the renal collecting duct to reabsorb water and correlates in C57BL/6 mice with the level of excreted prostaglandin E2 (PGE2). Lithium-induced hypercalcemia is ascribed to a decreased sensitivity of the parathyroid gland to blood Ca2+ levels. As lithium-induced NDI and hypercalcemia only occur in a subset of patients, we investigated whether these processes are linked by analyzing their development in 15 different inbred mouse strains. Starting at 10 weeks of age, female mice had access to normal chow or lithium diet (40mmol/kg). At day 8 and 26 mice were housed for 48 hours in metabolic cages to collect urine and at day 10 and 28 blood was isolated. Lithium treatment significantly elevated the urine production of several strains while other strains were not affected. Interestingly, in none of the strains lithium significantly affected urinary PGE2 levels. Furthermore, lithium did not significantly alter blood pH, while urinary proton excretion was elevated in several strains. Lithium-induced hypercalcemia was observed in 4 strains, and did not correlate with the effect of lithium on urine production. Our results demonstrate that development of lithium-induced NDI, hypercalcemia and excretion of PGE2 varies among inbred strains, indicating an independent development of these lithium-related features. Moreover, the diversity in response reveals that inclusion of additional strains may allow identification of susceptibility genes for these lithium-induced disorders.

Elucidating the pathogenesis of focal segmental glomerulosclerosis using CRISPR/Cas9 mediated genome editing