Assessment of MALDI matrices for the detection and visualization of phosphatidylinositols and phosphoinositides in mouse kidneys through matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI).

Phosphatidylinositols and their phosphorylated derivatives, known as phosphoinositides, are crucial in cellular processes, with their abnormalities linked to various diseases. Thus, identifying and measuring phosphoinositide levels in tissues are crucial for understanding their contributions to cellular processes and disease development. One powerful technique for mapping the spatial distribution of molecules in biological samples is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). This technique allows for the simultaneous detection and analysis of multiple lipid classes in situ, making it invaluable for unbiased lipidomic studies. However, detecting phosphoinositides with MALDI-MSI is challenging due to their relatively low abundance in tissues and complex matrix effects. Addressing this, our study focused on optimizing matrix selection and thickness for better detection of phosphatidylinositols and their phosphorylated forms in mouse kidney tissues. Various matrices were assessed, including 9AA, DAN, CMBT, and DHA, adjusting their coating to improve ionization efficiency. Our results demonstrate that DAN, DHA, and CMBT matrices produced high-intensity chemical images of phosphatidylinositol distributions within kidney sections. These matrices, particularly DAN, DHA, and CMBT, allowed the identification of even low-abundance phosphoinositides, through tentative identifications. Notably, DAN and DHA served as optimal candidates due to their prominent detection and ability to map a majority of phosphatidylinositol species, while CMBT showed potential detection capability for phosphatidylinositol triphosphate compounds. These findings not only provide valuable insights for future research on the involvement of phosphoinositides in kidney pathophysiology, but also propose the use of the identified optimal matrices, particularly DAN and DHA, as the preferred choices for enhanced detection and mapping of these lipid species in future studies.

Sublimation application of 5-chloro-2-mercaptobenzothiazole matrix for matrix-assisted laser desorption/ionization mass spectrometry imaging of mouse kidney.

RATIONALE: Sublimation is a solvent-free technique used to apply a uniform matrix coating over a large sample plate, improving the matrix's purity and enhancing the analyte signal. Although the 5-chloro-2-mercaptobenzothiazole (CMBT) matrix was introduced years ago, there are no reports of its application via sublimation. We investigated the experimental parameters that are optimal for CMBT matrix sublimation on mouse kidney samples. We also evaluated the stability of the sublimed CMBT matrix under a vacuum environment. Using kidney samples prepared with a sublimated CMBT matrix, we conducted matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) analysis of specific phospholipids (phosphatidylcholine and phosphatidylglycerol in the positive ion mode and phosphatidylinositol in the negative ion mode). We also explored various spatial resolutions (50, 20, and 10 µm) and performed sequential MALDI-hematoxylin and eosin (H&E) staining. METHODS: The CMBT matrix was applied to kidney samples using a sublimation apparatus connected to a vacuum pump to achieve a pressure of 0.05 Torr. The matrix was then subjected to different temperatures and sublimation times to determine the optimal conditions for matrix application. Subsequently, a Q-Exactive mass spectrometer equipped with a Spectroglyph MALDI ion source was employed for MALDI-MSI experiments. Standard protocols were followed for H&E staining after MALDI analysis. RESULTS: A matrix thickness of 0.15 mg/cm2 yielded high-quality images. The sublimated matrix exhibited minimal loss after approximately 20 h of exposure to a vacuum of 7 Torr, indicating its stability under these conditions. Ion images were successfully obtained at spatial resolutions of 50, 20, and 10 µm. Furthermore, orthogonal histological information was obtained through sequential MALDI-H&E staining. CONCLUSIONS: We demonstrate that samples prepared for MALDI-MSI using sublimation to apply the CMBT matrix yield high-quality mass spectrometric images of mouse kidney sections. We also provide data for the impact of various experimental parameters on image quality (e.g., temperature, time, matrix thickness, and spatial resolution).

Novel Fanconi renotubular syndromes provide insights in proximal tubule pathophysiology.

The various forms of Fanconi renotubular syndromes (FRTS) offer significant challenges for clinicians and present unique opportunities for scientists who study proximal tubule physiology. This review will describe the clinical characteristics, genetic underpinnings, and underlying pathophysiology of the major forms of FRST. Although the classic forms of FRTS will be presented (e.g., Dent disease or Lowe syndrome), particular attention will be paid to five of the most recently discovered FRTS subtypes caused by mutations in the genes encoding for L-arginine:glycine amidinotransferase (GATM), solute carrier family 34 (type Ii sodium/phosphate cotransporter), member 1 (SLC34A1), enoyl-CoAhydratase/3-hydroxyacyl CoA dehydrogenase (EHHADH), hepatocyte nuclear factor 4A (HNF4A), or NADH dehydrogenase complex I, assembly factor 6 (NDUFAF6). We will explore how mutations in these genes revealed unexpected mechanisms that led to compromised proximal tubule functions. We will also describe the inherent challenges associated with gene discovery studies based on findings derived from small, single-family studies by focusing the story of FRTS type 2 (SLC34A1). Finally, we will explain how extensive alternative splicing of HNF4A has resulted in confusion with mutation nomenclature for FRTS type 4.

Rho-GTPase Activating Protein myosin MYO9A identified as a novel candidate gene for monogenic focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a podocytopathy leading to kidney failure, whose molecular cause frequently remains unresolved. Here, we describe a rare MYO9A loss of function nonsense heterozygous mutation (p.Arg701∗) as a possible contributor to disease in a sibling pair with familial FSGS/proteinuria. MYO9A variants of uncertain significance were identified by whole exome sequencing in a cohort of 94 biopsy proven patients with FSGS. MYO9A is an unconventional myosin with a Rho-GAP domain that controls epithelial cell junction assembly, crosslinks and bundles actin and deactivates the small GTPase protein encoded by the RHOA gene. RhoA activity is associated with cytoskeleton regulation of actin stress fiber formation and actomyosin contractility. Myo9A was detected in mouse and human podocytes in vitro and in vivo. Knockin mice carrying the p.Arg701∗MYO9A (Myo9AR701X) generated by gene editing developed proteinuria, podocyte effacement and FSGS. Kidneys and podocytes from Myo9AR701X/+ mutant mice revealed Myo9A haploinsufficiency, increased RhoA activity, decreased Myo9A-actin-calmodulin interaction, impaired podocyte attachment and migration. Our results indicate that Myo9A is a novel component of the podocyte cytoskeletal apparatus that regulates RhoA activity and podocyte function. Thus, Myo9AR701X/+ knock-in mice recapitulate the proband FSGS phenotype, demonstrate that p.R701X Myo9A is an FSGS-causing mutation in mice and suggest that heterozygous loss-of-function MYO9A mutations may cause a novel form of human autosomal dominant FSGS. Hence, identification of MYO9A pathogenic variants in additional individuals with familial or sporadic FSGS is needed to ascertain the gene contribution to disease.

The pupil: a window on social automatic processing in autism spectrum disorder children.

BACKGROUND: Faces are crucial social stimuli, eliciting automatic processing associated with increased physiological arousal in observers. The level of arousal can be indexed by pupil diameter (the 'Event-Related Pupil Dilation', ERPD). However, many parameters could influence the arousal evoked by a face and its social saliency (e.g. virtual vs. real, neutral vs. emotional, static vs. dynamic). A few studies have shown an atypical ERPD in autism spectrum disorder (ASD) patients using several kinds of faces but no study has focused on identifying which parameter of the stimulus is the most interfering with face processing in ASD. METHODS: In order to disentangle the influence of these parameters, we propose an original paradigm including stimuli along an ecological social saliency gradient: from static objects to virtual faces to dynamic emotional faces. This strategy was applied to 186 children (78 ASD and 108 typically developing (TD) children) in two pupillometric studies (22 ASD and 47 TD children in the study 1 and 56 ASD and 61 TD children in the study 2). RESULTS: Strikingly, the ERPD in ASD children is insensitive to any of the parameters tested: the ERPD was similar for objects, static faces or dynamic faces. On the opposite, the ERPD in TD children is sensitive to all the parameters tested: the humanoid, biological, dynamic and emotional quality of the stimuli. Moreover, ERPD had a good discriminative power between ASD and TD children: ASD had a larger ERPD than TD in response to virtual faces, while TD had a larger ERPD than ASD for dynamic faces. CONCLUSIONS: This novel approach evidences an abnormal physiological adjustment to socially relevant stimuli in ASD.

SON haploinsufficiency causes impaired pre-mRNA splicing of CAKUT genes and heterogeneous renal phenotypes.

Although genetic testing is increasingly used in clinical nephrology, a large number of patients with congenital abnormalities of the kidney and urinary tract (CAKUT) remain undiagnosed with current gene panels. Therefore, careful curation of novel genetic findings is key to improving diagnostic yields. We recently described a novel intellectual disability syndrome caused by de novo heterozygous loss-of-function mutations in the gene encoding the splicing factor SON. Here, we show that many of these patients, including two previously unreported, exhibit a wide array of kidney abnormalities. Detailed phenotyping of 14 patients with SON haploinsufficiency identified kidney anomalies in 8 patients, including horseshoe kidney, unilateral renal hypoplasia, and renal cysts. Recurrent urinary tract infections, electrolyte disturbances, and hypertension were also observed in some patients. SON knockdown in kidney cell lines leads to abnormal pre-mRNA splicing, resulting in decreased expression of several established CAKUT genes. Furthermore, these molecular events were observed in patient-derived cells with SON haploinsufficiency. Taken together, our data suggest that the wide spectrum of phenotypes in patients with a pathogenic SON mutation is a consequence of impaired pre-mRNA splicing of several CAKUT genes. We propose that genetic testing panels designed to diagnose children with a kidney phenotype should include the SON gene.

Neonatal stroke and haematuria: Questions.

This is a report of an infant born near term with neonatal stroke and haematuria. Changes were noted on foetal magnetic resonance images, and these persisted postnatally. A routine renal ultrasound scan during follow-up detected haematuria with no associated proteinuria. A likely pathogenic genetic mutation was identified. This case highlights a relatively newly discovered cause for hereditary nephropathy affecting the basement membrane, initially affecting the glomerular but later the renal tubular basement membranes. The renal phenotype, pathogenic genotype and pathological findings on renal biopsy are discussed.

Neonatal stroke and haematuria: Answers.

BACKGROUND: This is a report of an infant born near term with neonatal stroke and haematuria. The renal phenotype, pathogenic genotype and pathological findings on renal biopsy are discussed. CASE-DIAGNOSIS: Prenatal magnetic resonance imaging revealed anomalies which persisted postnatally. Haematuria was detected during follow-up. The posttnatal renal ultrasound scan was normal, and there was no associated proteinuria. A likely pathogenic genetic mutation was detected. CONCLUSIONS: This case highlights a relatively newly discovered cause of hereditary nephropathy in which the basement membrane is affected, with initial effects on the glomerular membranes and subsequent effects on the renal tubular basement membranes.

The Phenotypic Spectrum of Nephropathies Associated with Mutations in Diacylglycerol Kinase ε.

The recent discovery of mutations in the gene encoding diacylglycerol kinase ε (DGKE) identified a novel pathophysiologic mechanism leading to HUS and/or MPGN. We report ten new patients from eight unrelated kindreds with DGKE nephropathy. We combined these cases with all previously published cases to characterize the phenotypic spectrum and outcomes of this new disease entity. Most patients presented with HUS accompanied by proteinuria, whereas a subset of patients exhibited clinical and histologic patterns of MPGN without TMA. We also report the first two patients with clinical and histologic HUS/MPGN overlap. DGKE-HUS typically manifested in the first year of life but was not exclusively limited to infancy, and viral triggers frequently preceded HUS episodes. We observed signs of complement activation in some patients with DGKE-HUS, but the role of complement activation remains unclear. Most patients developed a slowly progressive proteinuric nephropathy: 80% of patients did not have ESRD within 10 years of diagnosis. Many patients experienced HUS remission without specific treatment, and a few patients experienced HUS recurrence despite complete suppression of the complement pathway. Five patients received renal allografts, with no post-transplant recurrence reported. In conclusion, we did not observe a clear genotype-phenotype correlation in patients with DGKE nephropathy, suggesting additional factors mediating phenotypic heterogeneity. Furthermore, the benefits of anti-complement therapy are questionable but renal transplant may be a feasible option in the treatment of patients with this condition.

A variant in a cis-regulatory element enhances claudin-14 expression and is associated with pediatric-onset hypercalciuria and kidney stones.

The greatest risk factor for kidney stones is hypercalciuria, the etiology of which is largely unknown. A recent genome-wide association study (GWAS) linked hypercalciuria and kidney stones to a claudin-14 (CLDN14) risk haplotype. However, the underlying molecular mechanism was not delineated. Recently, renal CLDN14 expression was found to increase in response to increased plasma calcium, thereby inducing calciuria. We hypothesized therefore that some children with hypercalciuria and kidney stones harbor a CLDN14 variant that inappropriately increases gene expression. To test this hypothesis, we sequenced the CLDN14 risk haplotype in a cohort of children with idiopathic hypercalciuria and kidney stones. An intronic SNP was more frequent in affected children. Dual luciferase and cell-based assays demonstrated increased reporter or CLDN14 expression when this polymorphism was introduced. In silico studies predicted the SNP introduced a novel insulinoma-associated 1 (INSM1) transcription factor binding site. Consistent with this, repeating the dual luciferase assay in the presence of INSM1 further increased reporter expression. Our data suggest that children with the INSM1 binding site within the CLDN14 risk haplotype have a higher likelihood of hypercalciuria and kidney stones. Enhanced CLDN14 expression may play a role in the pathophysiology of their hypercalciuria.

Diacylglycerol kinase epsilon suppresses expression of p53 and glycerol kinase in mouse embryo fibroblasts.

The incorporation of glycerol into lipid was measured using SV40 transformed mouse embryo fibroblasts (MEFs) from either wild-type (WT) mice or from mice in which the epsilon isoform of diacylglycerol kinase (DGKε) was knocked out (DGKε-/-). We present an explanation for our finding that DGKε-/- MEFs exhibited greater uptake of 3H-glycerol into the cell and a greater incorporation into lipids compared with their WT counterparts, with no change in the relative amounts of various lipids between the DGKε-/- and WT MEFs. Glycerol kinase is more highly expressed in the DGKε-/- cells than in their WT counterparts. In addition, the activity of glycerol kinase is greater in the DGKε-/- cells than in their WT counterparts. Other substrates that enter the cell independent of glycerol kinase, such as pyruvate or acetate, are incorporated into lipid to the same extent between DGKε-/- and WT cell lines. We also show that expression of p53, a transcription factor that increases the synthesis of glycerol kinase, is increased in DGKε-/- MEFs in comparison to WT cells. We conclude that the increased incorporation of glycerol into lipids in DGKε-/- cells is a consequence of up-regulation of glycerol kinase and not a result of an increase in the rate of lipid synthesis. Furthermore, increased expression of the pro-survival gene, p53, in cells knocked out for DGKε suggests that cells over-expressing DGKε would have a greater propensity to become tumorigenic.

Gender identity disorder and autism spectrum disorder in a 23-year-old female.

We describe the case of a 23-year-old woman with Gender Identity Disorder (GID) asking for a cross-sex hormonal treatment with sex reassignment surgery and who was recently diagnosed with Autism Spectrum Disorder (ASD). Gender identity clinics are now reporting an overrepresentation of individuals with ASD among GID patients. The prevalence of ASD is 10-fold higher among GID patients than in general population. However, few case reports or studies have explored the co-occurrence of ASD and GID. This co-occurrence is relevant for diagnostic and clinical management and also raises important theoretical issues.

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

A Guide for Adult Nephrologists and Hematologists to Managing Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy in Teens Transitioning to Young Adults.

Atypical hemolytic uremic syndrome and C3 glomerulopathy/immune complex membranoproliferative glomerulonephritis are ultra-rare chronic, complement-mediated diseases with childhood manifestation in a majority of cases. Transition of clinical care of patients from pediatric to adult nephrologists-typically with controlled disease in native or transplant kidneys in case of atypical hemolytic uremic syndrome and often with chronic progressive disease despite treatment efforts in case of C3 glomerulopathy/immune complex membranoproliferative glomerulonephritis-identifies a challenging juncture in the journey of these patients. Raising awareness for the vulnerability of this patient cohort; providing education on disease pathophysiology and management including the use of new, high-precision complement antagonists; and establishing an ongoing dialog of patients, families, and all members of the health care team involved on either side of the age divide will be inevitable to ensure optimal patient outcomes and a safe transition of these patients to adulthood.

Inherited Kidney Complement Diseases.

In the past 20 years, we have witnessed tremendous advances in our ability to diagnose and treat genetic diseases of the kidney caused by complement dysregulation. Staggering progress was realized toward a better understanding of the genetic underpinnings and pathophysiology of many forms of atypical hemolytic uremic syndrome (aHUS) and C3-dominant glomerulopathies that are driven by complement system abnormalities. Many of these seminal discoveries paved the way for the design and characterization of several innovative therapies, some of which have already radically improved patients' outcomes. This review offers a broad overview of the exciting developments that have occurred in the recent past, with a particular focus on single-gene (or Mendelian), complement-driven aHUS and C3-dominant glomerulopathies that should be of interest to both nephrologists and kidney researchers. The discussion is restricted to genes with robust associations with both aHUS and C3-dominant glomerulopathies (complement factor H, complement component 3, complement factor H-related proteins) or only aHUS (complement factor B, complement factor I, and membrane cofactor protein). Key questions and challenges are highlighted, along with potential avenues for future directions.

The Phenotypic Spectrum of Tuberous Sclerosis Complex: A Canadian Cohort.

OBJECTIVE: We aimed to further elucidate the phenotypic spectrum of Tuberous Sclerosis Complex (TSC) depending on genotype. METHODS: A retrospective review of patients seen in the TSC clinic at the Hospital for Sick Children was conducted and the frequency of TSC manifestations was compared based on genotype. RESULTS: Nineteen-patients had TSC1 mutations, 36 had TSC2 mutations and 11 had no mutation identified (NMI). Patients with TSC2 mutations had a higher frequency of early-onset epilepsy and more frequent systemic manifestations. The NMI group had milder neurologic and systemic manifestations. Our data did not demonstrate that intellectual disability and infantile spasms were more common in TSC2 mutations. CONCLUSIONS: This is the first Canadian pediatric cohort exploring the genotype-phenotype relationship in TSC. We report that some manifestations are more frequent and severe in TSC2 mutations and that NMI may have a milder phenotype. Disease surveillance and counseling should continue regardless of genotype until this is better elucidated.

Canadian Association of Paediatric Nephrologists COVID-19 Rapid Response: Guidelines for Management of Acute Kidney Injury in Children.

PURPOSE: This article provides guidance on managing acute kidney injury (AKI) and kidney replacement therapy (KRT) in pediatrics during the COVID-19 pandemic in the Canadian context. It is adapted from recently published rapid guidelines on the management of AKI and KRT in adults, from the Canadian Society of Nephrology (CSN). The goal is to provide the best possible care for pediatric patients with kidney disease during the pandemic and ensure the health care team's safety. INFORMATION SOURCES: The Canadian Association of Paediatric Nephrologists (CAPN) COVID-19 Rapid Response team derived these rapid guidelines from the CSN consensus recommendations for adult patients with AKI. We have also consulted specific documents from other national and international agencies focused on pediatric kidney health. We identified additional information by reviewing the published academic literature relevant to pediatric AKI and KRT, including recent journal articles and preprints related to COVID-19 in children. Finally, our group also sought expert opinions from pediatric nephrologists across Canada. METHODS: The leadership of the CAPN, which is affiliated with the CSN, solicited a team of clinicians and researchers with expertise in pediatric AKI and acute KRT. The goal was to adapt the guidelines recently adopted for Canadian adult patients for pediatric-specific settings. These included specific COVID-19-related themes relevant to AKI and KRT in a Canadian setting, as determined by a group of kidney disease experts and leaders. An expert group of clinicians in pediatric AKI and acute KRT reviewed the revised pediatric guidelines. KEY FINDINGS: (1) Current Canadian data do not suggest an imminent threat of an increase in acute KRT needs in children because of COVID-19; however, close coordination between nephrology programs and critical care programs is crucial as the pandemic continues to evolve. (2) Pediatric centers should prepare to reallocate resources to adult centers as needed based on broader health care needs during the COVID-19 pandemic. (3) Specific suggestions pertinent to the optimal management of AKI and KRT in COVID-19 patients are provided. These suggestions include but are not limited to aspects of fluid management, KRT vascular access, and KRT modality choice. (4) Considerations to ensure adequate provision of KRT if resources become scarce during the COVID-19 pandemic. LIMITATIONS: We did not conduct a formal systematic review or meta-analysis. We did not evaluate our specific suggestions in the clinical environment. The local context, including how the provision of care for AKI and acute KRT is organized, may impede the implementation of many suggestions. As knowledge is advancing rapidly in the area of COVID-19, suggestions may become outdated quickly. Finally, most of the literature for AKI and KRT in COVID-19 comes from adult data, and there are few pediatric-specific studies. IMPLICATIONS: Given that most acute KRT related to COVID-19 is likely to be required in the pediatric intensive care unit initial setting, close collaboration and planning between critical care and pediatric nephrology programs are needed. Our group will update these suggestions with a supplement if necessary as newer evidence becomes available that may change or add to the recommendations provided.