Kidney Glomeruli Research Articles

Renal toxicity of methylprednisolone in male Wistar rats and the potential protective effect by boldine supplementation

ObjectiveIntravenous corticosteroids, methylprednisolone (MPL), which is one of the drugs that induce glomerular kidney diseases, when used for a long time at high dose by doctors to treat acute renal inflammation, promote the development of reactive oxygen species (ROS)-induced renal toxicity. This study investigated the role of boldine, a natural antioxidant with anti-apoptotic and anti-inflammatory properties, against MPL-induced renal toxicity in male Wistar rats. Methods120 rats were divided into eight equal groups as follows: G1 (control), G2, 3, and 4 (rats orally administered 5, 10, and 50 mg boldine/kg/day; respectively, for 28 days), G5 (rats intramuscularly injected with 100 mg MPL/kg only on the last three days), G6, 7, and 8 (rats administered boldine + MPL). After the last MPL injection, rats were sacrificed at intervals of 1, 24, and 48 h. ResultsThere was a significant increase in body weight, blood glucose, and urea levels, as well as a decrease in albumin level in serum. Oxidative stress markers levels increased at all times, and gene expression of anti-oxidant enzymes increased at 24 h. According to immunohistochemistry, there was a considerable increase in P53 and TNF-α levels following MPL therapy. The cell death DNA assays revealed detectable necrosis and apoptosis, induced by MPL treatment. ConclusionsOral administration of boldine has a modulatory protective, anti-oxidant, anti-apoptotic, and anti-inflammatory effect against free radicals.

Mobilization of CD11b+/Ly6chi monocytes causes multi organ dysfunction syndrome in acute pancreatitis.

ObjectiveAcute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with multi-organ dysfunction and high mortality. The pathogenesis of life –threatening organ complications, such as respiratory and renal failure, is unknown.DesignOrgan dysfunction was investigated in a mouse model of AP. The influence of monocytes and neutrophils on multi organ dysfunction syndrome (MODS) was investigated in vivo by antibody depletion. Using real-time-fluorescence and deformability-cytometry (RT-DC) analysis we determined the mechanical properties of neutrophils and monocytes during AP. Furthermore, blood samples of pancreatitis patients were used to characterize severity-dependent chemokine profiles according to the revised Atlanta classification.ResultsSimilar to AP in humans, severe disease in the mouse model associates with organ dysfunction mainly of lung and kidney, which is triggered by a mobilisation of Ly6g-/CD11b+/Ly6c hi monocytes, but not of Ly6g+/CD11b+ neutrophils. Monocyte depletion by anti-CCR2 antibody treatment ameliorated lung function (oxygen consumption) without interfering with the systemic immune response. RT-DC analysis of circulation monocytes showed a significant increase in cell size during SAP, but without a compensatory increase in elasticity. Patient chemokine profiles show a correlation of AP severity with monocyte attracting chemokines like MCP-1 or MIG and with leukocyte mobilisation.ConclusionIn AP, the physical properties of mobilized monocytes, especially their large size, result in an obstruction of the fine capillary systems of the lung and of the kidney glomeruli. A selective depletion of monocytes may represent a treatment strategy for pancreatitis as well as for other inflammation-related disorders.

Mechanism of Chlorpyrifos Induced Chronic Nephrotoxicity

INTRODUCTION: Chlorpyrifos is one of the most commonly used organophosphates in agriculture. Report on animal models of chronically exposed chlorpyrifos showed significant histo-structural renal damage. Moreover, evidence of high incidence of diabetes mellitus were observed among agricultural workers exposed to organophosphates. Available evidence linking derangement of glucose metabolism pathway with renal damage in this chronically exposed chlorpyrifos is lacking. The aim of this study was to determine the involvement of advanced glycation end-products formation in the development of nephrotoxicity in animals with chronic subcutaneous exposure to chlorpyrifos. MATERIALS AND METHODS: Eighteen rats were divided into three groups, with six rats in each group. Group 1 served as a control group, while Groups 2 and 3 received subcutaneous 3% dimethyl sulfoxide and 97% volume per volume soy oil as a vehicle or chlorpyrifos, respectively, alternate day for 180 days. Blood samples were taken for biochemical analysis. Kidney tissues were examined for immunohistochemistry and gene expression of pathways related to oxidative stress and advanced glycation end-products. RESULTS: Serum fasting glucose, creatinine, advanced glycation end-products and malondialdehyde levels were significantly increased (p<0.05), whereas paraoxonase-1 level was decreased in chlorpyrifos-treated rats compared with the control groups (p<0.05). The proximal tubular cells and the glomeruli of chlorpyrifos-exposed kidney showed strong malondialdehyde and advanced glycation end-products expression respectively. Gene expression of catalase and glutathione reductase were downregulated in the chlorpyrifos-treated rats. CONCLUSION: Glucose derangement and oxidative stress are the possible mechanisms of organophosphate-induced kidney damage.

Plasminogen activator-coated nanobubbles targeting cellbound β2-glycoprotein I as a novel thrombus-specific thrombolytic strategy.

β2-glycoprotein I (β2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with antiphospholipid syndrome (APS). β2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NB) coated with recombinant tissue plasminogen activator (rtPA) and a recombinant antibody specific for cell-bound β2-GPI. The therapeutic efficacy of targeted NB was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: i) thrombosis developed in a rat model of APS; ii) ferric chloride-induced mesenteric thrombosis in rats, and iii) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted NB bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through β2-GPI expressed on activated cells. In vitro, rtPA-targeted NB (rtPA-tNB) induced greater lysis of platelet-rich blood clots than untargeted NB. In a rat model of APS, administration of rtPA-tNB caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNB, but not untargeted NB and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNB, that target β2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NB. Our findings suggest that targeting cell-bound β2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APS-unrelated thrombotic conditions.

The NALCN channel regulates metastasis and nonmalignant cell dissemination

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium—a NALCN channel blocker—similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.

Asparagus racemosus improves immune-related parameters in Nile tilapia (Oreochromis niloticus) and mitigates deltamethrin-induced toxicity

Deltamethrin (DM) is one of the most toxic but widely used pyrethroid insecticides. Even though a non-target animal, fish are at high risk as they are deficient in the enzyme system that hydrolyses pyrethroids. Enhancing the immune system is a potential method in preventing fish diseases. The present investigation aims to study the modulations in the immune response-related parameters in Oreochromis niloticus that were exposed to DM, by dietary supplementation of aqueous root extract of Asparagus racemosus (ARE). The experiment compared fish in control, DM (1 μg/L) exposed (added to water), ARE (10 g, 20 g, and 30 g ARE/kg of feed) supplemented, and DM-ARE cotreated groups. After 21 days of experimental period, serological, histopathological, and immune response related-gene and protein analysis were carried out. The DM-ARE cotreated group showed significant increase in weight gain, specific growth rate, and decreased feed conversion ratio compared to the DM exposed group. The ARE cotreatment could significantly revert the alteration induced by DM in lysozyme, respiratory burst, myeloperoxidase, C-reactive protein, glucose, cortisol, total protein, albumin, and triglyceride levels. The liver histopathology showed membrane breakage, severe necrosis, infiltration of inflammatory cells, melano-macrophages, and nuclear atrophy, and the kidney showed tubular necrosis, hematopoietic necrosis, Bowman's capsule edema, and glomerulus degeneration in DM exposed group. In ARE cotreated group, the liver showed regenerative cellular changes and only mild to moderate cellular damages, and the kidney tubules and glomerulus had intact structure. ARE discernibly regulated the expression of immune-related genes and proteins (IgM, TNFα, IFN-γ, IL-1β, and IL-8) in fish. The DM-ARE cotreated groups showed reduced cumulative mortality and higher relative percent survival on experimental challenge with Aeromonas hydrophila compared to the DM group. Thus, ARE possess protective potential against DM-induced toxicity, and can be used as a cost-effective technique in aquafarming.

An ex vivo culture model of kidney podocyte injury reveals mechanosensitive, synaptopodin-templating, sarcomere-like structures.

Chronic kidney diseases are widespread and incurable. The biophysical mechanisms underlying them are unclear, in part because material systems for reconstituting the microenvironment of relevant kidney cells are limited. A critical question is how kidney podocytes (glomerular epithelial cells) regenerate foot processes of the filtration apparatus following injury. Recently identified sarcomere-like structures (SLSs) with periodically spaced myosin IIA and synaptopodin appear in injured podocytes in vivo. We hypothesized that SLSs template synaptopodin in the initial stages of recovery in response to microenvironmental stimuli and tested this hypothesis by developing an ex vivo culture system that allows control of the podocyte microenvironment. Results supported our hypothesis. SLSs in podocytes that migrated from isolated kidney glomeruli presented periodic synaptopodin-positive clusters that nucleated peripheral, foot process–like extensions. SLSs were mechanoresponsive to actomyosin inhibitors and substrate stiffness. Results suggest SLSs as mechanobiological mediators of podocyte recovery and as potential targets for therapeutic intervention.

A protective role for <i>Drosophila</i> Filamin in nephrocytes via Yorkie mediated hypertrophy.

Podocytes are specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Because of their position, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury. It is likely podocytes have adaptative responses to help buffer against deleterious mechanical force and thus reduce injury. However, these responses remain largely unknown. Here, using the <i>Drosophila</i> model, we show the mechanosensor Cheerio (dFilamin) provides a key protective role in nephrocytes. We found expression of an activated mechanosensitive variant of Cheerio rescued filtration function and induced compensatory and hypertrophic growth in nephrocytes depleted of the nephrocyte diaphragm proteins Sns or Duf. Delineating the protective pathway downstream of Cheerio we found repression of the Hippo pathway induces nephrocyte hypertrophy, whereas Hippo activation reversed the Cheerio-mediated hypertrophy. Furthermore, we find Yorkie was activated upon expression of active Cheerio. Taken together, our data suggest that Cheerio acts via the Hippo pathway to induce hypertrophic growth, as a protective response in abnormal nephrocytes.

Relationship between age at initiation of cysteamine treatment, adherence with therapy, and glomerular kidney function in infantile nephropathic cystinosis.

Infantile nephropathic cystinosis, due to impaired transport of cystine out of lysosomes, occurs with an incidence of 1 in 100-200,000 live births. It is characterized by renal Fanconi syndrome in the first year of life and glomerular dysfunction progression to end-stage kidney disease by approximately 10 years of age. Treatment with oral cysteamine therapy helps preserve glomerular function, but affected individuals eventually require kidney replacement therapy. This is because glomerular damage had already occurred by the time a child is diagnosed with cystinosis, typically in the second year of life. We performed a retrospective multicenter study to investigate the impact of initiating cysteamine treatment within the first 2 months of life in some infants and comparing two different levels of adherence in patients diagnosed at the typical age. We collected 3983 data points from 55 patients born between 1997 and 2020; 52 patients with 1592 data points could be further evaluated. These data were first analyzed by dividing the patient cohort into three groups: (i) standard treatment start with good adherence, (ii) standard treatment start with less good adherence, and (iii) early treatment start. At every age, mean estimated glomerular filtration rate (eGFR) was higher in early-treated patients than in later-treated patients. Second, a generalized additive mixed model (GAMM) was applied showing that patients with initiation of treatment before 2 months of age are expected to have a 34 ml/min/1.73 m2 higher eGFR than patients with later treatment start while controlling for adherence and patients' age. These data strongly suggest that oral cysteamine treatment initiated within 2 months of birth preserves kidney function in infantile nephropathic cystinosis and provide evidence of the utility of newborn screening for this disease.

PSTAT1 Is Activated during the Progression of IgA Nephropathy

Introduction: IgA nephropathy is the most common primary glomerular disease. Its pathogenesis is still poorly understood. Alterations of the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway may play an important role in IgA nephropathy. Methods: We evaluated the clinical features, pathology, and tissue staining for lymphocytes and phosphorylated STAT1 (pSTAT1) in 43 patients with biopsy proven IgA nephropathy. They were followed to determine their disease outcomes. All had biopsy tissue and multiple laboratory measurements to assess their kidney disease progression. Sixteen patients underwent repeat kidney biopsy to further assess their clinical status. Results: The median eGFR at baseline was 61 mL/min/1.73 m² and the median proteinuria was 2,600 mg/d. The median follow-up was 5 years with an average annual decline in eGFR of 2.25 mL/min/1.73 m². There was significant inflammation and atrophy seen in the first biopsy, which progressed among those who undertook a 2nd biopsy. Compared to healthy kidney tissue, glomeruli and tubulointerstitium demonstrated increased lymphocyte (CD3+) infiltrates and increased pSTAT1 staining by immunohistochemistry. Increased CD3 (p = 0.001) staining and increased pSTAT1 (p = 0.03) correlated with reduced eGFR levels. In repeat biopsy samples, increasing pSTAT1 staining correlated with loss of eGFR over time (p = 0.02). Conclusion: These findings support the hypothesis that pSTAT1 is activated in IgA nephropathy and may play a role in the progression toward kidney failure.

Lifting the Veil: Characteristics, Clinical Significance, and Application of β-2-Microglobulin as Biomarkers and Its Detection with Biosensors.

Because β-2-microglobulin (β2M) is a surface protein that is present on most nucleated cells, it plays a key role in the human immune system and the kidney glomeruli to regulate homeostasis. The primary clinical significance of β2M is in dialysis-related amyloidosis, a complication of end-stage renal disease caused by a gradual accumulation of β2M in the blood. Therefore, the function of β2M in kidney-related diseases has been extensively studied to evaluate its glomerular and tubular functions. Because increased β2M shedding due to rapid cell turnover may indicate other underlying medical conditions, the possibility to use β2M as a versatile biomarker rose in prominence across multiple disciplines for various applications. Therefore, this work has reviewed the recent use of β2M to detect various diseases and its progress as a biomarker. While the use of state-of-the-art β2M detection requires sophisticated tools, high maintenance, and labor cost, this work also has reported the use of biosensor to quantify β2M over the past decade. It is hoped that a portable and highly efficient β2M biosensor device will soon be incorporated in point-of-care testing to provide safe, rapid, and reliable test results.

Thrombomodulin and von willebrand factor as markers of endothelial dysfunction in patients with chronic kidney disease

The aim of research was to study the levels of thrombomodulin (TM) and von Willebrand factor (VWF) in the serum and urine of patients with chronic kidney disease (CKD)as diagnostic markers of endothelial dysfunction. The study involved 140 patients with CKD. The clinical diagnosis was determined based on standard methods of patients examination according to the kidney diseases classification and protocols of CKD patients management. The concentrations of TM and VWF in serum and urine were quantified by ELISA. A generalized endothelial dysfunction in the vessels of the whole body, including the kidneys and high concentration of TM and FVF in the serum and urine of patients with a diabetic nephropathy have been found. The concentration of TM and VWF in the serum of patients with a chronic glomerulonephritis was at the same level as in the serum of healthy individuals, while those in urine significantly exceeded the control values, indicating endothelial damage in the glomeruli of the kidneys due to exposure to pro-inflammatory cytokines. In our opinion, the studied markers will contribute to the timely diagnosis of endothelial dysfunction in patients with CKD and to the development of criteria for prescribing antiplatelet agents in glomerular kidney disease.

Genome-wide meta-analysis and omics integration identifies novel genes associated with diabetic kidney disease

Aims/hypothesisDiabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets.MethodsWe performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets.ResultsThe meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR<60 ml/min per 1.73 m2) and DKD (microalbuminuria or worse) phenotype (p=9.8×10−9; although not withstanding correction for multiple testing, p>9.3×10−9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN–RESP18, GPR158, INIP–SNX30, LSM14A and MFF; p<2.7×10−6). Integration of GWAS with human glomerular and tubular expression data demonstrated higher tubular AKIRIN2 gene expression in individuals with vs without DKD (p=1.1×10−6). The lead SNPs within six loci significantly altered DNA methylation of a nearby CpG site in kidneys (p<1.5×10−11). Expression of lead genes in kidney tubules or glomeruli correlated with relevant pathological phenotypes (e.g. TENM2 expression correlated positively with eGFR [p=1.6×10−8] and negatively with tubulointerstitial fibrosis [p=2.0×10−9], tubular DCLK1 expression correlated positively with fibrosis [p=7.4×10−16], and SNX30 expression correlated positively with eGFR [p=5.8×10−14] and negatively with fibrosis [p<2.0×10−16]).Conclusions/interpretationAltogether, the results point to novel genes contributing to the pathogenesis of DKD.Data availabilityThe GWAS meta-analysis results can be accessed via the type 1 and type 2 diabetes (T1D and T2D, respectively) and Common Metabolic Diseases (CMD) Knowledge Portals, and downloaded on their respective download pages (https://t1d.hugeamp.org/downloads.html; https://t2d.hugeamp.org/downloads.html; https://hugeamp.org/downloads.html).Graphical abstract

Nephrin expression in human epidermal keratinocytes and its implication in poor wound closure.

Nephrin is a type-1 transmembrane protein and a component of the slit diaphragm renal-filtration barrier. It has several functions in actin remodeling and cell-cell adhesion. Nephrin is principally located in the kidney glomerulus, but several studies have reported that nephrin is found in the pancreas, brain, and placenta. However, nephrin expression and its role in human skin have not yet been reported. First, using single-cell RNA sequencing, immunohistochemistry, and immuno-electron microscopy, nephrin expression was confirmed in human-skin epidermal keratinocytes. Nephrin expression colocalized with the expression of zonula occludens-1 in keratinocytes and was closely related to keratinocyte cell density, proliferation, and migration. High glucose treatment decreased nephrin expression and compromised keratinocyte cell migration without yes-associated protein nuclear entry. This reduced cell migration under high glucose conditions was improved in nephrin-overexpressing keratinocytes. Nephrin was highly expressed on the margins of re-epithelized epidermis based on in vivo mice and ex vivo human skin wound models. The results demonstrate that nephrin is expressed in human-skin keratinocytes and functions in cell adhesion, proliferation, and migration. In conclusion, this study suggests that nephrin may have a variety of physiological roles in human skin.

Establishment of type 2 diabetes mellitus models using streptozotocin after 3 months high-fat diet in Bama minipigs

In the past twenty years, the number of adults with diabetes has tripled. Most studies have been conducted using rodent models of type 2 diabetes mellitus (T2DM), and the developed drugs have low clinical conversion efficiency. Therefore, it is urgent to establish a more human-like large animal model to explore T2DM pathogenesis and formulate new disease prevention and control strategies. This study was designed to establish and validate a T2DM model using minipigs fed a high-fat or high-cholesterol/high-fat diet and injected with low-dose streptozotocin (STZ). We examined the influence of the STZ injection timing with a diet high in fat (HFD) compared with one high in cholesterol and fat (HCFD) on the atherosclerotic lesions accelerated by T2DM. Male Bama minipigs (n = 24) were randomly divided into five groups. The control group was fed a normal diet for 9 months. The STZ + HFD and STZ + HCFD groups were infused with 90 mg/kg STZ and then fed a high-fat diet or high-cholesterol and high-fat diet for 9 months, respectively. The HFD + STZ and HCFD + STZ groups were fed a high-fat diet or a high-cholesterol and high-fat diet, respectively, for 9 months (after 3 months, these pigs were injected intravenously with 90 mg/kg STZ). During the induction period, animal body weight, BMI, and serum GLU, INS, TG, TC, HDL-C, LDL-C, FFA, ALT, AST, CRE, and BUN were detected monthly intervals. IVGTT and insulin release tests were performed at 3-month intervals. At the end of the test, the coronary artery and abdominal aorta were examined by computed tomography and pathological observations, and the thickness of the basement membrane of the capillary of the retina and kidney glomerulus was measured under a transmission electron microscope. The serum glucose concentrations were normal in all groups except the HFD + STZ and HCFD + STZ groups. Animals fed an HFD for 9 months did not develop apparent atherosclerotic lesions, but atherosclerotic lesions were seen in the animals fed an HCFD. Hyperglycemia accelerated the formation of atherosclerotic lesions on the intimal surface of the abdominal aorta. Low-dose STZ after 3 months of HFD or HCFD successfully established a T2DM model in minipigs. The HFD did not induce apparent atherosclerotic lesions, but these were seen with the HCFD. Hyperglycemia accelerated atherosclerosis in the minipigs.

Age-related Changes in Renal Arterio-Arteriolosclerosis in Kidney Disease: Renal Biopsy-based Study

Age-related Changes in Renal Arterio-Arteriolosclerosis in Kidney Disease: Renal Biopsy-based Study

Effect of subacute poisoning with lambdacyhalothrin on vascular endothelial growth factor 2 receptor in mice kidneys

BackgroundPyrethroids are used for insect control. They act on voltage-gated sodium channels in neurons. Lambdacyhalothrin (LCH) is a type II pyrethroid producing choreoatetosis and salivation in rodents. Vascular endothelial growth factor (VEGF) expression in the kidney is high in the glomerular podocytes and kidney tubules. VEGF receptor 2 (VEFGR2) is the main mediator in angiogenesis and it regulates blood vessel permeability. Lack of VEGF in podocytes impairs filtration. The nuclear factor κB (NFκB) is widely known as an important mediator of inflammation.The aim of the study was to check if subacute oral intoxication with 0.1LD50 of LCH affects kidney size, function and VEGFR2 and NFκB in mice kidneys.MethodsA total of 32 Albino Swiss mice was used: females controls, males controls, females receiving 2 mg/kg LCH, males receiving 2 mg/kg LCH orally for 7 days. On day 8 animals were sacrificed, blood and kidneys were obtained. Kidney mass was determined, creatinine concentration was measured in blood sera, VEGFR2 and NFκB in kidney homogenate supernatant with ELISA kit.ResultsThere was no statistically significant differences in kidney mass, creatinine concentration in blood sera nor NFκB but mean VEGFR2 concentration in the kidneys of females exposed to LCH was 128.01 ng/ml and showed statistically significant difference in comparison with control females. There was no statistically significant difference between VEGFR2 concentration in the kidneys of males exposed to LCH and control males.ConclusionThe VEGFR2 increases in the course of LCH intoxication in females probably due to the protective effect of oestrogens.

MO046: Exome sequencing of Israeli Druze individuals on dialysis reveals common as well as population- specific monogenic etiologies in ∼30%

Abstract BACKGROUND AND AIMS Genetic etiologies are estimated to affect ∼10% of adults with advanced CKD. However, significant population disparities in genetic kidney disease exist and population-based screening are lacking especially among minority groups. The Druze population is a middle-eastern minority with high rates of end-stage renal disease (ESRD) and consanguinity, which suggests high rates of population-specific monogenic CKD etiologies. We therefore hypothesized that by exome sequencing we will identify a unique distribution of monogenic ESRD causes as compared with prior studies and that important ramifications for clinical practice may ensue. METHOD We initiated a national multicenter study of all Israeli dialysis units in order to provide comprehensive evidence for ESRD's genetic basis (Israeli ESRD Genetic Consortium Cohort). Specifically, during 2020, we recruited 97% (n = 94) of Israeli Druze individuals on dialysis from 12 different hospitals and community-based units. We conducted exome sequencing and diagnostic analysis for all patients. We assessed the diagnostic yield of genetic analysis and its relation to baseline clinical phenotypes. RESULTS Overall, the cohort consists of 94 individuals from 91 different families with first-degree consanguinity rate of 28%. Participants mean age was 62 years (ranging from 18 to 88 years). Most common primary clinical diagnoses were diabetic kidney disease, nephropathy of unknown origin, glomerular or cystic kidney disease, together encompassing 90% of all cases. None had previous molecular diagnosis. Using exome sequencing, we identified a genetic etiology in 27 out of 94 (28.7%) participants. We identified WDR19 (NPHP13) homozygous mutation c.878G &amp;gt; A (p.C293Y) as the most common genetic diagnosis (5.3%). Follow-up clinical characterization revealed that all affected individuals exhibited non-syndromic adulthood-onset ESRD, supporting a profound mutation-dependent phenotypic heterogeneity and weak pre-exome phenotypic specificity. Other prevalent genetic diagnoses included type IV collagen, PKD1, PKD2 and UMOD mutations as well as monogenic forms of diabetes and hyperlipidemia. Molecular diagnosis corresponded to the pre-exome clinical diagnosis in only one-third of the participants. CONCLUSION Exome sequencing in Druze with ESRD yields a genetic diagnosis in just &amp;lt;30% of cases with WDR19 mutation being the most prevalent single-gene etiology. These results, which remarkably inform clinical management, emphasize the importance of revealing population-specific mutations given the underutilization of genetic testing, particularly among adult minorities.

FC014: Target Deconvolution from Phenotypic Screens—Identification of Ataxia Telangiectasia Mutated as a Potential Therapeutic Target for Podocyte Protection

Abstract BACKGROUND AND AIMS As part of the renal filtration barrier, podocytes are critical to the function of kidney glomeruli. Their loss is one of the hallmarks of chronic kidney disease, including diabetic kidney disease (DKD). Indeed, many drug discovery efforts focus on preventing podocyte apoptosis triggered by diabetic stressors to delay kidney disease progression. In order to discover and characterize new therapeutic agents, phenotypic screens are a powerful tool to directly screen for the desired cellular outcome. These need to be complemented by target-centric assays to support the chemical optimization of compounds. Generally, the deconvolution and confirmation of molecular targets are considered challenging, but necessary in the follow-up of phenotype-based drug discovery programmes to allow progression towards the clinic. We have combined novel technologies like high-throughput next-generation sequencing (NGS) and Cell Painting [1] to develop an unbiased approach for deconvolution of targets from phenotypic screens. As an outcome, we identified a small molecule compound that can protect conditionally immortalized human podocytes (CIHP) from apoptosis in vitro. In a second step, we were able to identify the target responsible for this effect. METHOD Phenotypic screen: CIHP [2] were treated with high glucose and palmitate, a common DKD injury model, as the main driver for apoptosis as evaluated by caspase 3/7 activation [3]. Potentially protective agents were screened against apoptosis from a library of 50 000 small molecules. We selected a hit compound (CpdX) for subsequent target deconvolution to identify the target causing the protective effect. Target deconvolution and compound profiling: We compared the molecular signatures triggered by CpdX treatment to those of a reference panel of known, bio-annotated compounds (#L3500; Selleckchem). Dimensional reduction based on UMAP and PCA applied to transcriptomic data (NGS) as well as Cell Painting fingerprints were used to identify known compounds exhibiting a similar transcriptomic and morphological profile as the CpdX. We hypothesized that the molecular targets of these compounds also constitute likely targets for the new CpdX. RESULTS We developed a screening cascade (Figure 1A) to identify novel targets for kidney disease. A first-pass phenotypic podocyte apoptosis screen delivered a potent compound, able to rescue CIHP from a diabetic challenge in vitro (Figure 1B, C). Integrated analysis and alignment of cellular changes in transcriptomic profile and morphology following CpdX or reference compound treatment enabled us to identify the serine/threonine-protein kinase Ataxia Telangiectasia Mutated (ATM) as one of the putative targets of the orphan compound. Both KU-60019, a known inhibitor of ATM [4] (Figure 1D) and CpdX showed remarkably similar effects in NGS and Cell Painting (Figure 2A, B) and were also equally protecting the cells from apoptosis in vitro (Figure 1B). From this data, we inferred that ATM is a likely effector of CpdX treatment. CONCLUSION The initial lack of specific target candidates following primary phenotypic screens is a widespread problem in drug discovery. In the absence of a target deconvolution strategy, many potential candidates cannot be progressed easily to optimization. Here, we demonstrated that a combination of state-of-the-art technologies used to benchmark hit compounds from phenotypic screens versus known bio-annotated compound libraries represent a viable approach to perform target deconvolution of hits from phenotypic screening approaches. In this work, a phenotypic screen and subsequent deconvolution identified a novel target in combination with a novel compound, making a suitable starting point for progression into a potential kidney therapeutic agent.

MO042: Biallelic variants in TTC21B as a rare cause of early-onset arterial hypertension and tubuloglomerular kidney disease

Abstract BACKGROUND Massively parallel sequencing (MPS) has propelled precision medicine into the next generation. With genetic data more accessible than ever, the focus has now shifted from obtaining data to converting the acquired data into applicable clinical information. A major obstacle to such task arises when there is limited knowledge of the phenotypic spectrum of a genetic entity, limiting the application of virtual panels and prioritization of genetic candidates. METHOD We performed in-depth clinical, imaging and histological phenotyping of an index family presenting with extremely early-onset hypertension and kidney disease. We then performed genetic investigations on the proband, starting with a limited MPS panel testing for monogenic causes of arterial hypertension. This was followed by Genomics England 100 000 Genomes Project whole genome sequencing (WGS) and application of a 26-gene virtual panel for ‘extreme early-onset hypertension’. Subsequently, we reviewed the 100 000 Genomes Project rare disease data for patients recruited with extreme early-onset hypertension. We also conducted a literature review to identify any existing studies correlating TTC21B mutations and systemic hypertension. RESULTS The proband (Table 1, II.1) was a 3.5-year-old girl presenting with severe hypertension, proteinuria, kidney failure, left ventricular hypertrophy, liver function test abnormalities and growth retardation. A kidney ultrasound scan showed bilateral small kidneys with loss of corticomedullary differentiation. Kidney biopsy showed sclerosed glomeruli, severe tubular atrophy with tubulointerstitial fibrosis in addition to arteriolar changes in secondary to systemic hypertension. Her sibling (Table 1, II.2) equally presented with early-onset hypertension and progressive kidney disease. MPS panel testing and WGS virtual panel for early-onset hypertension both failed to identify pathogenic variants. Manual curation of WGS data then revealed a heterozygous nonsense variant p.(Gln834Ter) in conjunction with a heterozygous missense variant p.(Pro209Leu) in TTC21B, both of which were predicted to be pathogenic according to ACMG criteria. This was missed as the gene did not form part of the hypertension virtual panel. TTC21B mutations have been previously associated with typical ciliopathies such as nephronophthisis (NPHP) and Jeune asphyxiating thoracic dystrophy, but only recently implicated in glomerular kidney disease such as focal segmental glomerulosclerosis (FSGS). Literature review suggests roles for TTC21B (IFT139) both in the primary cilium and in podocyte cytoskeleton, revealing a striking association between TTC21B missense variants and early-onset hypertension. Searching the 100 000 Genomes Project revealed one additional case of arterial hypertension and mild proteinuria explained by biallelic mutations in TTC21B. CONCLUSION In conclusion, biallelic mutations in TTC21B produce a spectrum of phenotypes from typical ciliopathies to kidney-limited phenotypes. The latter are mostly encountered in patients carrying two missense, often hypomorphic, variants, and are characterized by lesions of the glomerular as well as the tubulointerstitial compartment, resembling both FSGS and NPHP. Increased clinical recognition of this mixed glomerular and tubulointerstitial disease with often mild or absent features of a typical ciliopathy as well as inclusion of TTC21B on gene panels for early-onset arterial hypertension may shorten the diagnostic odyssey for patients affected by this rare tubuloglomerular kidney disease.