De novo TRIM8 variants impair its protein localization to nuclear bodies and cause developmental delay, epilepsy, and focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10-11). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10-15). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.

DAAM2 Variants Cause Nephrotic Syndrome via Actin Dysregulation.

The discovery of >60 monogenic causes of nephrotic syndrome (NS) has revealed a central role for the actin regulators RhoA/Rac1/Cdc42 and their effectors, including the formin INF2. By whole-exome sequencing (WES), we here discovered bi-allelic variants in the formin DAAM2 in four unrelated families with steroid-resistant NS. We show that DAAM2 localizes to the cytoplasm in podocytes and in kidney sections. Further, the variants impair DAAM2-dependent actin remodeling processes: wild-type DAAM2 cDNA, but not cDNA representing missense variants found in individuals with NS, rescued reduced podocyte migration rate (PMR) and restored reduced filopodia formation in shRNA-induced DAAM2-knockdown podocytes. Filopodia restoration was also induced by the formin-activating molecule IMM-01. DAAM2 also co-localizes and co-immunoprecipitates with INF2, which is intriguing since variants in both formins cause NS. Using in vitro bulk and TIRF microscopy assays, we find that DAAM2 variants alter actin assembly activities of the formin. In a Xenopus daam2-CRISPR knockout model, we demonstrate actin dysregulation in vivo and glomerular maldevelopment that is rescued by WT-DAAM2 mRNA. We conclude that DAAM2 variants are a likely cause of monogenic human SRNS due to actin dysregulation in podocytes. Further, we provide evidence that DAAM2-associated SRNS may be amenable to treatment using actin regulating compounds.

Treatment and outcome of IgA nephropathy in children from one single center experience.

BACKGROUND: There is no standard recommendation for IgA nephropathy treatment in children. METHODS: This is a retrospective study. From 2012 to 2020, newly diagnosed primary IgAN followed up for at least 1 year were enrolled. The correlation of MESTC scores and clinical index including proteinuria, gross hematuria and renal dysfunction was analyzed. Treatment and clinical response of 6 month, 1year and 3 year at follow up were also analyzed. Complete renal remission was calculated with Kaplan-Meier analysis. RESULTS: The median follow up was 36 months, from 12 months to 87months in 40 IgAN children. Angiotensin-converting enzyme inhibitor (ACEI) was applied to all patients. 30% received ACEI alone; 15% received glucocorticoids; 37.5% received glucocorticoids plus cyclophosphamide, 17.5% received glucocorticoids plus mycophenolate mofetil. Individuals with diffuse mesangial hypercellularity (M1) were more likely to have nephrotic range proteinuria compared to patients with M0 (80% vs. 20%, P < 0.01). Complete renal remission at 6-month, 1-year and 3-year follow up is 50.25%, 70% and 87.5% respectively. Five-year complete renal remission calculated by Kaplan-Meier analysis is 58.4%. Although without significant difference, there is trend of better survival with complete renal remission in group of nephrotic range proteinuria onset. There is no severe adverse effect. CONCLUSION: This study supports the use of glucocorticoids plus immunosuppressive in addition to ACEI in IgA nephrology pediatric patients with proteinuria. We suggest proactive immunosuppressive treatment in IgA nephropathy in children. This is from a single center in China as may not same results in other population.

Reverse phenotyping facilitates disease allele calling in exome sequencing of patients with CAKUT.

PURPOSE: Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the leading cause of chronic kidney disease in children. In total, 174 monogenic causes of isolated or syndromic CAKUT are known. However, syndromic features may be overlooked when the initial clinical diagnosis of CAKUT is made. We hypothesized that the yield of a molecular genetic diagnosis by exome sequencing (ES) can be increased by applying reverse phenotyping, by re-examining the case for signs/symptoms of the suspected clinical syndrome that results from the genetic variant detected by ES. METHODS: We conducted ES in an international cohort of 731 unrelated families with CAKUT. We evaluated ES data for variants in 174 genes, in which variants are known to cause isolated or syndromic CAKUT. In cases in which ES suggested a previously unreported syndromic phenotype, we conducted reverse phenotyping. RESULTS: In 83 of 731 (11.4%) families, we detected a likely CAKUT-causing genetic variant consistent with an isolated or syndromic CAKUT phenotype. In 19 of these 83 families (22.9%), reverse phenotyping yielded syndromic clinical findings, thereby strengthening the genotype-phenotype correlation. CONCLUSION: We conclude that employing reverse phenotyping in the evaluation of syndromic CAKUT genes by ES provides an important tool to facilitate molecular genetic diagnostics in CAKUT.

Whole exome sequencing identifies potential candidate genes for spina bifida derived from mouse models.

Spina bifida (SB) is the second most common nonlethal congenital malformation. The existence of monogenic SB mouse models and human monogenic syndromes with SB features indicate that human SB may be caused by monogenic genes. We hypothesized that whole exome sequencing (WES) allows identification of potential candidate genes by (i) generating a list of 136 candidate genes for SB, and (ii) by unbiased exome-wide analysis. We generated a list of 136 potential candidate genes from three categories and evaluated WES data of 50 unrelated SB cases for likely deleterious variants in 136 potential candidate genes, and for potential SB candidate genes exome-wide. We identified 6 likely deleterious variants in 6 of the 136 potential SB candidate genes in 6 of the 50 SB cases, whereof 4 genes were derived from mouse models, 1 gene was derived from human nonsyndromic SB, and 1 gene was derived from candidate genes known to cause human syndromic SB. In addition, by unbiased exome-wide analysis, we identified 12 genes as potential candidates for SB. Identification of these 18 potential candidate genes in larger SB cohorts will help decide which ones can be considered as novel monogenic causes of human SB.

Mutations in PRDM15 Are a Novel Cause of Galloway-Mowat Syndrome.

BACKGROUND: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. METHODS: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. RESULTS: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. CONCLUSIONS: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.

Mutations in transcription factor CP2-like 1 may cause a novel syndrome with distal renal tubulopathy in humans.

BACKGROUND: An underlying monogenic cause of early-onset chronic kidney disease (CKD) can be detected in ∼20% of individuals. For many etiologies of CKD manifesting before 25 years of age, >200 monogenic causative genes have been identified to date, leading to the elucidation of mechanisms of renal pathogenesis. METHODS: In 51 families with echogenic kidneys and CKD, we performed whole-exome sequencing to identify novel monogenic causes of CKD. RESULTS: We discovered a homozygous truncating mutation in the transcription factor gene transcription factor CP2-like 1 (TFCP2L1) in an Arabic patient of consanguineous descent. The patient developed CKD by the age of 2 months and had episodes of severe hypochloremic, hyponatremic and hypokalemic alkalosis, seizures, developmental delay and hypotonia together with cataracts. We found that TFCP2L1 was localized throughout kidney development particularly in the distal nephron. Interestingly, TFCP2L1 induced the growth and development of renal tubules from rat mesenchymal cells. Conversely, the deletion of TFCP2L1 in mice was previously shown to lead to reduced expression of renal cell markers including ion transporters and cell identity proteins expressed in different segments of the distal nephron. TFCP2L1 localized to the nucleus in HEK293T cells only upon coexpression with its paralog upstream-binding protein 1 (UBP1). A TFCP2L1 mutant complementary DNA (cDNA) clone that represented the patient's mutation failed to form homo- and heterodimers with UBP1, an essential step for its transcriptional activity. CONCLUSION: Here, we identified a loss-of-function TFCP2L1 mutation as a potential novel cause of CKD in childhood accompanied by a salt-losing tubulopathy.

Pediatric Dent disease presenting with rickets and end-stage renal disease: case report and literature review.

Dent disease is a rare disease with proximal renal tubular dysfunction, and is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and chronic kidney disease. Renal failure slowly progresses and end-stage renal disease may develop in the late decades of life. We report a case of a 15-year-old boy who was diagnosed with Dent disease 1 with a CLCN5 truncating mutation. The patient presented with arthralgia and rickets at the onset of Dent disease and he was diagnosed with end-stage renal disease at the age of 15 years. His only symptoms were arthralgia and rickets during the disease course. The findings in this case suggest that patients with arthralgia and rickets could have a rare cause such as Dent disease.

Extragastrointestinal stromal tumor in a kidney transplant recipient.

The occurrence of de novo malignant neoplasms has been shown in post-transplant recipients receiving immunosuppressive treatment. We present a case of a rare extragastrointestinal stromal tumor (EGIST) located in the pelvic cavity of a kidney transplant patient. A 57-year-old female patient was admitted to our department because of non-specific lower abdominal pain 6 months after renal transplantation. An abdominal computed tomography scan showed a 4.5 cm diameter pelvic tumor mass. The tumor was resected en bloc and confirmed as not being connected to the gastrointestinal wall. Microscopically, the tumor consisted of typical spindle cells with 2-3 mitotic figures per 50 high-power fields. Immunohistochemically, the tumor cells were strongly positive for CD117 (c-kit), and negative for CD34, SMA, s-100 protein, and desmin. Genetically, the tumor showed a silent mutation in exon 18 of the PDGFRA gene at codon 824 GTC > GTT (V824V) [rs2228230]. No recurrence was noted 24 months after the operation. This case draws our attention to the importance of considering EGISTs (including GISTs), even though they are extremely uncommon, in the differential diagnosis of mesenchymal neoplasms, especially in transplant patients.

A retrospective study of nine patients with progressive pseudorheumatoid dysplasia: to explore early diagnosis and further treatment.

OBJECTIVES: Most patients with progressive pseudorheumatoid dysplasia (PPRD) are initially misdiagnosed because of disease rarity and lack of awareness by most clinicians. The purpose of this study was to provide further early diagnostic options and potential treatment to patients with PPRD. METHODS: A retrospective study was performed by collecting and organizing clinical manifestations, radiographic features, laboratory test results, genetic test outcome, treatment, and follow-up records of the patients with PPRD. Age of diagnosis and genotype-phenotype correlation were further analyzed. RESULTS: Nine PPRD children with causative CCN6 mutation were included. There were 3 pairs of siblings and 1 patient from inbred family. Five patients were primarily misdiagnosed as juvenile idiopathic arthritis (JIA). The interval between onset of symptoms and definite diagnosis of 8 patients varied from 3.6 to 20 years. Symptoms at the onset included enlarged and stiff interphalangeal joints of the fingers, gait disturbance, or joint pain. Laboratory tests revealed normal range of inflammatory parameters. Radiographic findings disclosed different degrees of abnormal vertebral bodies and epiphyseal enlargement of the interphalangeal joints. After the treatment of calcitriol in 5 patients with low level 25-hydroxyvitamin D3 for around 1.25 years to 1.75 years, 2 patients kept stable, while 3 of them improved gradually. CONCLUSIONS: Combining the patient's family history, clinical features, normal inflammatory markers, and the characteristic radiographic findings, the clinical diagnosis of PPRD for the patients could be obtained at very early stage of the disease. The patients with PPRD carrying c.624dupA variant in CCN6 may have delayed onset. Underlying vitamin D deficiency should be sought and corrected in patients with PPRD.

Integrated genomic, transcriptomic and metabolomic analysis reveals MDH2 mutation-induced metabolic disorder in recurrent focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) has an over 30% risk of recurrence after kidney transplantation (Ktx) and is associated with an extremely high risk of graft loss. However, mechanisms remain largely unclear. Thus, this study identifies novel genes related to the recurrence of FSGS (rFSGS). Whole genome-wide sequencing and next-generation RNA sequencing were used to identify the candidate mutant genes associated with rFSGS in peripheral blood mononuclear cells (PBMCs) from patients with biopsy-confirmed rFSGS after KTx. To confirm the functional role of the identified gene with the MDH2 c.26C >T mutation, a homozygous MDH2 c.26C >T mutation in HMy2.CIR cell line was induced by CRISPR/Cas9 and co-cultured with podocytes, mesangial cells, or HK2 cells, respectively, to detect the potential pathogenicity of the c.26C >T variant in MDH2. A total of 32 nonsynonymous single nucleotide polymorphisms (SNPs) and 610 differentially expressed genes (DEGs) related to rFSGS were identified. DEGs are mainly enriched in the immune and metabolomic-related pathways. A variant in MDH2, c.26C >T, was found in all patients with rFSGS, which was also accompanied by lower levels of mRNA expression in PBMCs from relapsed patients compared with patients with remission after KTx. Functionally, co-cultures of HMy2.CIR cells overexpressing the mutant MDH2 significantly inhibited the expression of synaptopodin, podocin, and F-actin by podocytes compared with those co-cultured with WT HMy2.CIR cells or podocytes alone. We identified that MDH2 is a novel rFSGS susceptibility gene in patients with recurrence of FSGS after KTx. Mutation of the MDH2 c.26C >T variant may contribute to progressive podocyte injury in rFSGS patients.

CXCL10 and CXCL13 Expression were highly up-regulated in peripheral blood mononuclear cells in acute rejection and poor response to anti-rejection therapy.

BACKGROUND: Acute rejection is still one of the main complications which enhances the cost and the risk to renal graft failure. Chemokines, interacting with respective receptors, can recruit leukocytes into grafts and mediate allograft rejection. In this study, we aimed to analyze gene expression of chemokines including CCL5/RANTES, CXCL10/IP-10, CXCL13/BCA-1, and receptors of CCR5, CXCR3, CXCR5 in peripheral blood mononuclear cells (PBMCs) during acute renal allograft rejection METHODS: Gene expression of all these chemokines and receptors in PBMCs were analyzed by real-time PCR from 14 stable recipients, 32 biopsy-proven acute rejection (AR), and 5 acute tubular necrosis (ATN). RESULTS: Gene expression of CCL5, CXCL10, CXCL13, and CCR5 were up-regulated both in AR and ATN group compared to stable recipients (fold change>2, P<0.05). Serum creatinine recovered to baseline level after anti-rejection therapy was defined as AR-sensitive and creatinine maintained above 200 µmol/L as AR-resistant. Expression of CXCL10 and CXCL13 were 5.98-, 2.94-, and 20.5, 10.8-fold change in AR-resistant and AR-sensitive compared to stable recipients, respectively. The expression of CXCL10 and CXCL13 was a twofold change in AR-resistant compared to AR-sensitive recipients (P<0.05). Five out of ten AR-resistant recipients lost graft function in the follow-up. CONCLUSION: CXCL10 and CXCL13 expression were highly up-regulated in PBMCs in acute renal allograft rejection, especially in poor response to anti-rejection therapy and detrimental prognosis.

Evaluation of sphingolipid metabolism in renal cortex of rats with streptozotocin-induced diabetes and the effects of rapamycin.

BACKGROUND: Abnormal lipid metabolism contributes to the pathogenesis of diabetes, but it is uncertain whether it plays a role in the development of diabetic nephropathy (DN). While rapamycin was shown to prevent DN development in streptozotocin (STZ)-induced diabetic rats in our previous studies, it is unknown if it intervenes with lipid metabolism. METHODS: We divided the rats into four groups: normal control rats, rapamycin-treated normal rats, diabetic rats and rapamycin-treated DN rats. The apoptosis was evaluated by immunohistochemistry. The crude lipid and sphingolipid were extracted from rat renal cortex and analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The expression of the three key enzymes in sphingolipid metabolism including serine palmitoyltransferase, acid sphingomyelinase and sphingomyelin synthase was measured by western blot and immunohistochemistry in rat renal cortex. RESULTS: The level of apoptosis was increased in diabetic rats, and rapamycin treatment reduced apoptosis. STZ treatment significantly increased formation of many sphingolipids species through elevated de novo synthesis. These changes were inhibited by treatment with rapamycin. CONCLUSIONS: Accumulation of sphingolipids contributes to STZ-induced diabetes, and the therapeutic effect of rapamycin on diabetic nephropathy is partly through suppression of sphingolipid abnormality.

Effects of chlorpyrifos exposure on kidney Notch2-Jagged1 pathway of early prenatal embryo.

AIMS: To evaluate the effects of this insecticide on the embryonic development of kidney and to assess the important role of Notch2-Jagged1 pathway in this duration. METHODS AND RESULTS: Chlorpyrifos (CPF) 5 mg/kg/d were administrated on gestation 7.5-11.5 day by subcutaneous injection. On gestation 16.5 day, the normal embryo kidney developed through S shape duration to the original kidney, which had the nephrons and could start to secret the urine. But for the CPF-treated mice, the embryo kidney developed much more slowly, they did not show the S shape and the nephrons. The Notch2-Jagged1 pathway should be expressed stronger in the normal embryo kidney on gestation 16.5 day, but for the CPF-treated mice we found the obvious weak pathway staining. CONCLUSIONS: CPF broke the Notch2-Jagged1 pathway during the embryo kidney development, and the Notch2-Jagged1 pathway plays an important role in the S shape to original kidney formation duration.

Generation of Monogenic Candidate Genes for Human Nephrotic Syndrome Using 3 Independent Approaches.

INTRODUCTION: Steroid-resistant nephrotic syndrome (SRNS) is the second most common cause of chronic kidney disease during childhood. Identification of 63 monogenic human genes has delineated 12 distinct pathogenic pathways. METHODS: Here, we generated 2 independent sets of nephrotic syndrome (NS) candidate genes to augment the discovery of additional monogenic causes based on whole-exome sequencing (WES) data from 1382 families with NS. RESULTS: We first identified 63 known monogenic causes of NS in mice from public databases and scientific publications, and 12 of these genes overlapped with the 63 known human monogenic SRNS genes. Second, we used a set of 64 genes that are regulated by the transcription factor Wilms tumor 1 (WT1), which causes SRNS if mutated. Thirteen of these WT1-regulated genes overlapped with human or murine NS genes. Finally, we overlapped these lists of murine and WT1 candidate genes with our list of 120 candidate genes generated from WES in 1382 NS families, to identify novel candidate genes for monogenic human SRNS. Using this approach, we identified 7 overlapping genes, of which 3 genes were shared by all datasets, including SYNPO. We show that loss-of-function of SYNPO leads to decreased CDC42 activity and reduced podocyte migration rate, both of which are rescued by overexpression of wild-type complementary DNA (cDNA), but not by cDNA representing the patient mutation. CONCLUSION: Thus, we identified 3 novel candidate genes for human SRNS using 3 independent, nonoverlapping hypotheses, and generated functional evidence for SYNPO as a novel potential monogenic cause of NS.

Recessive Mutations in SYNPO2 as a Candidate of Monogenic Nephrotic Syndrome.

INTRODUCTION: Most of the approximately 60 genes that if mutated cause steroid-resistant nephrotic syndrome (SRNS) are highly expressed in the glomerular podocyte, rendering SRNS a "podocytopathy." METHODS: We performed whole-exome sequencing (WES) in 1200 nephrotic syndrome (NS) patients. RESULTS: We discovered homozygous truncating and homozygous missense mutation in SYNPO2 (synaptopodin-2) (p.Lys1124∗ and p.Ala1134Thr) in 2 patients with childhood-onset NS. We found SYNPO2 expression in both podocytes and mesangial cells; however, notably, immunofluorescence staining of adult human and rat kidney cryosections indicated that SYNPO2 is localized mainly in mesangial cells. Subcellular localization studies reveal that in these cells SYNPO2 partially co-localizes with α-actinin and filamin A-containing F-actin filaments. Upon transfection in mesangial cells or podocytes, EGFP-SYNPO2 co-localized with α-actinin-4, which gene is mutated in autosomal dominant SRNS in humans. SYNPO2 overexpression increases mesangial cell migration rate (MMR), whereas shRNA knockdown reduces MMR. Decreased MMR was rescued by transfection of wild-type mouse Synpo2 cDNA but only partially by cDNA representing mutations from the NS patients. The increased mesangial cell migration rate (MMR) by SYNPO2 overexpression was inhibited by ARP complex inhibitor CK666. SYNPO2 shRNA knockdown in podocytes decreased active Rac1, which was rescued by transfection of wild-type SYNPO2 cDNA but not by cDNA representing any of the 2 mutant variants. CONCLUSION: We show that SYNPO2 variants may lead to Rac1-ARP3 dysregulation, and may play a role in the pathogenesis of nephrotic syndrome.

Recessive NOS1AP variants impair actin remodeling and cause glomerulopathy in humans and mice.

Nephrotic syndrome (NS) is a leading cause of chronic kidney disease. We found recessive NOS1AP variants in two families with early-onset NS by exome sequencing. Overexpression of wild-type (WT) NOS1AP, but not cDNA constructs bearing patient variants, increased active CDC42 and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, formin proteins, reduced NOS1AP-induced filopodia formation. NOS1AP knockdown reduced podocyte migration rate (PMR), which was rescued by overexpression of WT Nos1ap but not by constructs bearing patient variants. PMR in NOS1AP knockdown podocytes was also rescued by constitutively active CDC42Q61L or the formin DIAPH3 Modeling a NOS1AP patient variant in knock-in human kidney organoids revealed malformed glomeruli with increased apoptosis. Nos1apEx3-/Ex3- mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement, and glomerulosclerosis. These findings demonstrate that recessive NOS1AP variants impair CDC42/DIAPH-dependent actin remodeling, cause aberrant organoid glomerulogenesis, and lead to a glomerulopathy in humans and mice.

[Preparation of a novel targeted MR contrast agent Gd-DTPA-Granzyme B monoclonal antibody].

OBJECTIVE: To prepare a novel MRI targeted contrast agent Gd-DTPA-Granzyme B monoclonal antibody (mAb) and to test its reaction conditions. METHODS: The Granzyme B mAb was coupled with DTPA,and then conjugated with Gd. The Gd-DTPA antibody was characterized using MALDI-TOF-MS. Cytotoxicity test was performed with MTT assay, and immune activation was examined with immunohistochemistry. RESULT: MALDI-TOF-MS demonstrated that the molecular weight shifted from granzyme B mAb (133986) to Gd-DTPA-GB mAb (139736), which indicated the conjugation of the antibody with Gd-DTPA. The molar ratio of Gd per IgG molecule was about 20. MTT assay showed that Gd, DTPA, Gd-DTPA and Gd-DTPA-GB mAb groups did not make an impact on cell viability, and there were no significant differences among 4 groups (P>0.05). Immunohistochemistry results showed that compared with the positive control group the targeted contrast agent had a high immune activity. CONCLUSION: The novel contrast agent Gd-DTPA-Granzyme B mAb prepared in this study keeps a good immune activity and has no significant cytotoxicity.

Immunometabolism in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a typical autoimmune disease characterized by chronic inflammation and pathogenic auto-antibodies. Apart from B cells, dysregulation of other immune cells also plays an essential role in the pathogenesis and development of the disease including CD4+T cells, dendritic cells, macrophages and neutrophils. Since metabolic programs control immune cell fate and function, they are critical checkpoints in an effective immune response and are involved in the etiology of autoimmune disease. In addition, mitochondria and oxidative stress are both involved in cellular metabolism and is also essential in immune response. In this review, apart from the disturbed immune system, we will discuss mitochondrial dysfunction, oxidative stress, abnormal metabolism (including glucose, lipid and amino acid metabolism) of immune cells as well as epigenetic control of metabolism reprogramming to elucidate the underlying pathogenic mechanisms of systemic lupus erythematosus.

Donor-derived hypouricemia in irrelevant recipients caused by kidney transplantation.

BACKGROUND: Hereditary renal hypouricemia (HRH) is a genetically heterogenetic disease. Patients with HRH are almost asymptomatic; but some may experience exercise-induced acute kidney injury (EAKI) and nephrolithiasis which may bring concerns regarding the risk-benefit ratio as marginal kidney donors. This study examined the pathogenic mutations of hypouricemia in two recipients after receiving kidney transplantation, providing preliminary evidence for the mechanism of hypouricemia. METHODS: Two participants underwent detailed biochemical examinations. DNA and RNA were extracted from transplant specimens for sequencing. The whole-genome sequencing and polymerase chain reaction (PCR) amplification were performed to confirm the pathogenic genes. Functional effects of mutant proteins were verified by bioinformatics analysis. RNA-sequencing (RNA-seq) was used to study the transcriptome of hypouricemia. RESULTS: Both of the recipients had the low serum uric acid (UA) (45-65 µmol/l), high fraction excretion of UA (44% and 75%) and an increase in the UA clearance (35.9 and 73.3 mL/min) with a functioning graft. The sequencing analyses revealed 7 kinds of potential mutational genes in this case, two novel mutations p.R89H and p.L181V in SLC22A12 gene which were revealed by bioinformatics could be pathogenic in nature. CONCLUSIONS: Two novel mutations of SLC22A12 were identified. Preliminary functional analysis revealed a potential deleterious effect of these mutations in the grafts derived from the donor and sequencing analysis expand the molecular mechanisms of renal hypouricemia.