Noriko Sugawara

Assembly of Staphylococcus aureus γ-hemolysin into a pore-forming ring-shaped complex on the surface of human erythrocytes

Staphylococcal γ‐hemolysin consists of Hlg1 (or Luk F) of 34 kDa and Hlg2 of 32 kDa, which cooperatively lyse human erythrocytes. Since γ‐hemolysin caused swelling of human erythrocytes prior to lysis, we studied pore‐forming nature of the toxin by use of polyethylene glycols as osmotic protectants and determined the functional diameter of the pore. To elucidate the molecular architecture of the membrane pore formed by γ‐hemolysin, we solubilized the pore complex with 2% sodium dodecyl sulfate, separated it from erythrocyte membrane proteins by sucrose gradient ultracentrifugation, and observed the isolated complex under an electron microscope. Our data showed that Hlg1 and Hlg2 of γ‐hemolysin assemble into a ring‐shaped 195 kDa complex in a molar ratio of 1 : 1, which may form a membrane pore with a functional diameter of 2.1–2.4 nm.

Generation and analyses of R8L barttin knockin mouse

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henles loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.

Decreased glomerular filtration as the primary factor of elevated circulating suPAR levels in focal segmental glomerulosclerosis

BackgroundCirculating factor(s) has been thought to be the underlying cause of focal segmental glomerulosclerosis (FSGS), and recent studies foster this idea by demonstrating increased soluble urokinase receptor (suPAR) levels in the serum of FSGS patients.MethodsTo explore the possible contribution of suPAR in FSGS pathogenesis, we analyzed serum suPAR levels in 17 patients with FSGS and compared them with those in patients with steroid-sensitive nephrotic syndrome, chronic glomerulonephritis, or non-glomerular kidney diseases.ResultsSerum suPAR levels in patients with FSGS were higher than those in patients with steroid-sensitive nephrotic syndrome or chronic glomerulonephritis, but not higher than those in patients with non-glomerular kidney diseases. suPAR levels negatively correlate with estimated glomerular filtration rate and were decreased after renal transplantation in patients with FSGS as well as in those with non-glomerular kidney diseases. Furthermore, 6 FSGS patients with post-transplant recurrence demonstrated that suPAR levels were not high during the recurrence.ConclusionsBased on our results, elevated suPAR levels in FSGS patients were attributed mainly to decreased glomerular filtration. These data warrant further analysis for involvement of possible circulating factor(s) in FSGS pathogenesis.

Novel mutations in five Japanese patients with 3-methylcrotonyl-CoA carboxylase deficiency

AbstractIsolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency appears to be the most frequent organic aciduria detected in tandem mass spectrometry (MS/MS) screening programs in the United States, Australia, and Europe. A pilot study of newborn screening using MS/MS has recently been commenced in Japan. Our group detected two asymptomatic MCC deficiency patients by the pilot screening and collected data on another three MCC deficiency patients to study the molecular bases of the MCC deficiency in Japan. Molecular analyses revealed novel mutations in one of the causative genes, MCCA or MCCB, in all five of the patients: nonsense and frameshift mutations in MCCA (c.1750C > T/c.901_902delAA) in patient 1, nonsense and frameshift mutations in MCCB (c.1054_1055delGG/c.592C > T) in patient 2, frameshift and missense mutations in MCCB (c.1625_1626insGG/c.653_654CA > TT) in patient 3, a homozygous missense mutation in MCCA (c.1380T > G/ 1380T > G) in patient 4, and compound heterozygous missense mutations in MCCB (c.569A > G/ c.838G > T) in patient 5. No obvious clinical symptoms were observed in patients 1, 2, and 3. Patient 4 had severe neurological impairment and patient 5 developed Reye-like syndrome. The increasing use of MS/MS newborn screening in Japan will further clarify the clinical and genetic heterogeneity among patients with MCC deficiency in the Japanese population.

SIRPα interacts with nephrin at the podocyte slit diaphragm

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero‐oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury.

Phylogenetic, ontogenetic, and pathological aspects of the urine-concentrating mechanism.

The urine-concentrating mechanism is one of the most fundamental functions of avian and mammalian kidneys. This particular function of the kidneys developed as a system to accumulate NaCl in birds and as a system to accumulate NaCl and urea in mammals. Based on phylogenetic evidence, the mammalian urine-concentrating mechanism may have evolved as a modification of the renal medullas NaCl accumulating system that is observed in birds. This qualitative conversion of the urine-concentrating mechanism in the mammalian inner medulla of the kidneys may occur during the neonatal period. Human kidneys have several suboptimal features caused by the neonatal conversion of the urine-concentrating mechanism. The urine-concentrating mechanism is composed of various functional molecules, including water channels, solute transporters, and vasopressin receptors. Abnormalities in water channels aquaporin (AQP)1 and AQP2, as well as in the vasopressin receptor V2R, are known to cause nephrogenic diabetes insipidus. An analysis of the pathological mechanism involved in nephrogenic diabetes insipidus suggests that molecular chaperones may improve the intracellular trafficking of AQP2 and V2R, and, in the near future, such chaperones may become a new clinical tool for treating nephrogenic diabetes insipidus.

Analysis of the genes responsible for steroid-resistant nephrotic syndrome and/or focal segmental glomerulosclerosis in Japanese patients by whole-exome sequencing analysis

Steroid-resistant nephrotic syndrome (SRNS) represents glomerular disease resulting from a number of different etiologies leading to focal segmental glomerulosclerosis (FSGS). Recently, many genes causing SRNS/FSGS have been identified. These genes encode the proteins associated with the formation and/or maintenance of glomerular filtration barrier. Next-generation sequencing is used to analyze large numbers of genes at lower costs. To identify the genetic background of Japanese patients, we studied 26 disease-causing genes using whole-exome sequencing analysis in 24 patients with SRNS and/or FSGS from 22 different Japanese families. We finally found eight causative gene mutations, four recessive and four dominant gene mutations, including three novel mutations, in six patients from five different families, and one novel predisposing mutation in two patients from two different families. Causative gene mutations have only been identified in ~20% of families and further analysis is necessary to identify the unknown disease-causing gene. Identification of the disease-causing gene would support clinical practices, including the diagnosis, understanding of pathogenesis and treatment.

Immunohistological study of a pediatric patient with plasma cell‐rich acute rejection

We report here the case of a girl who developed plasma cell‐rich acute rejection (PCAR), a condition characterized by the presence of mature plasma cells infiltrating a renal allograft. The patients creatinine level increased sharply to 4.3 mg/dL from 0.9 mg/dL at 19 months post‐renal transplantation. She showed no response to methylprednisolone pulse therapy at a dose of 500 mg for three d but did show an immediate clinical and histopathological response to muromonab‐CD3 (OKT3) administration. She had two episodes of PCAR recurrence and subsequently lost her graft. She had no evidences of antibody‐mediated rejection including C4d deposition in peritubular capillaries and donor‐specific antibodies during the entire follow‐up period. To elucidate the pathogenesis of PCAR, immunohistological examination of infiltrating cells was performed. CD3‐positive cells infiltration seemed to be associated with the CD138‐positive cells infiltration, and the number of CD3‐positive cells was increased preceding PCAR recurrence. Additionally, a rapid decrease in the number of CD138‐positive cells and CD3‐positive cells following the OKT3 administration was observed. This case suggests that T‐cell mediated immune mechanisms might play a role in the development of PCAR.

Diversity of renal phenotypes in patients with WDR19 mutations: Two case reports

WDR19 has been reported as a causative gene of nephronophthisis‐related ciliopathies. Patients with WDR19 mutations can show various extrarenal manifestations such as skeletal disorders, Caroli disease, and retinal dystrophy, and typically display nephronophthisis as a renal phenotype. However, there is limited information on the renal phenotypes of patients with WDR19 mutations. We report two Japanese infants with Sensenbrenner syndrome caused by WDR19 mutations who demonstrated different features in renal ultrasound and histopathological results, despite several common extrarenal manifestations. Patient 1 had normal sized and hyperechogenic kidneys with several small cysts and histopathological findings compatible with infantile nephronophthisis. Renal ultrasound of Patient 2 showed enlarged kidneys with diffuse microcysts resembling those of autosomal recessive polycystic kidney disease. Her renal histopathology revealed dysplastic kidney with diffuse glomerular cysts. Genetic testing identified compound heterozygous mutations in WDR19 in both patients (Patient 1: c.953delA, c.3533G > A, Patient 2: c.2645 + 1G > T, c.3533G > A). Our patients suggest that WDR19 mutations can cause dysplastic kidney in addition to nephronophthisis pathologically. In addition, differences in pathology of the kidneys from WDR19 mutations may result in heterogeneous features in renal ultrasound findings. Renal phenotypes from WDR19 mutations may thus be more diverse than previously reported. Extrarenal manifestations and genetic testing can therefore help to diagnosis this disease more precisely.

Identification of Serine138 Residue in the 4-residue Segment K135K136I137S138 of LukS-I Component of Staphylococcus…

Luk-I produced by Staphylococcus intermedius was found to be a new member of the staphylococcal bi-component pore-forming toxin family, in which staphylococcal leukocidin, Panton-Valentine leukocidin, and γ-hemolysin are included. Luk-I consists of LukS-I and LukF-I. From the deduced amino acid sequence of LukS-I, a 4-residue sequence, K135K136I137S138, at the root of the stem region was found to be identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. A mutant of LukS-I (MLSI-SA), in which the Ser138 residue was replaced by an alanine residue, was created, purified, and assayed for its leukocytolytic and pore-forming activities with LukF-I. Both LukS-I and MLSI-SA formed a ring-shaped complex with LukF-I on rabbit erythrocytes and human polymorphonuclear leukocytes (HPMNLs) membrane. However, MLSI-SA showed no leukocytolytic activity with LukF-I. LukS-I was phosphorylated by protein kinase A in the presence of [γ-32P] ATP in a cell-free system, but MLSI-SA was not phosphorylated significantly. A potent and selective inhibitor of protein kinase A (N- [2(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89)) showed 50% inhibition of the Luk-I-induced cell lysis at 0.5 nM. Thus, it is concluded that the phosphorylation of the Ser138 residue in the 4-residue segment K135K136I137S138 of LukS-I is important for the leukocytolysis of HPMNLs.