Masahito Miyamoto

Identification and Characterization of Major Proteins Carrying ABO Blood Group Antigens in the Human Kidney

Background. It is generally admitted that ABO(H) blood group antigens are linked to lipids and proteins. Although glycolipids carrying ABO antigens have been well characterized in human kidneys, glycoproteins carrying ABO antigens are largely unknown, and their molecular properties remain to be elucidated. Methods. All the blood group A antigen-linked proteins in human kidney could be solubilized and captured on immobilized Helix pomatia lectin that recognizes A antigens. These proteins were separated on SDS-PAGE gels. The gel pieces containing protein bands immunoreactive with anti-A antibody were excised, in-gel digested with trypsin, and analyzed by nanoLC tandem mass spectrometer. Protein candidates that carry ABO antigens were confirmed by immunoprecipitation and double-labeled immunofluorescense microscopy. Results. All the glycoproteins carrying ABO antigens were found to be Asn-linked glycoproteins, and presented as multiple bands on SDS-PAGE with molecular masses ranging from 60 to 270 kDa. The protein bands were subjected for mass spectrometric analysis, which identified 121 distinct proteins with high confidence. Of the identified proteins, 55 N-glycosylated, membrane proteins were selected as glycoprotein candidates that carry ABO antigens. Among them, most abundantly expressed proteins as estimated by the number of peptide matches in the MS spectrometric analysis, such as platelet endothelial cell adhesion molecule 1, plasmalemmal vesicle-associated protein, and von Willebrand factor, were further characterized. Conclusions. Several glycoproteins were identified that represented major glycoproteins carrying ABO antigens in the human kidney, which exhibited distinct features in localization to most of vascular endothelial cells.

Comparison of human glomerulus proteomic profiles obtained from low quantities of samples by different mass spectrometry with the comprehensive database

BackgroundWe have previously constructed an in-depth human glomerulus proteome database from a large amount of sample for understanding renal disease pathogenesis and aiding the biomarker exploration. However, it is usually a challenge for clinical research to get enough tissues for large-scale proteomic characterization. Therefore, in this study, we focused on high-confidence proteomics analysis on small amounts of human glomeruli comparable to those obtained from biopsies using different mass spectrometers and compared these results to the comprehensive database.ResultsOne microgram of human glomerular protein digest was analyzed each on five LC- combined mass spectrometers (LIT-TOF, LTQ-Orbitrap, Q-TOF, LIT and MALDI-TOF/TOF) yielding 139, 185, 94, 255 and 108 proteins respectively identified with strict criteria to ensure high confidence (> 99%) and low false discovery rate (FDR) (< 1%). An integrated profile of 332 distinct glomerular proteins was subsequently generated without discerned bias due to protein physicochemical properties (pI and MW), of which around 60% were detected commonly by more than two LC-MS/MS platforms. Comparative analysis with the comprehensive database demonstrated 14 proteins uniquely identified in this study and more than 70% of identified proteins in small datasets were concentrated to the top abundant 500 in the comprehensive database which consists of 2775 non-redundant proteins.ConclusionThis study showed representative human glomerulus proteomic profiles obtained from biopsies through analysis of comparable amounts of samples by different mass spectrometry. Our results implicated that high abundant proteins are more likely to be reproducibly identified in multiple mass spectrometers runs and different mass spectrometers. Furthermore, many podocyte essential proteins such as nephrin, podocin, podocalyxin and synaptopodin were also identified from the small samples in this study. Bioinformatic enrichment analysis results extended our understanding of the major glomerular proteins about their subcellular distributions and functions. The present study indicated that the proteins localized in certain cellular compartments, such as actin cytoskeleton, mitochondrial matrix, cell surface, basolateral plasma membrane, contractile fiber, proteinaceous extracellular matrix and adherens junction, represent high abundant glomerular proteins and these subcellular structures are also highly significantly over-represented in the glomerulus compared to the whole human background.

Identification and localization of novel genes preferentially expressed in human kidney glomerulus.

Aim:  To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a glomerular cDNA library and verified the reliability of our database by comparison with the Stanford Microarray Database (SMD), followed by reverse transcription polymerase chain reaction (RT‐PCR) and in situ hybridization (ISH).

Human kidney glomerulus proteome and biomarker discovery of kidney diseases

The kidney glomerulus is the site of plasma filtration and production of primary urine in the kidney. The structure not only plays a pivotal role in ultrafiltration of plasma into urine but also is the locus of kidney diseases progressing to chronic renal failure. Patients afflicted with these glomerular diseases frequently progress to irreversible loss of renal function and inevitably require replacement therapies. The diagnosis and treatment of glomerular diseases are now based on clinical manifestations, urinary protein excretion level, and renal pathology of needle biopsy specimens. The molecular mechanisms underlying the progression of glomerular diseases are still obscure despite a great number of clinical and experimental studies. Proteomics is a particularly promising approach for the discovery of proteins relevant to physiological and pathophysiological processes, and has been recently employed in nephrology. Although until now most efforts of proteomic analysis have been conducted with urine, the biological fluid that is easily collected without invasive procedures, proteomic analysis of the glomerulus, the tissue most proximal to the disease loci, is the most straightforward approach. In this review, we attempt to outline the current status of clinical proteomics of the glomerulus and provide a perspective of protein biomarker discovery of glomerular diseases.

Overview of Kidney and Urine Proteome Databases

With the completion or almost completion of genome sequences of many organisms in combination with the tremendous development of mass spectrometric analysis of proteins, several comprehensive proteomic studies, targeting whole organisms, body fluids, organs, tissues, cells, cellular organelles, or functional protein complexes, have produced valuable resources that can be shared and retrieved. In the present review, we provide current concept of construction of protein databases with special emphasis on high-throughput identification of protein using mass spectrometry, annotations, computational tools, and search engines to retrieve information of the identified proteins. We then update the current status of available protein databases of kidney and urine proteomes.

The first case report of peritoneal dialysis related peritonitis caused by Microbacterium paraoxydans.

Peritonitis is still the major complication associated with peritoneal dialysis (PD). Microbacterium spp., a type of coryneform bacteria, is an environmental bacterium isolated from soil, waste water and animals. Human infection is rare, and only few cases have so far been reported in immunocompromised hosts, such as PD patients. Microbacterium paraoxydans, one type of Microbacterium spp. was identified for the first time in 2003. Only two cases of infection of Microbacterium paraoxydans have so far been reported. We herein report the first case of PD-related peritonitis caused by Microbacterium paraoxydans, which was identified by a sequence determination of the 16S rRNA gene. Based on the results of antibiotic sensitivity, the intravenous administration of erythromycin (EM) and oral administration of sulfamethoxazole/trimethoprim (ST) were selected, and PD was interrupted. EM administration was stopped after a total of 14 days. ST was administered for a total of 21 days, and later PD was resumed. Thereafter, no recurrence or relapse of peritonitis without removal of the PD catheter was observed. Microbacterium spp. exhibits multidrug resistance and such an infection is refractory in many cases. We assume that both accurate species identification and the use of antibiotic sensitivity tests are essential to effectively treat this kind of infection.

Percutaneous Transluminal Angioplasty in Japan: Five-Center Investigation:

Purpose Percutaneous transluminal angioplasty (PTA) is the first-line treatment for vascular access stenosis. To our knowledge, multicenter clinical research of PTA has not been reported in Japan. We examined the efficacy and safety of PTA for arteriovenous fistula (AVF) and arteriovenous graft (AVG) in five centers of Japan. Methods Three hundred cases of angioplasty for AVF and 300 for AVG were examined in three centers each. A hundred consecutive patients from each center who underwent PTA for AVG or AVF prior to March 2014 and met the inclusion criteria were searched retrospectively. Primary patency rates were estimated using the Kaplan-Meier method. Results The mean age was 69.3 ± 11.2 years in the AVF group and 70.2 ± 11.9 years in the AVG group. The anatomical success rates were 51.7% (155 of 300) in the AVF group and 72.0% (216 of 300) in the AVG group. The clinical success rates were 99.7% (299 of 300) in the AVF group and 100% (300 of 300) in the AVG group. A total of 25 complications (4.17%) were encountered in both groups including one major complication (0.17%). The primary patency was 99.0% at 1 month, 87.9% at 3 months and 51.7% at 6 months in the AVF group, and 96.0% at 1 month, 64.8% at 3 months and 20.4% at 6 months in the AVG group. Conclusions The clinical success rate of PTA in five centers was relatively high and a major complication rate was only 0.17%. However, anatomical success rates were low comparing with the previous studies and the primary patency rates were inferior to the past data.

Complete resolution of tumoral calcinosis after renal transplantation.

A 41-year-old male receiving hemodialysis for 10 years was referred to our hospital for multiple masses progressively growing in multiple joints and buttocks, which were diagnosed as giant tumoral calcinosis (TC) by radiographic findings. He had been hypercalcemic and hyperphosphatemic with high doses of vitamin D for chronic kidney disease-mineral and bone disorder. We then stopped vitamin D to manage the hypercalcemia and hyperphosphatemia; however, the TC did not regress after 1.5 years, thus the patient underwent renal transplantation. Subsequently, the TC gradually but almost completely disappeared over the next 1.5 years. A renal transplantation was thus found to be useful for the successful treatment of TC.

Association Between Staphylococcus aureus Bacteremia and Hospital Mortality in Hemodialysis Patients With Bloodstream Infection: A Multicenter Cohort From Japanese Tertiary Care Centers

Multiple studies have shown that Staphylococcus aureus bacteremia (SAB) has been a major cause of death in hemodialysis patients. We examined whether SAB is a risk for mortality among chronic hemodialysis patients in Japan where the standard vascular access is arteriovenous fistula (AVF). This was a multicenter, retrospective study of maintenance hemodialysis patients with bloodstream infection (BSI) from 2011 to 2013 at tertiary care centers in Japan. The endpoint was hospital mortality. Our cohort contained 32 SAB cases (14 MRSA and 18 MSSA) and 42 non‐SAB cases. Hospital mortality was higher among SAB cases than non‐SAB cases (46.9% vs. 23.8%, P = 0.038). In patients with BSI, SAB was significantly associated with hospital mortality after adjustment for potential confounders, including type of vascular access (OR 3.26). S. aureus was the leading cause of BSI and hospital mortality among this cohort. Therefore, initial empiric treatment should cover for S. aureus.

Change in skin perfusion pressure after the creation of upper limb arteriovenous fistula for maintenance hemodialysis access

Arteriovenous fistula (AVF) is the most important vascular access method for hemodialysis (HD). However, ischemic steal syndrome occasionally develops. This study evaluated the change in skin perfusion pressure (SPP) after the creation of upper limb AVF and analyzed the relationship between blood flow measurements and the change in SPP. The subjects included 21 patients who underwent radiocephalic AVF creation for the first time between November 2012 and September 2013. We measured SPP on the palm side of the third finger of both hands and assessed blood flow measurements using ultrasound examination before and after the creation of AVF. The subjects consisted of 15 men and 6 women (average age: 65.3 ± 12.7 years, including 12 diabetic patients). Observational period between before and after surgery was 4.9 ± 5.2 days. None of the patients had ischemic steal syndrome after the creation of AVF. Skin perfusion pressure tended to decrease after creation of AVF on the finger of AVF side (100.0 ± 20.9 vs. 87.9 ± 26.5 mmHg, P = 0.063). In contrast, SPP did not change in the limb without AVF (97.9 ± 20.7 vs. 101.0 ± 19.4 mmHg, P = 0.615). The rate of change in SPP was significantly decreased on the finger of AVF side compared with that of limb without AVF (0.055% vs. −0.112%, P = 0.014). There was no correlation between the change in SPP and blood flow measurements. Skin perfusion pressure is possible to detect ischemic steal syndrome after the creation of upper limb AVF.