Kidney injury molecule-1 inhibits metastasis of renal cell carcinoma.

Metastasis is present in approximately 30% of patients diagnosed with renal cell carcinoma (RCC) and is associated with a 5-year survival rate of < 15%. Kidney injury molecule 1 (KIM-1), encoded by the HAVCR1 gene, is a proximal tubule cell-surface glycoprotein and a biomarker for early detection of RCC, but its pathophysiological significance in RCC remains unclear. We generated human and murine RCC cell lines either expressing or lacking KIM-1, respectively, and compared their growth and metastatic properties using validated methods. Surprisingly, KIM-1 expression had no effect on cell proliferation or subcutaneous tumour growth in immune deficient (Rag1-/-) Balb/c mice, but inhibited cell invasion and formation of lung metastasis in the same model. Further, we show that the inhibitory effect of KIM-1 on metastases was observed in both immune deficient and immune competent mice. Transcriptomic profiling identified the mRNA for the pro-metastatic GTPase, Rab27b, to be downregulated significantly in KIM-1 expressing human and murine RCC cells. Finally, analysis of The Cancer Genome Atlas (TCGA) data revealed that elevated HAVCR1 mRNA expression in the two most common types of RCC, clear cell and papillary RCC, tumours correlated with significantly improved overall patient survival. Our findings reveal a novel role for KIM-1 in inhibiting metastasis of RCC and suggests that tumour-associated KIM-1 expression may be a favourable prognostic factor.

Identification of a Cyclospora cayetanensis Oocyst Antigens and Their Validity in the Detection of Immunogenic Patterns of Cyclosporiasis Patients.

INTRODUCTION: The diagnosis of cyclosporiasis is currently based on the microscopic detection of oocysts, which may provide invalid results. The availability of simple, objective immunological screening tests would facilitate epidemiological studies of cyclosporiasis. Therefore, the present study aimed to identify the antigens of Cyclospora cayetanensis oocysts and their validity in serodiagnosis. METHODS: According to parasitological and molecular diagnoses, three study groups were specified. Group (G) I included 30 patients with cyclosporiasis, GII included 12 patients with other parasitic infections, and GIII included 16 healthy subjects. SDS-PAGE was used to analyse C. cayetanensis antigens, and the validity of western blotting and enzyme-linked immunosorbent assays (ELISAs) was then assessed amongst the sera of all study groups. RESULTS: The C. cayetanensis antigenic profile showed eight characteristic bands with molecular weights ranging from 14 to 175 kDa. Western blot analysis of sera revealed 93.3% (28/30 of GI) and 92.8% (26/28 of GII and III) sensitivity and specificity, respectively, dividing the patients in GI into four subgroups. The most frequent diagnostic bands (71.4% of GI sera) showed weights of 26-28 kDa, followed by 71 kDa (53.6%). ELISA sensitivity was 90% (27/30), and specificity was 78.6%. Validation showed perfect agreement between the PCR and western blot results, and ELISA presented substantial agreement with both the PCR and western blot results. CONCLUSIONS: Our findings suggest the existence of high immunogenic diversity in C. cayetanensis and indicate that the 26-28 kDa immunogenic groups may potentially be used as a diagnostic marker of cyclosporiasis. Due to the high validity of ELISA, it might be the test of choice for the routine serodiagnosis of cyclosporiasis.

A Multicenter Assessment of the Sensitivity and Specificity for a Single High-Sensitivity Cardiac Troponin Test at Emergency Department Presentation for Hospital Admission.

BACKGROUND: Studies have illustrated how a low or undetectable high-sensitivity cardiac troponin (hs-cTn) concentration at emergency department (ED) presentation can rule out myocardial infarction (MI). A problem with using an undetectable hs-cTn cutoff is that this value may be defined differently among hospitals and is also difficult to monitor. In the present study, we assess the diagnostic performance of a clinical chemistry score (CCS) vs hs-cTn alone in the presentation blood sample in the ED for patient hospital admission in a multicenter setting. METHODS: From January 1 to June 30, 2018, consecutive patients with random glucose, creatinine (for an estimated glomerular filtration rate calculation), and hs-cTnI (Abbott, 2 hospitals, Hamilton, Ontario, n = 10496) or hs-cTnT (Roche, 4 hospitals, Calgary, Alberta, n = 25177) were assessed for hospital admission with the CCS (range of scores, 0-5) or hs-cTn alone. Sensitivity, specificity, predicative values, and likelihood ratios were calculated for a CCS of 0 and 5 and for hs-cTn alone (hs-cTnI cutoffs, 5 and 26 ng/L; hs-cTnT cutoffs, 6 and 14 ng/L). RESULTS: The CCS of 0 (CCS <1) identified approximately 10% of all patients as low risk and had a sensitivity for hospital admission of nearly 98% as compared to <93% when hs-cTnT (<6 ng/L) or hs-cTnI (<5 ng/L) cutoffs alone were used. A CCS ≥5 had a specificity for hospital admission >95%, with approximately 14% of patients at high risk. CONCLUSIONS: An ED disposition (admit or send home) using the presentation blood sample could occur in nearly 25% of all patients by use of the CCS.

Reducing red blood cell folate testing: a case study in utilisation management.

Mandatory enrichment of wheat flour in Canada with folic acid since 1998 has caused folate deficiency to be rare. There were 3019 red blood cell (RBC) folate tests performed during an 18-month period at London Health Sciences Centre (LHSC)/St. Joseph's Healthcare London (SJHC) without any folate deficiency detected. We implemented a quality improvement initiative to reduce RBC folate testing at LHSC/SJHC. We began with a retrospective review of RBC folate tests performed during the previous 18 months. We identified physicians who had ordered more than five tests during this period and sent them an educational email to inform them of our intentions and solicit their input. We then discontinued RBC folate testing in-house and a pop-up window was introduced to the computerised physician order entry system stating that biochemist approval would be needed before samples would be sent out for testing. During the audited 18-month period, the average monthly test volume was 168 (SD 20). The three departments ordering the most RBC folate testing were nephrology (15%), haematology (7%) and oncology (7%). Physician feedback was supportive of the change, and during the 2 months after targeted email correspondence, the average monthly test volume decreased 24% (p<0.01) to 128 (SD 1). On discontinuation of the test in-house and implementation of the pop-up, the average monthly test volume decreased another 74% (p<0.01) to 3 (SD 2). In the 10 months following discontinuation of the test on-site, there were only 39 RBC folate tests performed with no deficiency detected. This initiative significantly reduced unnecessary RBC folate orders. The change in ordering on email contact suggests that physician education was an important factor reducing overutilisation. However, the most significant decrease came from restricting the test so that only orders approved by a biochemist would be performed.

Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.

Ischemia-reperfusion injury during kidney transplantation predisposes to delayed graft function, rejection, and premature graft failure. Exacerbation of tissue damage and alloimmune responses may be explained by necroinflammation: an autoamplification loop of cell death and inflammation, which is mediated by the release of damage-associated molecular patterns (eg, high-mobility group box-1; HMGB1) from necrotic cells that activate both innate and adaptive immune pathways. Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is upregulated on injured proximal tubular epithelial cells and enables them to clear apoptotic and necrotic cells. Here we show a pivotal role for clearance of dying cells in regulating necroinflammation in a syngeneic murine kidney transplant model. We found persistent KIM-1 expression in KIM-1+/+ kidney grafts posttransplantation. Compared to recipients of KIM-1+/+ kidneys, recipients of KIM-1-/- kidneys exhibited significantly more renal dysfunction, apoptosis and necrosis, tubular obstruction, and graft failure. KIM-1-/- grafts also had more inflammatory cytokines, infiltrating neutrophils, and macrophages compared to KIM-1+/+ grafts. Most significantly, passive release of HMGB1 from apoptotic and necrotic cells led to dramatically higher serum HMGB1 levels and increased proinflammatory macrophages in recipients of KIM-1-/- grafts. Our data identify an endogenous protective mechanism against necroinflammation in kidney grafts that may be of therapeutic relevance in transplantation.

Tctex-1, a novel interaction partner of Kidney Injury Molecule-1, is required for efferocytosis.

Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is specifically upregulated on proximal tubular epithelial cells (PTECs) during acute kidney injury and mitigates tissue damage by mediating efferocytosis (the phagocytic clearance of apoptotic cells). The signaling molecules that regulate efferocytosis in TECs are not well understood. Using a yeast two-hybrid screen, we identified the dynein light chain protein, Tctex-1, as a novel KIM-1-interacting protein. Immunoprecipitation and confocal imaging studies suggested that Tctex-1 associates with KIM-1 in cells at baseline, but, dissociates from KIM-1 within 90 min of initiation of efferocytosis. Interfering with actin or microtubule polymerization interestingly prevented the dissociation of KIM-1 from Tctex-1. Moreover, the subcellular localization of Tctex-1 changed from being microtubule-associated to mainly cytosolic upon expression of KIM-1. Short hairpin RNA-mediated silencing of endogenous Tctex-1 in cells significantly inhibited efferocytosis to levels comparable to that of knock down of KIM-1 in the same cells. Importantly, Tctex-1 was not involved in the delivery of KIM-1 to the cell-surface. On the other hand, KIM-1 expression significantly inhibited the phosphorylation of Tctex-1 at threonine 94 (T94), a post-translational modification which is known to disrupt the binding of Tctex-1 to dynein on microtubules. In keeping with this, we found that KIM-1 bound less efficiently to the phosphomimic (T94E) mutant of Tctex-1 compared to wild type Tctex-1. Surprisingly, expression of Tctex-1 T94E did not influence KIM-1-mediated efferocytosis. Our studies uncover a previously unknown role for Tctex-1 in KIM-1-dependent efferocytosis in epithelial cells.

Lipase or amylase for the diagnosis of acute pancreatitis?

Acute pancreatitis is a rapid onset of inflammation of the pancreas causing mild to severe life threatening conditions [1, 2]. In Canada, acute pancreatitis is the 5th most expensive digestive disease in Canada with a considerable economic burden on the health care system [3]. The diagnosis of acute pancreatitis is usually based on the presence of abdominal pain and elevated levels of serum amylase and/or lipase. Many health care centers use either serum amylase, lipase or both to diagnose acute pancreatitis without considering which one could provide a better diagnostic accuracy. The aim of this review is to investigate whether serum lipase alone is a sufficient biomarker for the diagnosis of acute pancreatitis. We have examined various studies looking at the utilization, sensitivity, specificity and cost associated savings of lipase and amylase in the diagnosis of acute pancreatitis. When comparing different studies, serum lipase offers a higher sensitivity than serum amylase in diagnosing acute pancreatitis. Lipase also offers a larger diagnostic window than amylase since it is elevated for a longer time, thus allowing it to be a useful diagnostic biomarker in early and late stages of acute pancreatitis. Several recent evidence-based guidelines recommend the use of lipase over amylase. Nevertheless, both lipase and amylase alone lack the ability to determine the severity and etiology of acute pancreatitis. The co-ordering of both tests has shown little to no increase in the diagnostic sensitivity and specificity. Thus, unnecessary testing and laboratory expenditures can be reduced by testing lipase alone.

Nested PCR targeting intergenic spacer (IGS) in genotyping of Giardia duodenalis isolated from symptomatic and asymptomatic infected Egyptian school children.

Distinct sequences of Giardia duodenalis assemblages raised the hypothesis that certain assemblages may contribute to its clinical outcome. However, sequences analysis is time consuming, expensive, and needs many manual operations. Nested PCR targeting intergenic spacer (IGS) region was applied successfully to genotype G. duodenalis. This study aimed to identify the prevalence of G. duodenalis assemblages among giardiasis school children and its relation to the presence of symptoms using nested IGS/PCR. Of 65 microscopically confirmed Giardia-positive samples, 65 samples were genotyped proving high sensitivity (92.3%) of IGS/PCR. Negative IGS/PCR samples were also negative for ß-giardin gene. Subassemblage AI was the commonest with 66.6% (20/30) among asymptomatic children compared to 53.3% (16/30) of symptomatic, while assemblage B was found in 40% (12/30) of symptomatic compared to 20% (6/30) of asymptomatic. The difference was significant. AII was only found in asymptomatic with 13.4% (4/30), while mixed infections (AI&B) were recorded only in 6.6% (2/30) of symptomatic group. A significant relation was found between younger children susceptibility for AI and B infections as presented in 77.7 (12/16) and 83.3% (10/12) of symptomatic, respectively, and 80 (16/80) and 33.4% (2/4) of asymptomatic, respectively. Significant relations were found between AI with intermittent diarrhea and B with chronic. A significant relation was found between assemblage distributions and heavy infection intensity. In conclusion, higher incidence of assemblage B among symptomatic children compared to asymptomatic could denote its possible pathogenic potential.

G protein α12 (Gα12) is a negative regulator of kidney injury molecule-1-mediated efferocytosis.

Kidney injury molecule-1 (KIM-1) is a receptor for the "eat me" signal, phosphatidylserine, on apoptotic cells. The specific upregulation of KIM-1 by injured tubular epithelial cells (TECs) enables them to clear apoptotic cells (also known as efferocytosis), thereby protecting from acute kidney injury. Recently, we uncovered that KIM-1 binds directly to the α-subunit of heterotrimeric G12 protein (Gα12) and inhibits its activation by reactive oxygen species during renal ischemia-reperfusion injury (Ismail OZ, Zhang X, Wei J, Haig A, Denker BM, Suri RS, Sener A, Gunaratnam L. Am J Pathol 185: 1207-1215, 2015). Here, we investigated the role that Gα12 plays in KIM-1-mediated efferocytosis by TECs. We showed that KIM-1 remains bound to Gα12 and suppresses its activity during phagocytosis. When we silenced Gα12 expression using small interefering RNA, KIM-1-mediated engulfment of apoptotic cells was increased significantly; in contrast overexpression of constitutively active Gα12 (QLGα12) resulted in inhibition of efferocytosis. Inhibition of RhoA, a key effector of Gα12, using a chemical inhibitor or expression of dominant-negative RhoA, had the same effect as inhibition of Gα12 on efferocytosis. Consistent with this, silencing Gα12 suppressed active RhoA in KIM-1-expressing cells. Finally, using primary TECs from Kim-1+/+ and Kim-1-/- mice, we confirmed that engulfment of apoptotic cells requires KIM-1 expression and that silencing Gα12 enhanced efferocytosis by primary TECs. Our data reveal a previously unknown role for Gα12 in regulating efferocytosis and that renal TECs require KIM-1 to mediate this process. These results may have therapeutic implications given the known harmful role of Gα12 in acute kidney injury.

Kidney injury molecule-1 protects against Gα12 activation and tissue damage in renal ischemia-reperfusion injury.

Ischemic acute kidney injury is a serious untreatable condition. Activation of the G protein α12 (Gα12) subunit by reactive oxygen species is a major cause of tissue damage during renal ischemia-reperfusion injury. Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein that is highly up-regulated during acute kidney injury, but the physiologic significance of this up-regulation is unclear. Here, we report for the first time that Kim-1 inhibits Gα12 activation and protects mice against renal ischemia-reperfusion injury. We reveal that Kim-1 physically interacts with and inhibits cellular Gα12 activation after inflammatory stimuli, including reactive oxygen species, by blocking GTP binding to Gα12. Compared with Kim-1(+/+) mice, Kim-1(-/-) mice exhibited greater Gα12 and downstream Src activation both in primary tubular epithelial cells after in vitro stimulation with H2O2 and in whole kidneys after unilateral renal artery clamping. Finally, we show that Kim-1-deficient mice had more severe kidney dysfunction and tissue damage after bilateral renal artery clamping, compared with wild-type mice. Our results suggest that KIM-1 is an endogenous protective mechanism against renal ischemia-reperfusion injury through inhibition of Gα12.

Accelerated receptor shedding inhibits kidney injury molecule-1 (KIM-1)-mediated efferocytosis.

Efficient clearance of apoptotic cells (efferocytosis) prevents inflammation and permits repair following tissue injury. Kidney injury molecule-1 (KIM-1) is a receptor for phosphatidylserine, an "eat-me" signal exposed on the surface of apoptotic cells that marks them for phagocytic clearance. KIM-1 is upregulated on proximal tubule epithelial cells (PTECs) during ischemic acute kidney injury (AKI), enabling efferocytosis by surviving PTECs. KIM-1 is spontaneously cleaved at its ectodomain region to generate a soluble fragment that serves a sensitive and specific biomarker for AKI, but the biological relevance of KIM-1 shedding is unknown. Here, we sought to determine how KIM-1 shedding might regulate efferocytosis. Using cells that endogenously and exogenously express KIM-1, we found that hydrogen peroxide-mediated oxidative injury or PMA treatment accelerated KIM-1 shedding in a dose-dependent manner. KIM-1 shedding was also accelerated when apoptotic cells were added. Accelerated shedding or the presence of excess soluble KIM-1 in the extracellular milieu significantly inhibited efferocytosis. We also identified that TNF-α-converting enzyme (TACE or ADAM17) mediates both the spontaneous and PMA-accelerated shedding of KIM-1. While accelerated shedding inhibited efferocytosis, we found that spontaneous KIM-1 cleavage does not affect the phagocytic efficiency of PTECs. Our results suggest that KIM-1 shedding is accelerated by worsening cellular injury, and excess soluble KIM-1 competitively inhibits efferocytosis. These findings may be important in AKI when there is severe cellular injury.

Prevalence and clinical features of Dientamoeba fragilis infections in patients suspected to have intestinal parasitic infection.

The present study was conducted to determine the prevalence and clinical features of dientamoebiasis in patients presumed to be infected with intestinal parasites. A total of 168 patients were examined for D. fragilis using microscopy (after Wheatley's trichrome staining) and culture (using modified Boeck and Drbohlav's medium). D. fragilis trophozoites were detected in 15 samples (8.9%) examined using trichrome staining and in 50 samples (29.8%) by culture method. Other enteric parasites were common in the study population as 48.8% of patients (82/168) were found harboring intestinal parasites. Blastocystis hominis was the most common, identified in 33.3% (56/168) of the samples. Giardia lamblia was detected in 17.9% (30/168) and E. histolytica/E. dispar in 11.9% (20/168). The symptoms most frequently encountered were diarrhea, abdominal pain and weight loss and fatigue. Diarrhea and abdominal pain were significantly more frequent in patients with dientamoebiasis compared to non pathogenic cases (P < 0.05). Diarrhea was 38.5% of patients infected with D. fragilis compared to 50% of patients infected with G. lamblia, while abdominal pain was encountered with D. fragilis in 41% compared to 33.3% with G. lamblia. These differences were insignificant (P > 0.05).