The role of urinary KIM-1, NGAL,CA19-9 and β2-microglobulin in the assessment of ureteropelvic junction obstruction in adults

Purpose: The objective of this study is to evaluate the diagnostic properties of urinary biomarkers in adults with ureteropelvic junction obstruction: KIM-1, NGAL, CA19-9, and β2-microglobulin. We also assessed urinary biomarker concentrations following pyeloplasty.

Material and methods: We prospectively studied adults from December 2013 to February 2015. We included 47 patients with a mean age of 38.6?±?12.7 years. Each patient provided four samples of voided urine for biomarker measurement, one at pre-operative consultation and the others at 1, 3, and 6 months of post-operative follow-up. The control group consisted of 40 healthy individuals with no hydronephrosis on ultrasound evaluation.

Results: KIM-1 had an area under the curve of 0.79 (95% CI 0.70–0.89), NGAL 0.71 (95% CI 0.61–0.83), CA19-9 0.70 (95% CI 0.60–0.81), and β2-microgloblin 0.61 (95% CI 0.50–0.73). KIM-1 was the most sensitive marker with a cut-off of 170.4?pg/mg creatinine (sensitivity 91.4%, specificity 59.1%), whereas CA19-9 was the most specific with a cut-off of 51.3?U/mg creatinine (sensitivity 48.9%, specificity 88.0%). Urinary concentrations of biomarkers decreased after pyeloplasty.

Conclusions: The evaluation of urinary biomarkers is useful in adults undergoing pyeloplasty. KIM-1, NGAL, and CA19-9 were elevated and significantly decreased after surgery.     

Association study of the β2-adrenergic receptor gene polymorphisms and hypertension in the Northern Han Chinese

Background

The β2-adrenergic receptor (ADRB2) gene has been widely researched as a candidate gene for essential hypertension (EH), but no consensus has been reached in different ethnicities. The aim of the present study was to evaluate the possible association between the ADRB2 gene polymorphisms and the EH risk in the Northern Han Chinese population.

Methodology/Principal Findings

This study included 747 hypertensive subjects and 390 healthy volunteers as control subjects in the Northern Han Chinese. Genotyping was performed to identify the C-47T, A46G and C79G polymorphisms of the ADRB2 gene. G allelic frequency of A46G polymorphism was significantly higher in hypertensive subjects (P = 0.011, OR = 1.287, 95%CI [1.059–1.565]) than that in controls. Significant association could also be found in dominant genetic model (GG+AG vs. AA, P = 0.006, OR = 1.497, 95%CI [1.121–1.998]), in homozygote comparison (GG vs. AA, P = 0.025, OR = 1.568, 95%CI [1.059–2.322]), and in additive genetic model (GG vs. AG vs. AA, P = 0.012, OR = 1.282, 95%CI [1.056–1.555]). Subgroup analyses performed by gender suggested that this association could be found in male, but not in female. Stratification analyses by obesity showed that A46G polymorphism was related to the prevalence of hypertension in the obese population (GG vs. AG vs. AA, P<0.001, OR = 1.645, 95%CI [1.258–2.151]). Significant interaction was found between A46G genotypes and body mass index on EH risk. No significant association could be found between C-47T or C79G polymorphism and EH risk. Linkage disequilibrium was detected between the C-47T, A46G and C79G polymorphisms. Haplotype analyses observed that the T-47-A46-C79 haplotype was a protective haplotype for EH, while the T-47-G46-C79 haplotype increased the risk.

Conclusions/Significances

We revealed that the ADRB2 A46G polymorphism might increase the risk for EH in the Northern Han Chinese population.     

The CD39-adenosinergic axis in the pathogenesis of renal ischemia–reperfusion injury

Hypoxic injury occurs when the blood supply to an organ is interrupted; subsequent reperfusion halts ongoing ischemic damage but paradoxically leads to further inflammation. Together this is termed ischemia–reperfusion injury (IRI). IRI is inherent to organ transplantation and impacts both the short- and long-term outcomes of the transplanted organ. Activation of the purinergic signalling pathway is intrinsic to the pathogenesis of, and endogenous response to IRI. Therapies targeting the purinergic pathway in IRI are an attractive avenue for the improvement of transplant outcomes and the basis of ongoing research. This review aims to examine the role of adenosine receptor signalling and the ecto-nucleotidases, CD39 and CD73, in IRI, with a particular focus on renal IRI.     

The role of TRPM2 in pancreatic β-cells and the development of diabetes

TRPM2 is a Ca²⁺-permeable non-selective cation channel that can be activated by adenosine dinucleotides, hydrogen peroxide, or intracellular Ca²⁺. The protein is expressed in a wide variety of cells, including neurons in the brain, immune cells, endocrine cells, and endothelial cells. This channel is also well expressed in β-cells in the pancreas. Insulin secretion from pancreatic β-cells is the primary mechanism by which the concentration of blood glucose is reduced. Thus, impairment of insulin secretion leads to hyperglycemia and eventually causes diabetes. Glucose is the principal stimulator of insulin secretion. The primary pathway involved in glucose-stimulated insulin secretion is the ATP-sensitive K⁺ (K_ATP) channel to voltage-gated Ca²⁺ channel (VGCC)-mediated pathway. Increases in the intracellular Ca²⁺ concentration are necessary for insulin secretion, but VGCC is not sufficient to explain [Ca²⁺]_i increases in pancreatic β-cells and the resultant secretion of insulin. In this review, we focus on TRPM2 as a candidate for a [Ca²⁺]_i modulator in pancreatic β-cells and its involvement in insulin secretion and development of diabetes. Although further analyses are needed to clarify the mechanism underlying TRPM2-mediated insulin secretion, TRPM2 could be a key player in the regulation of insulin secretion and could represent a new target for diabetes therapy.     

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

Summary This paper reports our experience of molecular screening and fetal diagnosis of -thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [39 (CT); 6 (-A); ⁺-87 (CG); ⁺ IVSI nt 110 (GA); IVSI nt 1 (GA); ⁺ IVSI nt 6 (TC); IVSII nt 1 (GA); ⁺ IVSII nt 745 (CG)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [76 (-C); ⁺ IVSI nt 5 (GA); ⁺ IVSI nt 5 (GC); ⁺ IVSI -1 (cod 30) (GC); ⁺-87 (CT), -290 bp del.; ⁺-101 (CT)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the -globin gene ( IVSII nt 850-1 bp). In the remaining four cases, the -globin gene showed entirely normal sequences and the -globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of -thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of -thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.     

Excretion of urinary cadmium, copper, and zinc in cadmium-exposed and nonexposed subjects, with special reference to urinary excretion of β2-microglobulin and metallothioneinand metallothionein

The objectives of this study were to examine the association between urinary excretion of cadmium (U-Cd), copper (U-Cu), and zinc (U-Zn) and the severity of two different indicators of renal toxicity (urinary excretion of beta2-microglobulin [U-beta2-MG] and metallothionein [U-MT]) in Cd-exposed subjects compared to controls, and to assess the physiologic mechanisms by which the exposure to environmental Cd affects U-Cd, U-Cu, and U-Zn. The target population included 3508 Cd-exposed and 294 nonexposed participants who received a health survey conducted among the population of the Kakehashi River basin. Increases of U-Cd, U-beta2-MG, and U-MT in the Cd-exposed population were observed relative to excretion of these substances in controls. Regression analysis using a general linear model revealed that the correlations between U-Cd or U-Cu, and U-beta2-MG and between U-Cd, U-Cu or U-Zn, and U-MT were statistically significant in both sexes, but the correlation between U-Zn and U-beta2-MG excretion was significant only in men. These results suggest U-Cd and U-Cu is affected by dysfunction in renal tubular absorption (indicated by U-beta2-MG), whereas not only U-Cd and U-Cu but also U-Zn appear to be a function of renal cellular desquamation (indicated by U-MT).     

Immunofluorescence and ultrastructural localization of β2-microglobulin in human renal allografts

Summary The localization of 2-microglobulin (2m) was studied in renal biopsies from 18 patients with pathological transplanted kidney using immunofluorescence and electron-immunohistochemical techniques; the renal biopsies of 4 cases with normal kidneys were used as controls. Using immunofluorescence, 2m was not observed in the normal kidneys, 2m was found in the glomeruli (7 cases) and the tubular epithelium (16 cases) of the transplanted kidneys. Using immunoelectron microscopy, some labelling of the normal kidneys was observed mainly along the cell coat of foot processes and in tubular-epithelial lysosomes. In the glomeruli of transplanted kidneys, particularly in cases of acute or chronic rejection, 2m was most frequently localized on the outer layer of the basement membrane and along the cell coat of foot processes. The brush border of the proximal tubules and the lysosomal structures were intensely labelled. Although immunoelectron-microscopy studies are unable to discriminate between the localization of 2m in normal and transplanted kidneys, these findings nevertheless suggest the glomerular filtration of 2m and its metabolism in the tubular epithelium.     

Nucleation of β-rich oligomers and β-barrels in the early aggregation of human islet amyloid polypeptide

The self-assembly of human islet amyloid polypeptide (hIAPP) into β-sheet rich amyloid aggregates is associated with pancreatic β-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into β-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than β-sheets, consistent with ensemble-based experimental measurements. However, in ~4–6% independent simulations, β-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These β-rich oligomers could adopt β-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these β-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. β-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.     

Is there a role for renal alpha 2-adrenoceptors in the pathogenesis of hypertension?

Current information suggests that alpha 2-adrenoceptors do not directly influence vascular resistance or Na reabsorption in the rat kidney. To reexamine the effects of alpha 2-agonists we used isolated rat kidneys perfused at 37.5 degrees C with precise measurement of renal artery pressure and flow. The recirculating perfusate contained pyruvate as the sole metabolic substrate which enabled us to use gluconeogenesis as an index of proximal tubular alpha 1-responses. Clonidine and guanfacine in 100 nM concentrations decreased phosphate excretion without altering Na, Cl, or K reabsorption or gluconeogenesis; 500 nM concentrations increased vascular resistance and decreased glomerular filtration rate and Na, Cl, and K excretion with no significant effect on gluconeogenesis. Prior thyroparathyroidectomy prevented the antiphosphaturic but not the antinatriuretic or vascular responses. Clonidine, an alpha 2-agonist with some alpha 1-activity, was a more potent vasoconstrictor than methoxamine or guanfacine. In the presence of prazosin (1 microM), norepinephrine (60 nM) stimulated phosphate reabsorption; norepinephrine alone did not stimulate phosphate reabsorption which indicates alpha 1-antagonism of this alpha 2-response to NE. These results and a literature review suggest that increased renal alpha 2-adrenoceptors could raise renal vascular resistance, reduce renin secretion, and antagonize parathyroid hormone effects on Pi, Ca, HCO3, and Na reabsorption to produce a low renin type of hypertension with increased proximal Na reabsorption and abnormal Ca and Pi excretion.     

Soluble guanylate cyclase isoenzymes: The expression of α1, α2, β1, and β2 subunits in the benign and malignant breast tumors

Soluble guanylate cyclase (sGC) encompasses α and β subunits. This study examined the expression of α1, α2, β1, and β2 subunits in the malignant and benign breast tumors using the Western blot analysis. Both benign and malignant tumors showed a significantly higher expression of the α1 subunit in comparison with normal tissues (p < 0.0001). In contrast, the expression of α2 and β2 sGC were significantly lower in these tumors than normal tissues (p < .0015 and p < .001, p < .007 and p < .0001, respectively). The expression level of α1 sGC was significantly correlated with ER + PR+ (p < .0001). A significant correlation was also detected for sGC-α1 and -α2 expression with c-erbB2-negative status (p < .01). However, the expression level of sGC was not associated with tumor stage, tumor grade, or other clinicopathological features. In conclusion, as the expression of α1 sGC is upregulated and α2 and β2 sGC are downregulated in malignant breast tumors. Variations in the expression of sGC isoenzymes may be suggested as an indicator to confirm the enzyme antitumor activity.     

Immunohistochemical and in situ hybridization analysis of galectin-3, a β-galactoside binding lectin, in the urinary system of adult mice

Galectin is an animal lectin that has high affinity to β-galactoside of glycoconjugates. In the present study, cellular expression of galectin subtypes in the urinary system of adult mice was examined by in situ hybridization and immunohistochemistry. The major subtype expressed in the murine urinary system was galectin-3, which was expressed continuously from the kidney to the distal end of the urethra. The renal cortex expressed galectin-3 more intensely than the medulla. Renal galectin-3 immunoreactivity was strongest in the cortical collecting ducts, where principal cells were the sole cellular source. All cell layers of the transitional epithelium from the renal pelvis to the urethra strongly expressed galectin-3 at the mRNA and protein levels. An electron microscopic study demonstrated diffuse cytoplasmic localization of galectin-3 in principal cells of the collecting ducts and in the bladder epithelial cells. Urethral galectin-3 expression at the pars spongiosa decreased in intensity near the external urethral orifice, where the predominant subtype of galectin was substituted by galectin-7. The muscular layer of the ureter and urinary bladder contained significant signals for galectin-1. Taken together, the observations indicate that the adult urinary system shows intense and selective expression of galectin-3 in epithelia of the uretic bud- and cloaca-derivatives.     

The role of β-dimethylsulphoniopropionate,glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis

The tertiary sulphonium compound, -dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organic solutes from the cells. The ions K⁺, Na⁺, Cl^- and the above organic solutes can account for the osmotic balance of the cells.Abbreviations DMSP -dimethylsulphoniopropionate - _i intracellular osmolality - _o extracellular osmolality     

Combination of urinary kidney injury molecule-1 and interleukin-18 as early biomarker for the diagnosis and progressive assessment of acute kidney injury following cardiopulmonary bypass surgery: a prospective nested case–control study

The aim of this nested case–control study was to assess the combined use of urinary kidney injury molecule (KIM)-1 and interleukin (IL)-18 for acute kidney injury (AKI) after cardiopulmonary bypass surgery (CPB). From a cohort of 122 subjects who underwent CPB, serial urinary KIM-1 and IL-18 concentrations were determined in 30 AKI and 92 non-AKI patients. An increased level of urinary KIM-1 was associated with the occurrence of AKI, whereas an increased level of IL-18 was related to progressive AKI. The combination of these two biomarkers facilitates the early diagnosis and assessment of the likely progression of AKI after CPB.     

The Effect of Dietary β-Sitosterol and β-Sitostanol on the Metabolism of Cholesterol in Rats

Effects of dietary β-sitosterol (S) and β-sitostanol (HS) on the metabolism and fate of labeled cholesterol intravenously injected were compared in rats fed diets high in cholesterol. Kinetic behavior of the decay curve for serum cholesterol in the HS supplemented (C + HS) group approximated to that in the cholesterol-free (control) group. The largest dilution of the label was observed in rats of the cholesterol (C) group and the least in the C + HS group, the C + S group being intermediate. The specific activity of hepatic cholesterol was in the decreasing order of the C + HS, C + S and C groups, while the situation was reversed when expressed in terms of net incorporation. Thus, cholesterol pool seemed to be much smaller in the C + HS group than in the C + S group.

In a long term feeding experiment with diets free of cholesterol, HS exhibited significantly greater hypocholesterolemic activity than S did.

These data, together with those reported previously, indicated that inhibitory effect on the absorption of both endogenous and exogenous cholesterol was much more greater in HS than in S.     

Alterations in the expression of the β-cytoplasmic and the γ-smooth muscle actins in hypertrophied urinary bladder smooth muscle

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.     

The β-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans

The Wnt and Src pathways are widely used signal transduction pathways in development. β-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans β-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.     

Increased urinary excretion of nephrin, podocalyxin, and βig-h3 in women with preeclampsia

Emerging evidence has shown that podocyte injury and reduced specific podocyte protein expressions contribute to proteinuria in preeclampsia. We collected urine specimens from women with preeclampsia to study whether podocyte-specific protein shedding is associated with renal barrier dysfunction. Urine specimens from women with normal pregnancies and from pregnant women complicated by chronic hypertension were used for comparison. We determined soluble podocyte slit protein nephrin levels in the urine specimens. Podocalyxin, βig-h3, and VEGF concentrations were also measured. We found that nephrin and podocalyxin were barely detectable in the urine specimens from normal pregnant women and from women with chronic hypertension. In preeclampsia, urinary nephrin and podocalyxin concentrations were significantly increased and highly correlated to each other, r(2) = 0.595. Nephrin and podocalyxin were also correlated with urine protein concentrations. βig-h3 was detected in the urine specimens from women with preeclampsia, and it is highly correlated with nephrin and podocalyxin concentrations in preeclampsia. βig-h3 was undetectable in normal pregnancy and pregnancy complicated by chronic hypertension. Elevated VEGF levels were also found in women with preeclampsia compared with those of normal pregnancy and pregnancy complicated by chronic hypertension. These results provide strong evidence that podocyte protein shedding occurs in preeclampsia, and their levels are associated with proteinuria. The finding of urinary βig-h3 excretion in preeclampsia suggests that increased transforming growth factor activity might also be involved in the kidney lesion in this pregnancy disorder.     

The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on βB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of βB2-crystallin Greek-key motif 4. All of the three mutations promoted βB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected βB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of β-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between β-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of β-crystallins are achieved by the precisely regulated oligomeric equilibrium.