Assessment of two glycated hemoglobin immunoassays / Evaluación de 2 inmunoensayos para hemoglobina glucosilada

INTRODUCTION: Glycated hemoglobin (HbA1c) level reflects chronic glycemic status if reliable tests are used, however, in some regions worldwide high performing assays might not be readily available. This study aimed to asses two HbA1c immunoassays, comparing them with high-performance liquid chromatography (HPLC) assay, three methods available in Ecuador. MATERIAL AND METHODS: HbA1c were measured in 114 fresh whole blood-samples by DCA-Vantage point-of-care analyzer, I-Chroma portable fluorescent scanner immunoassay and BioRad Variant II Turbo HPLC. Normal and pathological HbA1c ranges were included. Blood samples with variants of hemoglobin were excluded. HbA1c values were expressed in National Glycohemoglobin Standardization Program percentages and mmol/mol, as mean±standard deviation. RESULTS: HbA1c results by HPLC and DCA-Vantage were similar: 6.3 ± 1.7% (45 ± 18.6 mmol/mol) vs.6.3 ± 1.8% (45 ± 19.7 mmol/mol), respectively, P = 0.057; while HbA1c values by I-Chroma were lower than HPLC, 5.8 ± 1.9% (40 ± 20.8 mmol/mol), P < 0.001. The coefficient of variation was below 2% for high and low HbA1c levels, in all methods studied. HbA1c values by HPLC and DCA-Vantage were highly correlated (Spearman's Rank Correlation [SRC]: 0.916), while the correlation among HPLC and I-Chroma was weak (SRC: 0.368). The mean bias between DCA-Vantage and HPLC was -0.02 ± 0.29% (-0.2 ± 3.2 mmol/mol), while for I-Chroma and HPLC mean bias was -0.50 ± 1.62% (- 5.5 ± 17.7mmol/mol). CONCLUSIÓN: HbA1c immunoassays DCA-Vantage was comparable to HPLC assay, showing good correlation, appropriate precision and low bias, whereas I-Chroma assay was precise but inaccurate. Therefore, DCA-Vantage has better performance than I-Chroma. These findings suggest that is convenient to assess the HbA1c immunoassays commercially available in our country, Ecuador

INTRODUCCIÓN: El nivel de hemoglobina glucosilada (HbA1c) refleja el estado glucémico crónico si se utilizan pruebas confiables. En algunas regiones del mundo los métodos de alto desempeño para medir la HbA1c no son fácilmente accesibles. Nuestros objetivos fueron evaluar 2 inmunoensayos, comparándolos con la cromatografía líquida de alta resolución (HPLC, por sus siglas en inglés), 3 ensayos disponibles en Ecuador. MATERIALES Y MÉTODOS: En 114 muestras de sangre entera medimos la HbA1c por DCA Vantage (R), escáner fluorescente i-Chroma (R) y HPLC Bio-Rad Variant II® Turbo. Incluimos valores normales y patológicos de HbA1c. Excluimos muestras con variantes de la hemoglobina. La HbA1c fue expresada en porcentaje según el Programa Nacional de Estandarización de la Glicohemoglobina y en mmol/mol (media± desviación estándar). RESULTADOS: La HbA1c medida por HPLC y DCA Vantage (R) fue semejante: 6,3 ± 1,7% (45 ± 18,6 mmol/mol) y 6,3 ± 1,8% (45 ± 19,7 mmol/mol), respectivamente, p = 0,057; pero la cuantificada por i-Chroma(R) fue menor a HPLC, 5,8 ± 1,9% (40 ± 20,8 mmol/mol), p < 0,001. El coeficiente de variación fue menor al 2% en los 3 ensayos estudiados. Los valores de HbA1c obtenidos por HPLC y DCA Vantage(R) estuvieron fuertemente correlacionados (correlación de Spearman [CS]: 0,916), mientras que la correlación entre HPLC y i-Chroma (R) fue débil (CS: 0,368). El sesgo medio entre DCA Vantage ® y HPLC fue -0,02 ± 0,29% (- 0,2 ± 3,2 mmol/mol), en cambio, entre i-Chroma(R) y HPLC fue -0,50 ± 1,62% (-5,5 ± 17,7mmol/mol). CONCLUSIÓN: El inmunoensayo DCA Vantage (R) fue comparable a HPLC, mostrando buena correlación, apropiada precisión y bajo sesgo, mientras que i-Chroma (R) fue preciso, pero inexacto. Por lo tanto, DCA Vantage (R) tiene mejor desempeño que i-Chroma (R). Estos hallazgos sugieren que es conveniente evaluar los inmunoensayos comercialmente disponibles en nuestro país, Ecuador

Assessment of two glycated hemoglobin immunoassays.

INTRODUCTION: Glycated hemoglobin (HbA1c) level reflects chronic glycemic status if reliable tests are used, however, in some regions worldwide high performing assays might not be readily available. This study aimed to asses two HbA1c immunoassays, comparing them with high-performance liquid chromatography (HPLC) assay, three methods available in Ecuador. MATERIAL AND METHODS: HbA1c were measured in 114 fresh whole blood-samples by DCA-Vantage point-of-care analyzer, I-Chroma portable fluorescent scanner immunoassay and BioRad Variant II Turbo HPLC. Normal and pathological HbA1c ranges were included. Blood samples with variants of hemoglobin were excluded. HbA1c values were expressed in National Glycohemoglobin Standardization Program percentages and mmol/mol, as mean±standard deviation. RESULTS: HbA1c results by HPLC and DCA-Vantage were similar: 6.3±1.7% (45±18.6mmol/mol) vs. 6.3±1.8% (45±19.7mmol/mol), respectively, P=0.057; while HbA1c values by I-Chroma were lower than HPLC, 5.8±1.9% (40±20.8mmol/mol), P<0.001. The coefficient of variation was below 2% for high and low HbA1c levels, in all methods studied. HbA1c values by HPLC and DCA-Vantage were highly correlated (Spearman's Rank Correlation [SRC]: 0.916), while the correlation among HPLC and I-Chroma was weak (SRC: 0.368). The mean bias between DCA-Vantage and HPLC was -0.02±0.29% (-0.2±3.2mmol/mol), while for I-Chroma and HPLC mean bias was -0.50±1.62% (-5.5±17.7mmol/mol). CONCLUSION: HbA1c immunoassays DCA-Vantage was comparable to HPLC assay, showing good correlation, appropriate precision and low bias, whereas I-Chroma assay was precise but inaccurate. Therefore, DCA-Vantage has better performance than I-Chroma. These findings suggest that is convenient to assess the HbA1c immunoassays commercially available in our country, Ecuador.

Podocyte vascular endothelial growth factor (Vegf164) overexpression causes severe nodular glomerulosclerosis in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: The pathogenic role of excessive vascular endothelial growth factor (VEGF)-A in diabetic nephropathy has not been defined. We sought to test whether increased podocyte VEGF-A signalling determines the severity of diabetic glomerulopathy. METHODS: Podocyte-specific, doxycycline-inducible Vegf164 (the most abundant Vegfa isoform) overexpressing adult transgenic mice were made diabetic with low doses of streptozotocin and examined 12 weeks after onset of diabetes. We studied diabetic and non-diabetic transgenic mice fed a standard or doxycycline-containing diet. VEGF-A and albuminuria were measured by ELISA, creatinine was measured by HPLC, renal morphology was examined by light and electron microscopy, and gene expression was assessed by quantitative PCR, immunoblotting and immunohistochemistry. RESULTS: Podocyte Vegf164 overexpression in our mouse model of diabetes resulted in advanced diabetic glomerulopathy, characterised by Kimmelstiel-Wilson-like nodular glomerulosclerosis, microaneurysms, mesangiolysis, glomerular basement membrane thickening, podocyte effacement and massive proteinuria associated with hyperfiltration. It also led to increased VEGF receptor 2 and semaphorin3a levels, as well as nephrin and matrix metalloproteinase-2 downregulation, whereas circulating VEGF-A levels were similar to those in control diabetic mice. CONCLUSIONS/INTERPRETATION: Collectively, these data demonstrate that increased podocyte Vegf164 signalling dramatically worsens diabetic nephropathy in a streptozotocin-induced mouse model of diabetes, resulting in nodular glomerulosclerosis and massive proteinuria. This suggests that local rather than systemic VEGF-A levels determine the severity of diabetic nephropathy and that semaphorin3a signalling and matrix metalloproteinase-2 dysregulation are mechanistically involved in severe diabetic glomerulopathy.

Semaphorin3a disrupts podocyte foot processes causing acute proteinuria.

Semaphorin3a was discovered as a secreted guidance protein that acts as a chemorepellent to migrating axons and endothelial cells. In the adult mouse kidney, it is expressed in podocytes and collecting tubules. Here, we show that exogenous semaphorin3a caused acute nephrotic range proteinuria associated with podocyte foot process effacement and fusion, endothelial cell damage, decreased vascular endothelial growth factor-A receptor expression, and downregulation of the slit-diaphragm proteins podocin, nephrin, and CD2-associated protein. When vascular endothelial growth factor 165 was administered at the same time as Semaphorin3a, no proteinuria or renal ultrastructural abnormalities occurred, suggesting that semaphorin3a effects may be mediated, in part, by downregulation of vascular endothelial growth factor receptor 2 signaling. Our findings indicate that a balance of semaphorin3a to vascular endothelial growth factor-A may be important for glomerular filtration barrier homeostasis.

Autocrine class 3 semaphorin system regulates slit diaphragm proteins and podocyte survival.

Class 3 semaphorins are guidance proteins that play crucial roles during development. Semaphorins 3A (sema 3A) and 3F are expressed by podocytes in vivo throughout ontogeny and their function is unknown. Here we examined the expression of class 3 semaphorins (3A, 3B, 3C, 3D, 3E, and 3F) and their receptors (neuropilins 1 and 2, plexins A1, A2, A3, B2, and D1) in undifferentiated and differentiated mouse podocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). All class 3 semaphorins, neuropilins 1 and 2 are expressed by undifferentiated and differentiated podocytes at similar levels. Differentiated podocytes expressed 2-4-fold higher plexin A1, A2, and A3 mRNA levels than undifferentiated podocytes. To examine semaphorin regulation, we exposed podocytes to recombinant sema 3A. Sema 3A decreased semaphorin 3B, plexin A1, A3, and D1 >/=50% and reduced plexin A2 mRNA to undetectable levels. To identify sema 3A function in podocytes, we examined whether sema 3A regulates slit diaphragm proteins and podocyte survival. Sema 3A induced a dose-response podocin downregulation and decreased its interaction with CD2-associated protein and nephrin, as determined by Western analysis and co-immunoprecipitation. To evaluate sema 3A role in podocyte survival, we quantified podocyte apoptosis using a caspase 3 activity marker. Sema 3A induced a 10-fold increase in podocyte apoptosis and significantly decreased the activity of the Akt survival pathway. Our data indicate that (1) immortalized podocytes in culture have a functional autocrine semaphorin system that is regulated by differentiation and ligand availability; (2) sema 3A signaling regulates the expression and interactions of slit-diaphragm proteins and decreases podocyte survival.

VEGF spatially directs angiogenesis during metanephric development in vitro.

Vascular endothelial growth factor (VEGF) is required for endothelial cell differentiation, vasculogenesis, and normal glomerular vascularization. To examine whether VEGF plays a role as a chemoattractant for the developing kidney vasculature, avascular metanephric kidneys from rat embryos (E14) were cocultured with endothelial cells. To determine whether VEGF directly provides chemoattractive guidance for migration, we examined migration of endothelial cells toward VEGF-coated beads. Mouse glomerular endothelial cells expressing beta-galactosidase (MGEC) were isolated from Flk-1(+/-) heterozygous mice and passaged 4-12 times. Upon 24 h culture on collagen I gels MGEC formed a lattice or capillary-like network. Embryonic metanephroi were cocultured with MGEC on collagen I gels for 1-6 days in defined media, stained for beta-galactosidase, and examined by light microscopy. Metanephric organs induced a rearrangement of the endothelial cell lattice and attracted MGEC. MGEC invaded the metanephric organs forming capillary-like structures within and surrounding the forming nephrons. This process was accelerated and amplified by low oxygen (3% O(2)) and was prevented by anti-VEGF neutralizing antibodies. MGECs migrated toward VEGF-coated beads, whereas PBS-coated beads did not alter MGEC networks. We conclude that VEGF produced by the differentiating nephrons acts as a chemoattractant providing spatial direction to developing capillaries toward forming nephrons during metanephric development in vitro.

Vascular endothelial growth factor induces nephrogenesis and vasculogenesis.

The expression of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 in the rat kidney was examined during ontogeny using Northern blot analysis and immunocytochemistry. In prevascular embryonic kidneys (embryonic day 14 [E14]), immunoreactive Flt-1 and Flk-1 were observed in isolated angioblasts, whereas VEGF was not detected. Angioblasts aligned forming cords before morphologically differentiating into endothelial cells. In late fetal kidneys (E19), immunoreactive VEGF was detected in glomerular epithelial and tubular cells, whereas Flt-1 and Flk-1 were expressed in contiguous endothelial cells. To determine whether VEGF induces endothelial cell differentiation and vascular development in the kidney, the effect of recombinant human VEGF (5 ng/ml) was examined on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 d in culture in serum-free, defined media, metanephric kidney growth and morphology were assessed. DNA content was higher in VEGF-treated explants (1.9 +/- 0.17 microg/kidney, n = 9) than in paired control explants (1.4 +/- 0.10 microg/kidney, n = 9) (P < 0.05). VEGF induced proliferation of tubular epithelial cells, as indicated by an increased number of tubules and tubular proliferating cell nuclear antigen-containing cells. VEGF induced upregulation of Flk-1 and Flt-1 expression, as assessed by Western blot analysis. Developing endothelial cells were identified and localized using immunocytochemistry and electron microscopy. Flt-1, Flk-1, and angiotensin-converting enzyme-containing cells were detected in VEGF-treated explants, whereas control explants were negative. These studies confirmed previous reports indicating that the expression of VEGF and its receptors is temporally and spatially associated with kidney vascularization and identified angioblasts expressing Flt-1 and Flk-1 in prevascular embryonic kidneys. The data indicate that VEGF expression is downregulated in standard culture conditions and that VEGF stimulates growth of embryonic kidney explants by expanding both endothelium and epithelium, resulting in vasculogenesis and enhanced tubulogenesis. These data suggest that VEGF plays a critical role in renal development by promoting endothelial cell differentiation, capillary formation, and proliferation of tubular epithelia.

Renal functional reserve in children with a previous episode of haemolytic-uraemic syndrome.

Renal function [creatinine clearance (CCr)] and renal functional reserve (RFR) was measured in 16 children who had had haemolytic-uraemic syndrome (HUS) an average of 6.6 +/- 0.72 years previously. All patients had normal plasma creatinine and blood pressure and only 3 had proteinuria, which was mild in every instance. Patients were studied whilst ingesting three diets which provided an average of 1.5, 2.1 and 3.1 g protein/kg body weight per day, respectively. Diets were administered over three consecutive periods of 7 days each and CCr was measured on the 7th day of each diet. Values tended to correlate with protein intake. They were in the normal range when patients were taking 1.5 and 2.1 g protein diets and increased markedly in 13 of the 16 patients (P less than 0.001) when they ingested the high-protein diet (3.1 g). The effect on glomerular filtration rate (GFR)--measured by CCr and inulin clearance (Cin)--of an acute oral protein load was studied in 12 of the HUS patients and four control subjects. In the control periods, prior to the protein load, values for CCr were similar in the HUS and control subjects (104.0 +/- 11.0 vs 121.6 +/- 10.1 ml/min per 1.73 m2, NS). However Cin values were significantly reduced in HUS patients (59.5 +/- 9.2 vs 102.7 +/- 12.4 ml/min per 1.73 m2, (P less than 0.025). The CCr/Cin ratio in the patients averaged 2.10 compared with 1.13 in controls. Acute protein loading was accompanied by an increase in Cin in all controls but in only 8 of the 12 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal response to a protein meal in children with single kidneys.

The renal response of healthy adults to an oral protein load results in a significant increase of renal plasma flow (RPF) and glomerular filtration rate (GFR) 100 to 150 min after the meal. The renal response to protein loading of single kidney adults is unclear and it has not been evaluated yet in children. Therefore, we studied 8 children 10.2 +/- 1.1 years old, 6.7 +/- 1.41 years after nephrectomy (SK) and 4 healthy children (C). RPF was estimated by para-amino-hippurate clearance (Cpah) and GFR by inulin clearance (Cin) before and after an oral protein load of 1.5 g protein/kg body wt and expressed in ml/min/1.73 m2 BSA. Mean baseline Cin were similar in SK and in C (90 +/- 8 vs 103 +/- 12) while baseline Cpah was lower in SK (339 +/- 19 vs 481 +/- 36, p less than 0.005). After the meal GFR and RPF increased significantly in C (155 +/- 18 and 783 +/- 68, p less than 0.005 and p less than 0.05 vs baseline values, respectively) whereas no significant GFR and RPF changes were seen in SK (81 +/- 9 and 350 +/- 42, respectively). However, the 3 SK children with lower protein intake showed a mild vasodilating response. We conclude that in single kidney children hyperfiltration occurs at baseline conditions and the renal response to acute protein loading is partially or completely blunted, being modulated by protein intake.