Levels of albuminuria and risk of developing macroalbuminuria in type 2 diabetes: historical cohort study.

Although increased urinary albumin excretion may increase the risk of adverse renal outcomes in patients with diabetes, it remains unclear whether microalbuminuria is associated with a higher incidence of macroalbuminuria in the absence of non-diabetic kidney events that frequently develop during the long-term course of type 2 diabetes. This historical cohort study included patients with type 2 diabetes, spot urine albumin:creatinine ratio (ACR) <300 mg/gCr and normal serum creatinine concentrations treated between August 1988 and April 2015. Patients with any evidence suggesting non-diabetic kidney diseases at baseline were excluded. Over a median follow-up of 50 months, 70 of the 1760 included patients developed macroalbuminuria. Twenty-one of these patients were diagnosed with non-diabetic renal events. The five-year cumulative incidence of macroalbuminuria in patients with ACRs of 0-7.5 mg/gCr, 7.5-30 mg/gCr, 30-150 mg/gCr, and 150-300 mg/gCr were 0%, 0.53%, 3.5%, and 36.0%, respectively, with significant differences between each pair of ACR categories. In type 2 diabetes, higher urinary ACR, even within a level of normoalbuminuria, was associated with a greater incidence of macroalbuminuria when non-diabetic renal events were excluded. These results conflict with findings suggesting that microalbuminuria is a poor indicator for the progression of diabetic nephropathy.

Serum monomeric α2-macroglobulin as a clinical biomarker in diabetes.

OBJECTIVE: Despite the number of potential biomarker proteins for diabetes, very few of them have proven useful as clinically beneficial indicators, because of the technical difficulties associated with their identification among highly abundant serum proteins. We attempted to identify a protein with distinguishable expression in human diabetes. METHODS: We applied a highly efficient strategy for the purification of endogenous low abundance proteins from diabetic and non-diabetic serum samples. Extracted sera were fractionated by SDS-PAGE and protein bands were isolated and analyzed by mass spectrometry using an ion-trap mass spectrometer. The identities of the proteins were confirmed by western blotting and the serum levels evaluated. RESULTS: A significantly upregulated protein in diabetic patients was identified as monomeric α2-macroglobulin. Its tetramer, another dominant circulating molecular form, was only marginally increased in diabetes. CONCLUSION: Serum monomeric α2-macroglobulin is highly expressed in many diabetic subjects. It is identical to the human 'cardiac isoform of α2-macroglobulin' described in the literature, a well-known acute phase serum biomarker protein mechanistically involved in cardiac and atherosclerotic diseases.

Reduced angiogenesis and delay in wound healing in angiotensin II type 1a receptor-deficient mice.

Angiotensin II (Ang II) is a bioactive peptide that plays important roles in blood pressure regulation and salt-water homeostasis. Recently, Ang II was reported to function in the promotion of angiogenesis. Since the wound healing process is highly dependent upon angiogenesis, we employed Ang II receptor knockout mice (AT1a(-/-)) to investigate whether or not Ang II facilitates angiogenesis and wound healing via AT1a receptor signaling. In comparison to wild-type (WT) mice, wound healing and wound-induced angiogenesis were significantly suppressed in AT1a(-/-) mice, and these mice exhibited reduced expression of CD31 in wound granulation tissues. In comparison to vehicle-treated mice, wound healing was delayed significantly in mice treated with an AT1-R antagonist and this delay was accompanied by the reduced expression of vascular endothelial growth factor in wound granulation tissues. These findings suggest that Ang II-AT1a signaling plays a crucial role in wound healing and wound-induced angiogenesis.

An ATP-sensitive potassium channel blocker suppresses sodium-induced hypertension through increased secretion of urinary kallikrein.

It is suggested that an ATP-sensitive potassium channel blocker suppresses sodium-induced hypertension through increased secretion of urinary kallikrein. We reported that glibenclamide, an ATP-sensitive potassium channel blocker, accelerated dose-dependent secretion of renal kallikrein in sliced kidney cortex and in vivo in rats. In vehicle-treated normal Brown- Norway-Kitasato (nBN-Ki) rats, the administration of glibenclamide increased urinary kallikrein secretion, but changed neither the systolic blood pressure nor the urinary sodium on low (0.3%) NaCl diets. Although on high (8%) NaCl diets, the systolic blood pressure of the nBN-Ki rats administrated glibenclamide was significantly lower (P<0.05). The urinary levels of kallikrein and sodium of the nBN-Ki rats administrated glibenclamide were significantly increased (P<0.05, glibenclamide vs. vehicle). A similar result was obtained with a kidney-selective ATP-sensitive potassium blocker, N,N'-dicyclohexyl-4-morpholinecarboxamidine (U18177), in SD rats. Mutant kininogen-deficient Brown-Norway Katholiek (muBN-Ka) rats fed high (8%) NaCl diets showed an increase in urinary kallikrein levels, but showed neither hypotensive nor natriuretic actions by glibenclamide. A bradykinin B(2) receptor antagonist, 8-[3-[N-(E)-3-(6-acetamidopyridin-3-yl) acryloylglyycyl]-N-methylamino]-2,6-dichlorobenzyloxy]-2-methylquinoline (FR173657), which was administrated to SD rats, together with glibenclamide, abolished the hypotensive and natriuretic effects of glibenclamide in high-sodium (8%NaCl) hypertension, despite an accelerated secretion of urinary kallikrein. Therefore, these results indicate that glibenclamide, an ATP-sensitive potassium channel blocker suppressed sodium-induced hypertension through sodium excretion from the kidney resulting from accelerated secretion of urinary kallikrein.

Renal structure as an indicator for development of albuminuria in normo- and microalbuminuric type 2 diabetic patients.

Baseline glomerular structure in microalbuminuric (MA) and proteinuric Caucasian type 2 diabetic patients predicted progressive glomerular filtration rate decline while baseline urinary albumin excretion (UAE) did not. Little is known about whether or not renal structure at the early stages of diabetic nephropathy (DN) in type 2 diabetic patients can predict further functional development of DN. Baseline renal structure and function and follow-up data of renal function were examined in 17 type 2 diabetic patients (11 men, 45+/-7 (mean+/-S.D.) years old) with known diabetes duration 11+/-8 years without definable renal disease other than DN. Six patients showed normoalbuminuria (NA), 11 microalbuminuria (MA), and were followed up for 6.4+/-1.8 years after the baseline renal biopsy. Light and electron microscopic morphometric analyses provided quantitative glomerular and tubulointerstitial structural changes. No statistically significant difference was observed in hemoglobin A1c (HbA1c) values or mean blood pressure (MBP) between baseline and follow-up, even though the number of patients placed on antihypertensive drugs increased from 3 to 7. Follow-up UAE was not significantly different from the baseline UAE although 13 of 17 cases showed an increase. Baseline UAE did not correlate with the follow-up UAE or morphometric measures. Glomerular basement membrane width and volume fraction of the mesangium and mesangial matrix positively correlated with follow-up UAE. In NA and MA Japanese type 2 diabetic patients, baseline renal structural measures are more reliable indicators for the development of UAE than baseline UAE.

Detailed glomerular ultrastructure in Japanese type 2 diabetic patients by the quick-freezing and deep-etching method.

Mesangial expansion and glomerular basement membrane (GBM) thickening did not correlate with urinary albumin excretion (UAE) in type 2 diabetic patients in our previous studies; therefore, it was necessary to elucidate more detailed ultrastructural changes in the early stages of diabetic nephropathy (DN) in type 2 diabetic patients. The quick-freezing and deep-etching (QF-DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. The QF-DE method was applied to six type 2 diabetic patients without definable renal diseases other than DN. Four patients were normoalbuminuric (NA) and the other two were microalbuminuria (MA). Three control specimens were the normal parts from nephrectomies due to renal cell carcinomas. Electron microscopic morphometric analyses provided quantitative glomerular structural changes. Replica membranes were prepared by the QF-DE method, and diameters of mesh structures at the GBM and mesangial matrix (MM) were measured on electron micrographs as previously described. By the QF-DE method, both the GBM middle layer and MM were composed of polygonal meshwork structures. The mesh pores of the GBM and MM were more enlarged and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The mesh diameters of the GBM and MM in the diabetic patients were also larger than those of the controls. Such a mesh diameter of the GBM was well correlated with the amount of UAE, while the mesh diameter of MM showed a slight correlation with UAE. Although there were small number of subjects in the present study, the detailed ultrastructural changes in NA and MA type 2 diabetic patients, which had not been disclosed by conventional electron microscopy, were revealed by the QF-DE method. Increased mesh diameters of GBM might be related with the increase of UAE.

Alterations in mRNA expression of myelin proteins in the sciatic nerves and brains of streptozotocin-induced diabetic rats.

Diabetic neuropathy is the most common complication of diabetes. We examined the levels and the mRNA expression of myelin proteins in the sciatic nerves and the brains of streptozotocin-induced diabetic rats. The diabetic rats exhibited a decrease in body weight, elevation of the blood glucose level and a decrease in motor nerve conduction velocity at 2 weeks after streptozotocin injection. In the sciatic nerves of diabetic rats, the level of P0 protein and its mRNA expression were markedly reduced at 20 weeks after the injection. In the brains, the levels of proteolipid protein and myelin-associated glycoprotein and their mRNA expression were selectively decreased at 20 weeks after the injection. This affected expression of myelin proteins was found even when no histological abnormalities were detectable. Considering the functional significance of myelin proteins, this impairment of protein expression is possibly involved in the pathogenesis of diabetic neuropathy, including that in brain disorders.

Clinical applications of the quick-freezing and deep-etching method to human diabetic nephropathy.

Mesangial expansion and glomerular basement membrane (GBM) thickening were not different between normoalbuminuric (NA) and microalbuminuric (MA) type 2 diabetic patients. The quick-freezing and deep-etching (QF-DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. In the present study, the QF-DE method was applied to the renal biopsy from 6 type 2 diabetic patients without definable renal diseases other than diabetic nephropathy. Four patients were NA and the other two were MA. Three control specimens were normal parts in surgically resected kidneys of renal cell carcinoma. Replica membranes were prepared by the QF-DE method as previously described. By the QF-DE method, both GBM middle layer and mesangial matrix (MM) were composed of polygonal meshwork structures. The mesh pores of GBM and MM were more enlarged in size and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The diameters of mesh pores in the diabetic patients were significantly larger than those in the control subjects. In conclusion, the QF-DE method could be applied to needle renal biopsy and the present study has firstly clarified the difference of ultrastructural changes between NA and MA type 2 diabetic patients, which had not been disclosed by the conventional electron microscopy, were revealed by the QF-DE method.

Determination of antihyperglycemic biguanides in serum and urine using an ion-pair solid-phase extraction technique followed by HPLC-UV on a pentafluorophenylpropyl column and on an octadecyl column.

An HPLC-UV method was established for the determination of metformin and buformin in biological fluids. Metformin was not retained on particles packed in conventional solid-phase extraction cartridges; in contrast, buformin was retained too firmly and not eluted with a solvent for recovery. However, both drugs were retained on particles that had been treated with an ion-pair reagent of heptanesulfonate or dodecylsulfate and recovered almost completely. The recovered fraction was subjected to HPLC on a pentafluorophenylpropyl column which was suitable for the determination of both biguanides in serum and in urine. Limits of quantitation were low enough for clinical use, and reproducibility was high with an RSD of 0.9-2.3%. HPLC on a conventional octadecyl column was suitable only for the determination of buformin in serum since interfering peaks appeared on the chromatograms of urine samples. The method was applied to analysis of some clinical specimens.

Glomerular hyperfiltration in non-proteinuric and non-hypertensive Japanese type 2 diabetic patients.

Glomerular hyperfiltration (GHF) may be an important factor in the initiation of glomerular damage and in predisposing diabetic patients to the later development of diabetic nephropathy (DN). Previous reports show wide range of prevalence of GHF in type 2 diabetic patients. This cross-sectional study was designed to determine the prevalence of GHF at an early stage of DN in Japanese type 2 diabetic patients and to investigate the relationships between clinical variables and GHF. We measured the glomerular filtration rate (GFR) using the plasma clearance of iohexol in 56 control subjects and 93 type 2 diabetic patients without hypertension or overt proteinuria. We used Altman's method to calculate the age-adjusted 95% reference ranges for GFR from the data of control subjects and classified GHF in type 2 diabetics from the reference ranges. Hyperfiltrators (defined as GFR > mean GFR + 1.96 S.D. of control subjects) was found in 17% patients (16/93). Other 77 patients (83%) were normofiltrators (defined as GFR< or = mean GFR + 1.96 S.D. of control subjects). GFR values, both in hyperfiltrators and normofiltrators, were 140.5 +/- 14.6 and 98.8 +/- 14.0 ml/min/1.73 m2, respectively. Age, sex, BMI, blood pressure, albumin excretion rate, and frequency of microalbuminuria did not differ between the hyperfiltrators and normofiltrators. Fasting plasma glucose and hemoglobin A1c were significantly higher in the hyperfiltrators than the normofiltrators (P<0.001 and 0.004, respectively). GHF exists among Japanese type 2 diabetic patients with no evidence of overt proteinuria or hypertension. Glycemic control might be a significant determinant of GHF in these patients.

Cyclooxygenase-2 inhibitors attenuate increased blood pressure in renovascular hypertensive models, but not in deoxycorticosterone-salt hypertension.

COX-2 is an inducible cyclooxygenase (COX) that has been reported to be expressed in the macula densa and surrounding cortical thick ascending limb in normotensive rats. The present study assessed the contribution of COX-2 in three different rat models of hypertension, each characterized by a different activation of the renal renin-angiotensin system. Mean blood pressure (MBP) in the rat 2 kidney-1 clip (2K1C) model was significantly reduced with a COX-2 selective inhibitor, NS-398 (10 mg/kg, p.o., twice a day) (vehicle-administered rats (n = 8): 154 +/- 6 mmHg; NS-398-administered rats (n = 5): 128 +/- 10 mmHg). By contrast, a COX-1 selective inhibitor, mofezolac, did not lower MBP. Increased plasma renin activity (23 +/- 8 ng/kg/h (n = 6) vs. sham operation, 2.4 +/- 0.9 ng/kg/h (n = 4)) was markedly reduced to 6.8 +/- 2.7 ng/ml/h (n = 5) by NS-398, but not by mofezolac. The development of 1 kidney-1 clip (1K1C) hypertension was also inhibited by NS-398 (vehicle (n = 12): 133 +/- 1 mmHg; NS-398 (n = 7): 122 +/- 3 mmHg) accompanied by a reduction in plasma renin activity (3.0 +/- 0.3 ng/ml/h, n = 4) to 1.0 +/- 0.2 ng/ml/h (n = 5). The COX-2 inhibitor increased urinary excretions in the 1K1C model, but not in the 2K1C model. In a deoxycorticosterone acetate (DOCA)-salt model, plasma renin activity was markedly suppressed to less than 0.3 ng/ml/h. The COX-2 inhibitor caused no significant changes in MBP, plasma renin activity, or urinary excretion, suggesting that COX-2 made a lesser contribution in this model. Increased expression of COX-2 mRNA and protein was observed in the kidneys of 1K1C and 2K1C rats, but not in DOCA-salt rats. These results suggest that COX-2 plays a significant role in the development of 2K1C and 1K1C renovascular hypertension, in addition to making a substantial contribution to the diuretic effect in the 1K1C model.