Early Phase Of Diabetic Nephropathy Research Articles

Amadori-configurated albumin induces nitric oxide-dependent apoptosis of endothelial cells: a possible mechanism of diabetic vasculopathy.

We have demonstrated previously that Amadori-configurated glycated albumin (GA) enhances nitric oxide synthase (NOS) activity, and this action may modulate glomerular hyperfiltration in early phases of diabetic nephropathy. Since the late stage of diabetic vasculopathy is characterized by reductions in viable cells within an expanded and disorganized matrix, we tested the hypothesis that GA enhances endothelial cell (EC) apoptosis. Murine (t End.1) or human umbilical vein ECs (HUVECs) were incubated with graded GA concentrations (furosine 0.48-96 nmol/ml) at levels that approximated those reported in sera of diabetic patients (76 +/- 0.02 nmol/ml). Apoptosis was evaluated using terminal uridine nick end labelling (TUNEL) to detect DNA fragmentation in gel electrophoresis and p53 expression in immunoperoxidase. Transcription of the inducible (i) and constitutive (c) isoforms of NOS was detected by northern analysis, and total NOS activity was measured as [(3)H]citrulline production from [(3)H]arginine. Cells were also incubated with the NOS inhibitors L-nitromethylarginine (L-NAME) at 0.01 M and aminoguanidine (AMG) at 0.01 M, the protein synthesis inhibitor cycloheximide (CHX) at 1 micro g/ml, and the NO donor sodium nitroprusside (SNP) at 0.01 M. ECs cultured in the presence of GA at furosine concentrations corresponding to levels in diabetic patients showed a significant enhancement of apoptosis. GA also caused parallel dose-dependent increases in iNOS mRNA expression and total NOS activity. The pro-apoptotic effect of GA was inhibited by L-NAME, AMG and CHX, but enhanced by SNP. We found that Amadori-configurated GA at furosine concentrations similar to those in diabetic patients favoured EC apoptosis through enhancement of iNOS activity. We propose that this process may be involved in the progressive cellular loss occurring in vascular and glomerular diabetic sclerosis.

Advanced Glycation End Product-induced Apoptosis and Overexpression of Vascular Endothelial Growth Factor and Monocyte Chemoattractant Protein-1 in Human-cultured Mesangial Cells

Advanced glycation end products (AGE) have been implicated in the pathogenesis of glomerulosclerosis in diabetes. However, their involvement in the development of the early phase of diabetic nephropathy has not been fully elucidated. We investigated the effects of AGE on growth and on vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) expression in human cultured mesangial cells. We prepared three immunochemically distinct AGE by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or glycolaldehyde. When human mesangial cells were cultured with various types of AGE-BSA, viable cell numbers as well as DNA syntheses were significantly decreased. All of the AGE-BSA were found to significantly increase p53 and Bax protein accumulations and subsequently induce apoptotic cell death in mesangial cells. An antioxidant, N-acetylcysteine, significantly prevented the AGE-induced apoptotic cell death in mesangial cells. Human mesangial cells stimulated prostacyclin production by co-cultured glomerular endothelial cells. Furthermore, various types of AGE-BSA were found to up-regulate the levels of mRNAs for VEGF and stimulate the secretion of VEGF and MCP-1 proteins in mesangial cells. The results suggest that AGE disturbed glomerular homeostasis by inducing apoptotic cell death in mesangial cells and elicited hyperfiltration and microalbuminuria by stimulating the secretion of VEGF and MCP-1 proteins, thereby being involved in the pathogenesis of the early phase of diabetic nephropathy.

Activation of renal signaling pathways in db/db mice with type 2 diabetes

Altered regulation of signaling pathways may contribute to the pathogenesis of renal disease. We examined renal cortical signaling pathways in type 2 diabetes. The status of renal cortical signaling pathways was examined in control and db/db mice with type 2 diabetes in the early phase of diabetic nephropathy associated with renal matrix expansion and albuminuria. Tyrosine phosphorylation of renal cortical proteins was increased in diabetic mice. Renal cortical activities of phosphatidylinositol 3-kinase (PI 3-kinase) in antiphosphotyrosine immunoprecipitates, Akt (PKB), and ERK1/2-type mitogen-activated protein (MAP) kinase activities were significantly augmented sixfold (P < 0.01), twofold (P < 0.0003), and sevenfold (P < 0.001), respectively, in diabetic mice compared with controls. A part of the increased renal cortical PI 3-kinase activity was due to insulin receptor activation, as PI 3-kinase activity associated with beta chain of the insulin receptor was increased nearly fourfold (P < 0.0235). Additionally, the kinase activity of the immunoprecipitated insulin receptor beta chain was augmented in the diabetic renal cortex, and tyrosine phosphorylation of the insulin receptor was increased. In the liver, activities of PI 3-kinase in the antiphosphotyrosine immunoprecipitates and Akt also were increased threefold (P < 0.05) and twofold (P < 0.0002), respectively. However, there was no change in the hepatic insulin receptor-associated PI 3-kinase activity. Additionally, the hepatic ERK1/2-type MAP kinase activity was inhibited by nearly 50% (P < 0.01). These studies demonstrate that a variety of receptor signaling pathways are activated in the renal cortex of mice with type 2 diabetes, and suggest a role for augmented insulin receptor activity in nephropathy of type 2 diabetes.

Increased blood levels of platelet-activating factor in insulin-dependent diabetic patients with microalbuminuria.

Platelet-activating factor (PAF), a phospholipid mediator of inflammation, may induce an enhanced size- and charge-dependent glomerular permeability in experimental animals. Studies on the role of PAF in enhanced glomerular permeability in the early phase of diabetic nephropathy are still lacking. We evaluated the intravascular levels of PAF and its main catabolic enzyme, the PAF-specific plasma acetyl-hydrolase (PAF-AH), in basal conditions and after exercise, in normo- or micro-albuminuric insulin-dependent diabetic (IDD) patients and in normal subjects. The results obtained indicate that the concentration of PAF in whole blood was significantly enhanced in basal conditions, during and after exercise in all microalbuminuric IDD patients, but not in normoalbuminuric IDD or in control subjects. The increased concentration of PAF did not correlate with changes in the activity of PAF-AH, suggesting an enhanced production rather than a decreased catabolism of PAF. These results indicate an association between increased production of PAF and enhanced glomerular permeability in microalbuminuric IDD patients.

Diagnostic significance of urinary immunoglobulin G in diabetic nephropathy

An early manifestation of diabetic nephropathy, increased excretion of albumin, is now generally believed to be sufficiently specific, particularly in subjects with diabetes mellitus, to predict the subsequent development of clinically overt diabetic nephropathy. However, certain other proteins besides albumin may also be excreted in abnormal amounts during this early phase of diabetic nephropathy. We evaluated the diagnostic utility of urinary immunoglobulin G (IgG) in patients with diabetic nephropathy by comparing the findings with the clinical stage and renal biopsy specimen. Using 24-hour urine samples, IgG was measured by an enzyme-linked immunosorbent assay. In addition, urine samples were assayed for albumin, transferrin, beta 2-microglobulin and N-acetyl-beta-D-glucosaminidase. Serum IgG concentration and HbA1c were also evaluated. A total of 197 patients with non-insulin-dependent diabetes mellitus were enrolled in this study. Subjects were grouped according to the rate of urinary albumin excretion (clinical stage). Fifty of these cases were also divided into four groups according to the severity of diffuse glomerular lesions using Gellman's criteria. The urinary excretion of IgG was significantly increased in diabetic patients as compared with the healthy controls. Among diabetic patients, IgG level showed a significant increase with respect to the clinical stage of nephropathy and the progress of glomerular diffuse lesions. In the stage of normoalbuminuria, the urinary excretion of IgG showed a significant increase in parallel with the progress of glomerular diffuse lesions, whereas there was no relationship between the urinary excretion of albumin and the progress of glomerular diffuse lesions. While the excretion of IgG correlated with that of albumin and transferrin, there was no correlation between the excretion of IgG and the other laboratory indices evaluated. These findings indicate that measurement of urinary IgG may be more useful than albuminuria in detecting the early stage of diabetic nephropathy.

Renal functional reserve in insulin dependent diabetic children.

Microalbuminuria has been shown to be predictive for clinical diabetic nephropathy. Renal functional reserve (RFR), as a response to protein loading in a short period of time, is a parameter to assess the ability of kidneys to increase the glomerular filtration rate (GFR). The aim of this study was to predict the early phase of diabetic nephropathy by measuring urinary albumin level and RFR capacity in patients with insulin-dependent diabetes mellitus (IDDM). Twenty-two patients with IDDM were studied: 11 with a disease duration of less than 5 years (group 1) and 11 with a disease duration of more than 5 years (group 2). As the control group, 15 healthy children (group 3) were included in the study. At the beginning of the study, glucose was measured and the urinary albumin/creatinine ratio was calculated. Average glycosylated hemoglobin (HbA1c) over 1 year was determined. After protein loading (red meat containing 2 g/kg of protein), the creatinine clearance was calculated at each hour for a duration of 4 h. The RFR was accepted as the peak percentage increase in GFR over the baseline value. Although metabolic control in group 2 was better, the RFR in group 2 was significantly lower than in group 1 (P < 0.05). Urinary microalbumin levels between the groups did not differ (P > 0.05). In two patients in whom microalbuminuria was detected, the RFR was much lower. Detecting lower RFR levels in patients with normal urinary albumin excretion, as well as in patients with microalbuminuria, may support the idea that the RFR capacity is more sensitive than microalbuminuria in assessing the early phase of diabetic nephropathy.

Microalbuminuria in IDDM is associated with increased expression of monocyte procoagulant activity.

Microalbuminuria, the early phase of diabetic nephropathy, is associated with an increased risk of atherothrombosis. Monocytes play an important part in the pathogenesis of atherosclerosis and in the activation of haemostasis. However, procoagulant activity is poorly understood in Type I (insulin-dependent) diabetes mellitus, particularly in the presence of microalbuminuria. This study aimed to evaluate spontaneous and endotoxin-induced monocyte procoagulant activity in insulin-dependent diabetic patients with normoalbuminuria or microalbuminuria. Seventeen patients with microalbuminuria, 28 with normoalbuminuria and 26 healthy control subjects matched for age, sex, body mass index and smoking habit were studied. Mononuclear cells from peripheral venous blood were incubated with or without bacterial lypopolysaccharide. Spontaneous procoagulant activity and procoagulant activity after 3 h and 6 h of incubation were calculated. Spontaneous procoagulant activity values were similar in the three groups. After 3 h and 6 h incubation with bacterial lypopolysaccharide, procoagulant activity values were slightly, but not statistically significantly, higher in the normoalbuminuric diabetic group than in control group, and significantly higher in microalbuminuric diabetic group than in control group (p < 0.01). The increased endotoxin-induced monocyte procoagulant activity helps to explain the link between microalbuminuria and the increased risk of atherothrombosis in patients with Type I diabetes.

Nonenzymatically glycated albumin (Amadori adducts) enhances nitric oxide synthase activity and gene expression in endothelial cells

Hyperglycemia is considered to induce diabetic nephropathy through nonenzymatic glycation of proteins. Since hyperfiltration is likely to be the mechanism initiating the glomerular lesions, we investigated the effects of Amadori glucose adducts in serum albumin on the production of vasoactive mediators, including nitric oxide (NO) and eicosanoids, by endothelial cells (EC). Amadori adducts of glycated albumin induced a dose-response increase in NO synthase activity of murine endothelioma cells, up to 16.4 +/- 2.1-fold increase of basal values (P < 0.0001) at concentrations of 35 mg/ml mimicking physiological serum albumin concentration, and 4.6 +/- 0.8-fold increase at 17 mg/ml (P < 0.001). The effect was still detectable with glycated albumin 1.7 mg/ml, which approaches its estimated concentration in diabetic serum (1.6 +/- 0.3-fold increase, P < 0.05) The phenomenon was reproducible in human umbilical vein endothelial cells, though to a lesser extent, and further studies on murine EC were employed. The mRNA encoding for inducible NO synthase was overexpressed in EC incubated with Amadori adducts of glycated albumin in comparison to native albumin. Glycated albumin induced increased mRNA expression and synthesis of TNF-alpha. The stimulatory effect induced by glycated albumin on NO synthase activity was almost completely inhibited by anti TNF alpha antibodies. 3H-thymidine incorporation by EC was significantly inhibited when cells were grown in presence of glycated albumin (P < 0.001), and the phenomenon was abolished by the coincubation of the NO competitive inhibitor L-NAME. The early glycosylation products increased thromboxane production (P < 0.001), while prostaglandin E2 synthesis was unaffected. These data indicate that Amadori products of glycated albumin modulate NO synthase activity and eicosanoid balance in EC. These effects may be relevant to the hemodynamic changes in the early phases of diabetic nephropathy and in the lasting progression to sclerosis.

A quantitative ultrastructural study of juxtaglomerular arterioles in IDDM patients with micro- and normoalbuminuria.

Hyalinization of juxtaglomerular arterioles is prominent in advanced diabetic nephropathy and may have important functional consequences. We studied the early stages of diabetic renal disease using kidney biopsy material from insulin-dependent diabetic patients, 8 with normal albumin excretion rate (< 15 micrograms/min) and 16 with microalbuminuria (15-200 micrograms/min). Ten living non-diabetic kidney donors served as a control group. Median duration of diabetes was 9.5 years (range 5-31) in patients with normoalbuminuria, and 12 years (7-22) in patients with microalbuminuria (p = 0.27). The tissue was sectioned systematically, 1-micron thick sections for light microscopy at 10-micron intervals, and thin sections for electron microscopy taken at 60-micron intervals. The arterioles were identified as afferent or efferent, and total profiles were photographed (magnification 7500x), providing a systematic independent sample for measurements using standard stereological methods. Patients with microalbuminuria had significantly increased arteriole parameters compared with the control group: for afferent and efferent arterioles the volume fraction of matrix/media, means and (co-efficient of variation, CV), was 0.47 (0.16) vs 0.33 (0.19) (p = 0.0009), and 0.62 (0.14) vs 0.45 (0.23) (p = 0.0004) and matrix-T, expressing amount of matrix per unit arteriolar surface, 2.38 (0.38) micron vs 1.44 (0.30) micron (p = 0.004), and 1.62 (0.28) micron vs. 1.03 (0.34) (p = 0.0009). Patients with normoalbuminuria showed no significant differences from the control group, and had lower values than microalbuminuric patients for all parameters except the afferent matrix-T. In the normoalbuminuric group a correlation was found between parameters for afferent arterioles and those for glomerular structure. In conclusion there is arteriolar accumulation of extracellular material in the early phase of diabetic nephropathy, concomitant with early glomerulopathy.

Urinary kallikrein excretion in Type 1 (insulin-dependent) diabetes mellitus

Kidney haemodynamics appear to change after the early phases of diabetic nephropathy: increases in glomerular filtration rate and in renal plasma flow have been widely reported, while kidney size is increased. As the renal kallikrein-kinin system has been demonstrated to regulate kidney blood circulation, we have evaluated the excretion of urinary kallikrein in 87 Type 1 (insulin-dependent) diabetic patients with and without hyperfiltration. Urinary kallikrein excretion was measured in 24-h urine collections. The esterolytic activity was determined by fluorimetric assay. The excretion of urinary kallikreins was significantly higher in hyperfiltering patients (472 +/- 125 esterase units/24 h) than in normofiltering (168 +/- 77 esterase units/24 h) and control subjects (151 +/- 39 esterase units/24 h), p < 0.001. Furthermore, we observed a positive correlation between urinary kallikrein excretion and glomerular filtration rate (r = 0.785). These data suggest that variations of kallikrein and kinin concentrations may play a role in the alteration of renal haemodynamics in Type 1 diabetes. It is possible that the kinin-kallikrein system, the renin-angiotensin system and the prostaglandins may interact to determine the haemodynamic alterations which are present in the diabetic disease.

Glomerular response to hyperglycemia in human diabetic nephropathy

We have studied the effect of acute hyperglycemia on glomerular function in seven insulin-dependent diabetics with overt nephropathy during hyperglycemic or euglycemic clamp. In all patients glomerular filtration rate (GFR) was higher during hyperglycemia than during euglycemia (35.0 +/- 15.5 vs. 21.4 +/- 10.3 ml.min-1.1.73 m-2, P less than 0.01), whereas renal plasma flow did not change significantly. To establish which determinant of GFR is altered by hyperglycemia, fractional clearances of neutral dextrans of graded molecular size were determined in both glycemic states, and data were analyzed by a theoretical model of hindered transport of macromolecules through a porous membrane. Hyperglycemia significantly increased sieving coefficients of small dextran molecules (28-40 A in radius), whereas fractional clearances of large macromolecules (greater than 44 A) did not change. Theoretical analysis suggested that the ultrafiltration coefficient (Kf) and membrane permeability to small dextrans (less than 40 A) increased in hyperglycemia in respect to euglycemia. Because these results have been obtained in patients with severe renal failure and hypertension, our conclusions do not necessarily apply to the early phase of diabetic nephropathy.

Combining antihypertensive agents in early diabetic nephropathy

80 Antihypertensive treatment induces a fall in urinary protein and albumin excretion in insulin-dependent diabetic (IDDM) patients with both incipient,‘s2 and overt nephropathy. 3r4 These observations indicate that protein excretion in some way reflects the level of the systemic blood pressure, probably via parallel changes in intraglomerular pressure. Accordingly, antihypertensive agents may reduce high intraglomerular pressure during exercise in patients with diabetic nephropathy. 5,6 More importantly, clinical studies have shown that antihypertensive treatment can reduce the rate of fall in GFR considerably in the overt stage of diabetic nephropathy,3s4 and even more optimistic results have been published in diabetics with incipient nephropathy.‘v2 ACE inhibitors have been shown to have a beneficial effect on kidney function both in the early2s6 and more advanced stages of diabetic nephropathy. 7-9 However, studies using ACE inhibitors in combination with selective beta-blockers and diuretics in the early phase of diabetic nephropathy has not been performed, but may be rational, since both early cardiac hyperfunction1° and increased extracellular volume” may be present in these patients with abnormal renal hemodynamics.12 We have, therefore, undertaken a triple treatment study adding an ACE inhibitor to a conventional antihypertensive program consisting of a selective beta-blocker and a thiazide diuretic.