Development Of Early Diabetic Nephropathy Research Articles

Comparison the Role of Some Novel Biomarkers in Predicting Diabetic Nephropathy Between Diabetic Type 2 and Chronic Kidney Diseases Patients in Kirkuk City

Background: Diabetes mellitus most dangerous metabolic disorder and have high morbidity and mortality complications, the most DM complication is diabetic nephropathy. Diabetic nephropathy( DN) is an irreversible progressive disorder characterized by increased urine albumin excretion and/or impaired GFR. DM causes about 50 % of kidndiseasesase. An increase in prevalence of diabetes is projected to lead to a significant increase in patients with end-stage renal disease (ESRD) requiring renal replacement therapy (RRT), and up to 50% of the dialysis population is diabetic. We study 30 Diabetic patients with Chronic kidney diseases with hemodialysis ,30 diabetic patient with chronic kidney disease with out hemodialysis , 15 patient with diabetes only , and 15 control group .we measured kidney injury molecule-1 , liver type fatty acid binding protein ,zinc alpha 2 glycoprotein , serum creatinine ,uric acid .blood urea and random blood sugar .results showed that highest KIM-1 concentration in diabetic nephropathy with hemodialysis group (2.325± 1.095) followed by control group (1.842 ±0.866 ab )and followed with diabetes group (1.572 ±0.752 b ) and lowest mean level was diabetic nephropathy without hemodialysis group (1.344 ±0.623 b ) with highly significant differences (p < 0.001) of KIM-1 among groups .The highest mean level of L-FABP was recorded in diabetic nephropathy with hemodialysis group (254.6 ± 29.6 a ) followed by the control group (235.8 ± 28.9) and followed by diabetic group ( 9210.3 ± 28.7 ab )and the lowest mean level was diabetic nephropathy without hemodialysis group (149.5 ±25.9 a) with significant difference (P<0.002) of L-FABP among groups .The study showed that the highest mean level of ZAG recorded in diabetic nephropathy with hemodialysis group (140.1 ± 23.6 a) followed by control group( 130.9 ±11.5 a)and the lowest mean level was in diabetes group (88.3 ± 14.0 b ) and diabetic nephropathy without hemodialysis group (87.4 ± 19.60 b ) respectively with highly significant difference (P<0.0003). Aims: Role of biomarkers (KIM-1, L-FABP, ZAG) in detection early development of diabetic nephropathy .Correlation between biomarkers (KIM-1, L-FABP, ZAG) and progression of diabetic nephropathy to end stage of renal disease .Estimate how much relationship between biomolecules (creatinine , urea and uric acid ) with diabetes and its complication Methodology :The study included 90 individuals from Kirkuk city / Blood sample collected from patient and analysis was conducted to estimate concentration using Enzyme Linked Immunosorbent Assay (ELISA),and biochemicals using spectrophotometers . Keywords: Diabetes, diabetic nephropathy , chronic kidney disease ,kidney biomarkers

Growth Hormone and IGF1 Actions in Kidney Development and Function.

Growth hormone (GH) exerts multiple effects on different organs including the kidneys, either directly or via its main mediator, insulin-like-growth factor-1 (IGF-1). The GH/IGF1 system plays a key role in normal kidney development, glomerular hemodynamic regulation, as well as tubular water, sodium, phosphate, and calcium handling. Transgenic animal models demonstrated that GH excess (and not IGF1) may lead to hyperfiltration, albuminuria, and glomerulosclerosis. GH and IGF-1 play a significant role in the early development of diabetic nephropathy, as well as in compensatory kidney hypertrophy after unilateral nephrectomy. Chronic kidney disease (CKD) and its complications in children are associated with alterations in the GH/IGF1 axis, including growth retardation, related to a GH-resistant state, attributed to impaired kidney postreceptor GH-signaling and chronic inflammation. This may explain the safety of prolonged rhGH-treatment of short stature in CKD.

RNA-sequencing reveals genome-wide long non-coding RNAs profiling associated with early development of diabetic nephropathy.

BackgroundDiabetic nephropathy (DN) seriously threatens the lives of patients, and the mechanism of DN remains largely unknown because of the complex regulation between long non-coding RNA (lncRNA) and protein-coding genes. In early development of diabetic nephropathy (DN), pathogenesis remains largely unknown.ResultsWe used RNA-sequencing to profile protein-coding and lncRNA gene transcriptome of mouse kidney proximal tubular cells during early stage of DN at various time points. Over 7000 protein-coding and lncRNA genes were differentially expressed, and most of them were time-specific. Nearly 40% of lncRNA genes overlapped with functional element signals using CHIP-Seq data from ENCODE database. Disease progression was characterized by lncRNA expression patterns, rather than protein-coding genes, indicating that the lncRNA genes are potential biomarkers for DN. For gene ontologies related to kidney, enrichment was observed in protein-coding genes co-expressed with neighboring lncRNA genes. Based on protein-coding and lncRNA gene profiles, clustering analysis reveals dynamic expression patterns for kidney, suggesting that they are highly correlated during disease progression. To evaluate translation of mouse model to human conditions, we experimentally validated orthologous genes in human cells in vitro diabetic model. In mouse model, most gene expression patterns were repeated in human cell lines.ConclusionsThese results define dynamic transcriptome and novel functional roles for lncRNAs in diabetic kidney cells; these roles may result in lncRNA-based diagnosis and therapies for DN.

Frequency of microalbuminuria in newly diagnosed type 2 diabetics at Nishtar Hospital, Multan

Objectives: to describe frequency of microalbuminuria in newly diagnosed Type 2 Diabetics. Study Design: Descriptive and cross-sectional study. 50 indoor and outdoor newly diagnosed type 2 diabetics were screened for microalbuminuria. Setting: Medical Unit III of Nishtar Hospital Multan. Material And Methods: Both males and females with newly diagnosed type 2 diabetics were included in the study while Presence of overt proteinuria on routine urine analysis, Patients with evidence of congestive cardiac failure, urinary tract infection, and chronic obstructive pulmonary disease and Pregnant diabetics were excluded from the study. A detailed history was taken from every patient and meticulous clinical examination performed on each of them. Diabetes mellitus was confirmed by fasting and random hyperglycemia. Routine investigations like complete blood examination, complete urine examination serum urea, creatinine, X-ray chest, ECG, were obtained for each patient. Micral test (Boehringer Manhhem) was used for detection of microalbuminuria. Results: Average age of the patients was 45 years. 41 patients were males and 9 were females. 30% of these patients were found to be microalbuminuric. Both fasting blood sugar and random blood sugar levels were impaired in microalbuminurics. 38% of the total patients were smokers and 24% were hypertensives. Ischemic heart disease was found in 14% of patients on ECG where as LVH (Left Ventricular Hypertrophy) was evident in 4% of the patients. Renal parameters and Chest X-rays of all the patients were normal. None of them had CVA (Cerebrovascular Accident). Conclusion This study has documented higher frequency rate of microalbuminuria in newly diagnosed type 2 diabetics in our society. Poor glycemic control and delay in the diagnosis of diabetes mellitus were factors in the development of early diabetic nephropathy. Screening for early detection of diabetes mellitus is recommended.

The effects of hyperuricemia on early diabetic nephropathy in patients with type 2 diabetes mellitus

Objective To investigate the effect of hyperuricemia on early renal damage in patients with type 2 diabetic,and to explore the improvement of albumin-to-creatinine ratio (ACR) after lifestyle intervention to control the level of serum uric acid.Methods One hundred and sixty patients with type 2 diabetic mellitus from our hospital from January 2012 to December 2012 were recruited and divided into the high uric acid group (HUA,60 cases),and the normal uric acid group (NUA,100 cases).Fasting blood glucose (FBG),insulin (FINS),glycosylated hemoglobin (HbA1c),serum lipid levels,morning urine albumin-to-creatinine ratio (ACR) were detected in each group,and body mass index (BMI),insulin resistance index (HOMA-IR) were calculated.The HUA group was intervened by lifestyle for at least 6 months,and related indexes were measured again.Results The FINS,HOMA-IR,ACR and triglyceride(TG) of the patients in the HUA group were higher than that in the NUA group((110.33 ±38.73) pmol/L vs.(72.64 ±21.94) pmol/L,t =2.876; (7.0 ±2.3)vs.(4.6±1.3),t =2.364;13.6(5.1,53.2) mg/mmol vs.6.4(2.9,16.8) mg/mmol,H=3.390;(2.53± 1.45) mmol/L vs.(1.71 ± 1.32) mmol/L,t =2.626 ; P ＜ 0.05).By the Spearman correlation analysis,the level of serum uric acid was positively correlated with FINS,HOMA-IR,ACR,and BMI (r =0.304,0.225,0.248,0.271 respectively; P ＜ 0.05).After adjusting the factor of age,duration,obesity,blood glucose and insulin resistance,the level of serum uric acid was still positively correlated with ACR (r =0.312,P ＜ 0.01).The FINS,UA,HOMA-IR and ACR in group HUA were lower than those before lifestyle intervention ((463.7± 18.6) μmol/L vs.(380.3 ± 9.5) μmol/L,t =3.065 ; (110.33 ± 38.73) pmol/L vs.(83.30 ± 28.21)pmol/L,t =2.532; (7.0 ±2.3) vs.(5.0 ±0.9),t =2.165;13.6(5.1,53.2) mg/mmol vs.8.1(4.9,20.4)mg/mmol,H =2.912 ; P ＜ 0.05).Conclusion The patients with type 2 diabetes complicating hyperuricemia were likely to having abnormal ACR.Controlling the level of serum uric acid by lifestyle intervention may be benefit for delaying the development of early diabetic nephropathy in patients with type 2 diabetes. Key words: Hyperuricemia; Type 2 diabetes; Diabetic nephropathy; Albumin/creatinine; Lifestyle intervention

Decreased Activated Protein C Levels are Inversely Associated with the Urinary Albumin Excretion Rate in Patients with Type 2 Diabetes

Activated protein C (APC) plays a vital renoprotective role against diabetic nephropathy in STZ-induced diabetic mice by inhibiting endothelial cell and podocyte apoptosis. This study aimed to examine the relationship between the degree of albuminuria and APC levels in patients with type 2 diabetes. Ninety-four patients with type 2 diabetes and 38 healthy subjects were recruited into this study. The urinary albumin concentrations of urine collected over 24 hours were measured by ELISA to evaluate the mean urinary albumin excretion rate (UAER). The HbA1c levels were determined using an HPLC assay. There was no significant difference in the APC levels between diabetic patients with normoalbuminuria and controls, although APC levels were significantly lower in diabetic patients with microalbuminuria (23.06 +/- 19.82 vs. 54.58 +/- 53.63 pg/mL, p < 0.05) or macroalbuminuria (8.06 +/- 10.09 vs. 54.58 +/- 53.63 pg/mL, p < 0.05) as compared to control subjects. In addition, in comparison to diabetic patients with normoalbuminuria, APC levels were lower in diabetic patients with microalbuminuria (23.06 +/- 19.82 vs. 55.69 +/- 31.98 pg/mL, p < 0.05) and they were the lowest in diabetic patients with macroalbuminuria (8.06 +/- 10.09 vs. 55.69 +/- 31.98 pg/mL, p < 0.05). Multivariate regression and ROC analysis found that the levels of APC and HbA1c were independent risk factors for UAER levels (R2(ad) = 0.61, f = 25.69, p < 0.01) and the value of APC < or = 41.588 pg/mL indicated elevated albuminuria in patients with type 2 diabetes. Decreased APC levels in patients with type 2 diabetes may serve as a biomarker to indicate the early development of diabetic nephropathy.

Clinical effect of telmisartan combined with simvastatin in treatment of early diabetic nephropathy

Objective To evaluate the clinical efficacy and safty of combined treatment telmisartan plus simvastatin on early diabetic nephropathy. Methods 46 patients with early diabetic nephropathy were randomly divided into two groups. Control group ( n ＝ 23 ) received regular insulin therapy, captopril l0mg tid. Study group received telmisartan 80mg qd plus simvastatin 20mg qd on the basis of control group therapy. All patients received 16 weeks treatment. The clinical parameters were measured before and after therapy. Results After 16 weeks treatment,the urine MAU in both study group and control group were significantly decreased( all P ＜0. 01 ) ,however,the urine MAU in telmisartan plus simvastatin combination group decreased significantly than control group( P ＜ 0. 01 ). Improvement of lipid level in the study group better than the control group( P ＜0. 01 ). Kidney function,fasting blood glucose levels and HbAlc had no significant changes before and after treatment. Conclusion Telmisartan plus simvastatin combined treatment was better than the traditional therapy,which can delay the development of early diabetic nephropathy. Key words: Diabetic nephropathy; Telmisartan; Simvastatin

Angiogenesis and chronic kidney disease

The number of patients requiring renal replacement therapy due to end-stage renal disease (ESRD) is increasing worldwide. The prevalence of chronic kidney disease (CKD), and the importance of CKD as a risk factor in development of ESRD and in complicating cardiovascular disease (CVD) have been confirmed. In recent years, the involvement of angiogenesis-related factors in the progression of CKD has been studied, and the potential therapeutic effects on CKD of modulating these factors have been identified. Vascular endothelial growth factor (VEGF)-A, a potent pro-angiogenic factor, is involved in the development of the kidney, in maintenance of the glomerular capillary structure and filtration barrier, and in the renal repair process after injury. VEGF-A is also involved in the development of early diabetic nephropathy, demonstrated by the therapeutic effects of anti-VEGF-A antibody. Angiopoietin (Ang)-1 induces the maturation of newly formed blood vessels, and the therapeutic effects of Ang-1 in diabetic nephropathy have been described. In experimental models of diabetic nephropathy, the therapeutic effects of angiogenesis inhibitors, including angiostatin, endostatin and tumstatin peptides, the isocoumarin NM-3, and vasohibin-1, have been reported.Further analysis of the involvement of angiogenesis-related factors in the development of CKD is required. Determining the disease stage at which therapy is most effective and developing an effective drug delivery system targeting the kidney will be essential for pro-or anti-angiogenic strategies for patients with CKD.

Blockade of Endothelial-Mesenchymal Transition by a Smad3 Inhibitor Delays the Early Development of Streptozotocin-Induced Diabetic Nephropathy

OBJECTIVEA multicenter, controlled trial showed that early blockade of the renin-angiotensin system in patients with type 1 diabetes and normoalbuminuria did not retard the progression of nephropathy, suggesting that other mechanism(s) are involved in the pathogenesis of early diabetic nephropathy (diabetic nephropathy). We have previously demonstrated that endothelial-mesenchymal-transition (EndoMT) contributes to the early development of renal interstitial fibrosis independently of microalbuminuria in mice with streptozotocin (STZ)-induced diabetes. In the present study, we hypothesized that blocking EndoMT reduces the early development of diabetic nephropathy.RESEARCH DESIGN AND METHODSEndoMT was induced in a mouse pancreatic microvascular endothelial cell line (MMEC) in the presence of advanced glycation end products (AGEs) and in the endothelial lineage–traceble mouse line Tie2-Cre;Loxp-EGFP by administration of AGEs, with nonglycated mouse albumin serving as a control. Phosphorylated Smad3 was detected by immunoprecipitation/Western blotting and confocal microscopy. Blocking studies using receptor for AGE siRNA and a specific inhibitor of Smad3 (SIS3) were performed in MMECs and in STZ-induced diabetic nephropathy in Tie2-Cre;Loxp-EGFP mice.RESULTSConfocal microscopy and real-time PCR demonstrated that AGEs induced EndoMT in MMECs and in Tie2-Cre;Loxp-EGFP mice. Immunoprecipitation/Western blotting showed that Smad3 was activated by AGEs but was inhibited by SIS3 in MMECs and in STZ-induced diabetic nephropathy. Confocal microscopy and real-time PCR further demonstrated that SIS3 abrogated EndoMT, reduced renal fibrosis, and retarded progression of nephropathy.CONCLUSIONSEndoMT is a novel pathway leading to early development of diabetic nephropathy. Blockade of EndoMT by SIS3 may provide a new strategy to retard the progression of diabetic nephropathy and other diabetes complications.

Deletion of MK2 signalling in vivo inhibits small Hsp phosphorylation but not diabetic nephropathy

It is supposed that some stress-induced heat shock proteins (Hsps) are regulated through e.g. stimulation of the p38MAPK/MK(MAPKAP)-2 signalling pathway. It has been postulated from in vitro experiments that phosphorylation of Hsp25(rodents)/Hsp27(human), the major phosphorylation substrate of MK2, is responsible for mesangial contractility and glomerular hyperfiltration in the diabetic kidney. To verify this hypothesis in vivo we studied the renal function of nondiabetic and streptozotocin (STZ)-induced, diabetic MK2(-/-) mice in comparison to wild-type (WT) control mice. Following 8 weeks of hyperglycaemia, light microscopy showed increased glomerulosclerosis and tubulointerstitial renal fibrosis in both diabetic study groups. Protein analysis demonstrated that Hsp25 phosphorylation is stimulated upon high-glucose condition but inhibited in the diabetic MK2(-/-) mice. However, we found the kidney-body weight ratio significantly increased in diabetic WT and MK2(-/-) mice. No difference regarding the increased expression of the extracellular matrix proteins and TGF-beta1 between both diabetic study groups was observed. Importantly, diabetic MK2(-/-) mice showed no protection against renal hyperfiltration in the diabetic state and the development of diabetic albuminuria. Although activation of p38MAPK has been previously shown in diabetes mellitus, our results indicate that blockade of the downstream MK2/Hsp25 signalling pathway does not interfere with the development of early diabetic nephropathy.

YM440, a novel hypoglycemic agent, protects against nephropathy in Zucker fatty rats via plasma triglyceride reduction

The novel hypoglycemic agent, YM440 (( Z)-1,4-bis{4-[(3,5-dioxo-1,2,4-oxadiazolidin-2-yl) methyl] phenoxy}but-2-ene) is a ligand of the peroxisome proliferator-activated receptor, (PPAR) γ. YM440 has been shown to counteract insulin resistance in diabetic rodent models. However, it is not clear whether this compound has a significant effect on hyperlipidemia in vivo. Hyperlipidemia has been reported to be a risk factor for the early development of renal disease. The aim of this study is to examine the effects of chronic treatment with YM440 on hyperlipidemia and renal injury in obese Zucker fatty (ZF) rats. Treatment of 8-week-old ZF rats with YM440 (100 mg/kg/day) for 16 weeks decreased plasma triglyceride and cholesterol concentrations. YM440 markedly reduced the rate of progression of both albuminuria and proteinuria. YM440 normalized urinary N-acetyl-β- d-glucosaminidase (NAG) activity, which is a marker for renal proximal tubular damage, and ameliorated the rise in systolic blood pressure compared to the vehicle control. YM440 also blocked the development of nephromegaly. Histological analyses revealed that both glomerular area expansion and tubular cast accumulation gradually lessened in YM440-treated ZF rats. Regression analyses between the plasma triglyceride levels and the renal parameters (urinary protein excretion and albumin excretion) indicated that the renal parameters correlated positively with the plasma triglyceride levels. In conclusion, the hypolipidemic effects of YM440 prevent renal injury in ZF rats. YM440 might be useful for preventing the early development of diabetic nephropathy in subjects with type 2 diabetes by ameliorating metabolic control problems.

An imbalance between matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 contributes to the development of early diabetic nephropathy

High glucose and angiotensin-II (Ang-II) levels are the known important mediators of diabetic nephropathy. However, the effects of these mediators on matrix metalloproteinase-2 (MMP-2) and on tissue inhibitor of metalloproteinase-2 (TIMP-2) in proximal tubule cells have yet to be fully examined within the context of early stage diabetic nephropathy. In this study, we attempted to characterize changes in MMP-2 and TIMP-2 in streptozotocin-induced diabetic rats. To further examine the molecular mechanisms involved, we evaluated the effects of high glucose (30 mM) or Ang-II on MMP-2, TIMP-2 and collagen synthesis in proximal tubule cells, and investigated whether MMP-2 and TIMP-2 are regulated via the TGF-beta1 pathway. In streptozotocin-induced diabetic rats, TIMP-2 mRNA and protein levels were significantly higher than in controls. Urinary protein excretion also showed a significant positive correlation with glomerular and tubular TIMP-2 protein expressions, and a negative correlation with MMP-2 expression. In cultured cells, both high glucose and Ang-II induced significant increases in TGF-beta1, TIMP-2, and in collagen synthesis, and significant decreases in MMP-2 gene expression and activity, and thus disrupted the balance between MMP-2 and TIMP-2. Moreover, treatment with a selective angiotensin type 1 (AT1) receptor antagonist significantly inhibited Ang-II mediated changes in TGF-beta1, MMP-2, TIMP-2, and in collagen production, suggesting the role of the AT1 receptor. The addition of exogenous TGF-beta1 produced an effect similar to those of high glucose and Ang-II. Furthermore, the inhibition of TGF-beta1 protein prevented Ang-II-induced MMP-2 and TIMP-2 alterations, suggesting the involvement of a TGF-beta1 pathway. High glucose or Ang-II treatment induce alterations in MMP-2 and TIMP-2 balance, which favour TIMP-2 over-activity. Moreover, Ang-II-mediated changes in the productions of MMP-2 and TIMP-2 occur via AT1 receptors and a TGF-beta1-dependent mechanism. These results suggest that an imbalance between the MMP-2 and TIMP-2, caused primarily by an increase in TIMP-2 activity, contributes to the pathogenesis of diabetic nephropathy.

The Antioxidative Effects of Astragalus Saponin I Protect Against Development of Early Diabetic Nephropathy

It has been known that oxidative stress plays an important role in the development of diabetic nephropathy (DN). The antioxidative effects of Astragalus saponin I (AS I) were studied in vitro and in vivo. In the presence of high glucose and H2O2, the total antioxidative capability, catalase, reduced glutathione, and superoxide dismutase level of rat mesangial cells were significantly decreased, and transforming growth factor beta1 (TGF-beta1) mRNA level, collagen IV, and laminin level were significantly increased. When compared with those in the high glucose group, these 4 indexes of cells incubated in 2.0 and/or 20 micromol/L of AS I were significantly enhanced, and levels of TGF-beta1 mRNA, collagen IV and laminin were statistically decreased. By flowcytomery, percentages of S phase of cells incubated in high glucose and H2O2 were lowered, while those in AS I were increased. Furthermore, the physical behaviors of rats treated with 12 mg/kg of AS I restored with vigor and weight gaining, while the level of HbAlC was significantly reduced. Thus, AS I has antioxidative effects and is a potential compound worth further study because it may prevent the development of DN.

Circulating immune complexes among diabetic children.

Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease associated with the presence of different types of autoantibodies. The presence of these antibodies and the corresponding antigens in the circulation leads to the formation of circulating immune complexes (CIC). CIC are known to persist in the blood for long periods of time. Such CIC following deposition in the small blood vessels have the potential to lead to microangiopathy with debilitating clinical consequences. The aim of our pilot study was to investigate whether a correlation exists between CIC and the development of microvascular complications in diabetic children. Isolation of a new glycoprotein complement inhibition factor (CIF) from the parasitic plant Cuscuta europea seed, which appears to bind specifically to complement component C3 has provided an unique tool for the measurement of immune complexes by means of ELISA-type techniques (CIF-ELISA). We studied the levels of CIC (IgG, IgM and IgA) in 58 diabetic children (mean age 12.28±4.04 years, diabetes duration 5.3±3.7 years), 29 of them had vascular complications (group 1) and the other 29 were without vascular complications (group 2). As controls, we studied sera samples from 21 healthy children (mean age 13.54±4.03 years). Sera from the diabetic patients showed statistically significant higher levels of CIC IgG ( p=0.03) than sera from the control group. In sera from group 1 values of CIC IgG showed statistically significant higher levels than controls (0.720±0.31 vs. 0.46±0.045; p=0.011) Sera from 59% of the patients were positive for CIC IgG, 36% for CIC IgM and 9% for CIC IgA. Among 26 patients with microalbuminuria, sera from 17/26 (65%) were positive for CIC IgG, 8/26 (31%) for CIC IgM and 2/26 (8%) for CIC IgA. CIC IgG correlated with HbA1c (r=0.51; p=0.005) and microalbuminuria (r=0.42, p=0.033). CIC IgA correlated with age (r=0.44, p=0.03). CIC IgM correlated with the duration of diabetes (r=0.63, p=0.02). These findings suggest that elevated levels of CIC IgG are associated with the development of early diabetic nephropathy.

Early development of diabetic nephropathy in adolescent type 2 diabetes mellitus (T2DM)

Early development of diabetic nephropathy in adolescent type 2 diabetes mellitus (T2DM)

High glucose-induced intercellular adhesion molecule-1 (ICAM-1) expression through an osmotic effect in rat mesangial cells is PKC-NF-kappa B-dependent.

Infiltration of mononuclear cells and glomerular enlargement accompanied by glomerular cell proliferation are very early characteristics of the pathophysiology of diabetes. To clarify the mechanism of early diabetic nephropathy, we measured [3H]-thymidine incorporation and cell numbers to show the influence of a high ambient glucose concentration and the osmotic effect on rat mesangial cell proliferation. We also measured the effect of high glucose on the expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 by flow cytometry and semiquantitative RT-PCR in mesangial cells and the adhesion of leukocytes to mesangial cells. Cells exposed to high D-glucose (30 mmol/l) caused an increase in [3H]-thymidine incorporation and cell numbers at 24 and 48 h and normalized at 72 h (p < 0.05), whereas these changes were not found in high mannitol (30 mmol/l), IL-1 beta, or TNF alpha-stimulated mesangial cells. Cells exposed to high-glucose (15, 30, or 60 mmol/l) or osmotic agents (L-glucose, raffinose and mannitol) showed that intercellular adhesion molecule-1 expression began to increase after 24 h, reached its maximum at 24 and 48 h and gradually decreased afterwards. The stimulatory effects of high glucose and high mannitol on mRNA expression were observed as early as 6 h and reached its maximum at 12 h. Up-regulation of ICAM-1 protein and mRNA was also found in IL-1-beta and TNF-alpha-stimulated mesangial cells. Neither vascular adhesion molecule-1 protein nor mRNA expression was, however, affected by high glucose and high mannitol. Notably, the protein kinase C inhibitors calphostin C and staurosporine reduced high glucose- or high mannitol-induced intercellular adhesion molecule-1 mRNA expression and high glucose-induced proliferation. Furthermore, the NF-kappa B inhibitor N-tosyl-L-phenylalanine chloromethyl ketone reduced high glucose- or high mannitol-induced intercellular adhesion molecule-1 mRNA expression and high glucose-induced proliferation. Results showed that high glucose (15, 30 mmol/l) or high concentrations of osmotic agents remarkably increased the number of adherent leukocytes to mesangial cells (p < 0.01) compared with control cells (5 mmol/l D-glucose). Functional blocking of intercellular adhesion molecule-1 on mesangial cells with rat intercellular adhesion molecule-1 monoclonal antibody, calphostin C, staurosporine, or N-tosyl-L-phenylalanine chloromethyl ketone significantly inhibited high glucose- or high mannitol-induced increase in leukocyte adhesion (p << 0.05). These results suggest that high glucose can upregulate intercellular adhesion molecule-1 protein and mRNA expression but not vascular adhesion molecule-1 expression in mesangial cells and promote leukocyte adhesion through up-regulation of intercellular adhesion molecule-1 through osmotic effect, possibly depending on the protein kinase C nuclear factor-kappa B (PKC-NF-kappa B) pathway. High glucose itself can also promote mesangial cell proliferation through the PKC-NF-kappa B pathways. We conclude that hyperglycaemia in itself seems to be an important factor in the development of early diabetic nephropathy.

Initial Ultrastructural Changes in Pore Size and Anionic Sites of the Glomerular Basement Membrane in Streptozotocin-Induced Diabetic Rats and Their Prevention by Insulin Treatment

Background/Aim: The present study was conducted to elucidate the mechanism(s) of the development of early diabetic nephropathy, examining ultrastructural changes employing electron microscopy, especially changes in pore size of the glomerular basement membrane (GBM) of streptozotocin (STZ)-induced diabetics rats. Methods: Urinary albumin excretion rate (UAE), pore size of the lamina densa of the GBM visualized directly by the tissue negative staining method, and number of anionic sites (AS) in the corresponding portion of the lamina rara externa were determined for 6 weeks in diabetic rats without and with insulin treatment. Results: The UAE of the diabetic rats increased with time and was significantly greater than that of the nondiabetic control rats after 4 weeks (p < 0.01), while insulin treatment suppressed the increased UAE of diabetic rats. The median values in both short diameter and long dimension of the pores in the diabetic group were markedly increased at the 2nd week as compared with those in the nondiabetic control rats, whereas no significant change was found in the pore size of the diabetic rats with insulin treatment. Moreover, the number of AS in the GBM of the diabetic rats was significantly (p < 0.001) decreased from the 2nd week onward. Insulin treatment also prevented a decrease in AS number in diabetic rats. Conclusions: It is suggested from these results that an impairment of barrier size selectivity occurs at a very early stage of STZ-induced diabetes in rats, which may enhance the abnormality of the charge-selective properties of the GBM. In addition, insulin treatment may protect this barrier system through normalizing blood glucose control in STZ-diabetic rats.

Sodium-lithium Transport in Adolescents With IDDM: Relationship to incipient nephropathy and glycemic control

The aims of this study were to examine genetic and environmental influences in the development of early diabetic nephropathy and to assess the value of measuring membrane sodium transport as a marker for early nephropathy. We measured erythrocyte sodium-lithium (Na-Li) countertransport, blood pressure (BP), HbA1c, and microalbuminuria (MA) in 84 adolescents with insulin-dependent diabetes mellitus (IDDM), 29 of whom had MA. Twenty-nine non-MA patients were selected and matched for age, sex, and IDDM duration with the 29 diabetic subjects with MA. The 84 diabetic adolescents were also compared with 85 nondiabetic siblings. The erythrocyte Na-Li countertransport was significantly greater in the IDDM group than in the sibling group (mean +/- SD, 0.41 +/- 0.14 vs. 0.30 +/- 0.11 mmol Li.liters of erythrocytes-1.h-1, respectively, P < 0.0001), but a significant correlation was noted between the results in IDDM subjects and their siblings (r = 0.42, P < 0.0008). Na-Li countertransport was not different in the diabetic subjects with or without MA (0.43 +/- 0.13 vs. 0.37 +/- 0.13 mmol Li.liters of erythrocytes-1.h-1, respectively). There was a significant correlation in the IDDM group between recent diabetic control (HbA1c) and Na-Li countertransport (r = 0.37, P < 0.003). Diastolic BP was significantly higher in the IDDM group with MA than in those without MA (60 +/- 6 vs. 55 +/- 6 mmHg, respectively, P < 0.03). These results suggest that erythrocyte Na-Li countertransport is influenced by the diabetic milieu. However, there was also evidence in our subjects of a genetic contribution to Na-Li countertransport as seen by the correlation between levels in the IDDM subjects and their siblings. Using Na-Li countertransport, we were not able to segregate those IDDM adolescents with and without early nephropathy.

The Immunogenetics of Early Nephropathy in Insulin-dependent Diabetes Mellitus: Association Between the HLA-A2 Antigen and Albuminuria

One-hundred and seventy-two normotensive, insulin-dependent diabetic patients without clinical proteinuria (Albustix negative) were typed for the major histocompatibility complex class I (HLA-A, -B) and class II (HLA-DR) antigens. Urinary albumin excretion was measured as the albumin:creatinine ratio (UA/UC, mg/mmol) in an early morning sample. Patients expressing the HLA-A2 antigen had significantly higher UA/UC values than those not expressing the antigen. The observed ratio of geometric means was 1.77 (95 per cent confidence interval (CI) 1.18-2.67; p < 0.01); the relative risk of microalbuminuria (UA/UC > 3.0 mg/mmol) associated with expression of HLA-A2 was 2.52 (95 per cent CI 1.11-5.73; p < 0.05). There was no significant association between UA/UC and HLA-B8, -B15, -DR3, -DR4 or other antigens. Patients were re-studied after a mean period of 5.3 years: multiple linear regression analysis showed that the UA/UC at this time was positively related to the initial glycosylated haemoglobin level (p < 0.01) and expression of the HLA-A2 antigen (p < 0.05), but not to blood pressure or creatinine clearance. Fifteen patients developed macroalbuminuria at follow-up (UA/UC > 45.5 mg/mmol). Compared with a group matched for age, sex, duration of diabetes, and glycosylated haemoglobin who did not develop macroalbuminuria, macroalbuminuric patients had a higher frequency of HLA-A2 (p < 0.01). The odds ratio of progressing to macroalbuminuria associated with HLA-A2 had a 95 per cent CI of 1.71 to infinity. We conclude that an immunogenetic factor may play a role in the development of early diabetic nephropathy and that the risk associated with expression of the HLA-A2 antigen is independent of metabolic control and blood pressure.