Analysis of Serum Advanced Glycation Endproducts Reveals Methylglyoxal-Derived Advanced Glycation MG-H1 Free Adduct Is a Risk Marker in Non-Diabetic and Diabetic Chronic Kidney Disease.

Accumulation of advanced glycation endproducts (AGEs) is linked to decline in renal function, particularly in patients with diabetes. Major forms of AGEs in serum are protein-bound AGEs and AGE free adducts. In this study, we assessed levels of AGEs in subjects with and without diabetes, with normal renal function and stages 2 to 4 chronic kidney disease (CKD), to identify which AGE has the greatest progressive change with decline in renal function and change in diabetes. We performed a cross-sectional study of patients with stages 2-4 CKD, with and without diabetes, and healthy controls (n = 135). Nine protein-bound and free adduct AGEs were quantified in serum. Most protein-bound AGEs increased moderately through stages 2-4 CKD whereas AGE free adducts increased markedly. Methylglyoxal-derived hydroimidazolone MG-H1 free adduct was the AGE most responsive to CKD status, increasing 8-fold and 30-fold in stage 4 CKD in patients without and with diabetes, respectively. MG-H1 Glomerular filtration flux was increased 5-fold in diabetes, likely reflecting increased methylglyoxal glycation status. We conclude that serum MG-H1 free adduct concentration was strongly related to stage of CKD and increased in diabetes status. Serum MG-H1 free adduct is a candidate AGE risk marker of non-diabetic and diabetic CKD.

Familial aggregation of albuminuria and arterial hypertension in an Aboriginal Australian community and the contribution of variants in ACE and TP53.

BACKGROUND: Aboriginal Australians are at high risk of cardiovascular, metabolic and renal diseases, resulting in a marked reduction in life expectancy when compared to the rest of the Australian population. This is partly due to recognized environmental and lifestyle risk factors, but a contribution of genetic susceptibility is also likely. METHODS: Using results from a comprehensive survey of one community (N = 1350 examined individuals), we have tested for familial aggregation of plasma glucose, arterial blood pressure, albuminuria (measured as urinary albumin to creatinine ratio, UACR) and estimated glomerular filtration rate (eGFR), and quantified the contribution of variation at four candidate genes (ACE; TP53; ENOS3; MTHFR). RESULTS: In the subsample of 357 individuals with complete genotype and phenotype data we showed that both UACR (h2 = 64%) and blood pressure (sBP h2 = 29%, dBP, h2 = 11%) were significantly heritable. The ACE insertion-deletion (P = 0.0009) and TP53 codon72 polymorphisms (P = 0.003) together contributed approximately 15% of the total heritability of UACR, with an effect of ACE genotype on BP also clearly evident. CONCLUSIONS: While the effects of the ACE insertion-deletion on risk of renal disease (especially in the setting of diabetes) are well recognized, this is only the second study to implicate p53 genotype as a risk factor for albuminuria - the other being an earlier study we performed in a different Aboriginal community (McDonald et al., J Am Soc Nephrol 13: 677-83, 2002). We conclude that there are significant genetic contributions to the high prevalence of chronic diseases observed in this population.

The CTGF gene -945 G/C polymorphism is not associated with cardiac or kidney complications in subjects with type 2 diabetes.

BACKGROUND: Connective tissue growth factor (CTGF) has been implicated in the cardiac and kidney complications of type 2 diabetes, and the CTGF -945 G/C polymorphism is associated with susceptibility to systemic sclerosis, a disease characterised by tissue fibrosis. This study investigated the association of the CTGF -945 G/C promoter variant with cardiac complications (left ventricular (LV) hypertrophy (LVH), diastolic and systolic dysfunction) and chronic kidney disease (CKD) in type 2 diabetes. METHODS: The CTGF -945 G/C polymorphism (rs6918698) was examined in 495 Caucasian subjects with type 2 diabetes. Cardiac structure and function were assessed by transthoracic echocardiography. Kidney function was assessed using estimated glomerular filtration rate (eGFR) and albuminuria, and CKD defined as the presence of kidney damage (decreased kidney function (eGFR <60 ml/min/1.73 m2) or albuminuria). RESULTS: The mean age ± SD of the cohort was 62 ± 14 years, with a body mass index (BMI) of 31 ± 6 kg/m2 and median diabetes duration of 11 years [25th, 75th interquartile range; 5, 18]. An abnormal echocardiogram was present in 73% of subjects; of these, 8% had LVH alone, 74% had diastolic dysfunction and 18% had systolic ± diastolic dysfunction. CKD was present in 42% of subjects. There were no significant associations between the CTGF -945 G/C polymorphism and echocardiographic parameters of LV mass or cardiac function, or kidney function both before and after adjustment for covariates of age, gender, BMI, blood pressure and hypertension. CTGF -945 genotypes were not associated with the cardiac complications of LVH, diastolic or systolic dysfunction, nor with CKD. CONCLUSIONS: In Caucasians with type 2 diabetes, genetic variation in the CTGF -945 G/C polymorphism is not associated with cardiac or kidney complications.

Advanced glycation urinary protein-bound biomarkers and severity of diabetic nephropathy in man.

BACKGROUND/AIMS: The formation of advanced glycation end products (AGEs) is accelerated in patients with diabetic nephropathy. The aim of this study was to ascertain if the urinary excretion of proteins modified by advanced glycation can be used as biomarkers for albuminuria in individuals with type 1 or type 2 diabetes. METHODS: Community-based patients with type 1 (n = 68) or type 2 diabetes (n = 216) attending a diabetes clinic of a tertiary referral hospital were classified as having normoalbuminuria (Normo, albumin excretion rate (AER) <20 µg/min), microalbuminuria (Micro, AER 20-200 µg/min) or macroalbuminuria (Macro, AER ≥200 µg/min). Serum and urine AGE-modified proteins were measured. RESULTS: In patients with both type 1 diabetes and type 2 diabetes, there was a clear association between the degree of albuminuria and urinary AGE-modified proteins (p < 0.0001). Exclusive to patients with type 1 diabetes, urinary excretion of the AGE carboxymethyllysine correlated with AER, whereas patients with type 2 diabetes and macroalbuminuria had an increase in urinary methylglyoxal, an AGE intermediate. These changes were independent of isotopic glomerular filtration rate levels. Serum concentrations of AGEs or soluble receptor for AGEs were not consistently associated with albuminuria in either type 1 or type 2 diabetes. CONCLUSIONS: Urinary excretion of proteins modified by AGEs may be useful biomarkers of albuminuria in individuals with type 1 and type 2 diabetes, warranting prospective investigation in larger diabetic cohorts.

Study Protocol--accurate assessment of kidney function in Indigenous Australians: aims and methods of the eGFR study.

BACKGROUND: There is an overwhelming burden of cardiovascular disease, type 2 diabetes and chronic kidney disease among Indigenous Australians. In this high risk population, it is vital that we are able to measure accurately kidney function. Glomerular filtration rate is the best overall marker of kidney function. However, differences in body build and body composition between Indigenous and non-Indigenous Australians suggest that creatinine-based estimates of glomerular filtration rate derived for European populations may not be appropriate for Indigenous Australians. The burden of kidney disease is borne disproportionately by Indigenous Australians in central and northern Australia, and there is significant heterogeneity in body build and composition within and amongst these groups. This heterogeneity might differentially affect the accuracy of estimation of glomerular filtration rate between different Indigenous groups. By assessing kidney function in Indigenous Australians from Northern Queensland, Northern Territory and Western Australia, we aim to determine a validated and practical measure of glomerular filtration rate suitable for use in all Indigenous Australians. METHODS/DESIGN: A cross-sectional study of Indigenous Australian adults (target n = 600, 50% male) across 4 sites: Top End, Northern Territory; Central Australia; Far North Queensland and Western Australia. The reference measure of glomerular filtration rate was the plasma disappearance rate of iohexol over 4 hours. We will compare the accuracy of the following glomerular filtration rate measures with the reference measure: Modification of Diet in Renal Disease 4-variable formula, Chronic Kidney Disease Epidemiology Collaboration equation, Cockcroft-Gault formula and cystatin C- derived estimates. Detailed assessment of body build and composition was performed using anthropometric measurements, skinfold thicknesses, bioelectrical impedance and a sub-study used dual-energy X-ray absorptiometry. A questionnaire was performed for socio-economic status and medical history. DISCUSSION: We have successfully managed several operational challenges within this multi-centre complex clinical research project performed across remote North, Western and Central Australia. It seems unlikely that a single correction factor (similar to that for African-Americans) to the equation for estimated glomerular filtration rate will prove appropriate or practical for Indigenous Australians. However, it may be that a modification of the equation in Indigenous Australians would be to include a measure of fat-free mass.

Three Dimensional Glomerular Reconstruction: A Novel Approach to Evaluate Renal Microanatomy in Diabetic Kidney Disease.

Mesangial metrics reflect glomerular filtration surface area in diabetes. The point-sampled intercept (PSI) method is the conventional method to calculate these parameters. However, this is time consuming and subject to underestimation. We introduce a novel three-dimensional (3D) reconstruction method applicable to light microscopy to measure mesangial metrics. Transmission electron microscopy (TEM), PSI and our new 3D imaging methods were used to quantify mesangial metrics from 22 patients with type 2 diabetes, normo-, micro- and macroalbuminuria and an estimated glomerular filtration rate of <60 mL/min/1.73 m2. Repeated-measures ANOVA test was used to test the equality of the measurement means from the three methods and the degree of inter method variability. Repeated-measures and post-estimation ANOVA tests together with correlation coefficient measurements were used to compare the methods with TEM as reference. There was a statistically significant difference in mesangial volume measurements (F(2, 16) = 15.53, p = 0.0002). The PSI method underestimated measurements compared to TEM and 3D methods by 30% (p = 0.001) and 15%, respectively (p < 0.001). 3D and TEM measurements did not differ significantly. 3D reconstruction is a reliable and time efficient method for calculating mesangial metrics. It may prove to be a useful tool in clinical and experimental diabetic kidney disease.

Low testosterone levels are common and associated with insulin resistance in men with diabetes.

CONTEXT: Low testosterone levels are common in men with type 2 diabetes and may be associated with insulin resistance. OBJECTIVE: We investigated prevalence of testosterone deficiency and the relationship between testosterone and insulin resistance in a large cohort of men with type 2 and type 1 diabetes. DESIGN: The study was a cross-sectional survey of 580 men with type 2 diabetes and 69 men with type 1 diabetes. A subgroup of 262 men with type 2 diabetes was then reassessed after a median of 6 months. RESULTS: Forty-three percent of men with type 2 diabetes had a reduced total testosterone, and 57% had a reduced calculated free testosterone. Only 7% of men with type 1 diabetes had low total testosterone. By contrast, 20.3% of men with type 1 diabetes had low calculated free testosterone, similar to that observed in type 2 diabetes (age-body mass index adjusted odds ratio = 1.4; 95% confidence interval = 0.7-2.9). Low testosterone levels were independently associated with insulin resistance in men with type 1 diabetes as well as type 2 diabetes. Serial measurements also revealed an inverse relationship between changes in testosterone levels and insulin resistance. CONCLUSIONS: Testosterone deficiency is common in men with diabetes, regardless of the type. Testosterone levels are partly influenced by insulin resistance, which may represent an important avenue for intervention, whereas the utility of testosterone replacement remains to be established in prospective trials.

Association between intrarenal arterial resistance and diastolic dysfunction in type 2 diabetes.

BACKGROUND: In comparison to the well established changes in compliance that occur at the large vessel level in diabetes, much less is known about the changes in compliance of the cardiovascular system at the end-organ level. The aim of this study was therefore to examine whether there was a correlation between resistance of the intrarenal arteries of the kidney and compliance of the left ventricle, as estimated by measurements of diastolic function, in subjects with type 2 diabetes. METHODS: We studied 167 unselected clinic patients with type 2 diabetes with a kidney duplex scan to estimate intrarenal vascular resistance, i.e. the resistance index (RI = peak systolic velocity-minimum diastolic velocity/peak systolic velocity) and a transthoracic echocardiogram (TTE) employing tissue doppler studies to document diastolic and systolic ventricular function. RESULTS: Renal RI was significantly higher in subjects with diastolic dysfunction (0.72 +/- 0.05) when compared with those who had a normal TTE examination (0.66 +/- 0.06, p < 0.01). Renal RI values were correlated with markers of diastolic dysfunction including the E/Vp ratio (r = 0.41, p < 0.001), left atrial area (r = 0.36, p < 0.001), the E/A ratio (r = 0.36, p < 0.001) and the E/E' ratio (r = 0.31, p < 0.001). These associations were independent of systolic function, hypertension, the presence and severity of chronic kidney disease, the use of renin-angiotensin inhibitors and other potentially confounding variables. CONCLUSION: Increasing vascular resistance of the intrarenal arteries was associated with markers of diastolic dysfunction in subjects with type 2 diabetes. These findings are consistent with the hypothesis that vascular and cardiac stiffening in diabetes are manifestations of common pathophysiological mechanisms.

Lowering of proteinuria in response to antihypertensive therapy predicts improved renal function in late but not in early diabetic nephropathy: a pooled analysis.

In late diabetic nephropathy (DN) the initial lowering of albumin excretion rate (AER) with antihypertensive therapy is proportional to the degree of subsequent preservation of glomerular filtration rate (GFR). Whether a similar relationship exists between AER and GFR in early diabetes is not known. The present analysis has compared AER and GFR responses to antihypertensive therapy in 33 published studies (77 treatment groups) of early and late DN in type 1 (T1) and type 2 (T2) diabetes, analyzed on an intention-to-treat basis. Prospective trials were included if the initial change in AER during the first year of therapy and the change in GFR during at least 2 years of follow-up could be estimated from group mean data. The initial % decreases in AER were 5.9 +/- 4.3 (T1), 10.5 +/- 5.4 (T2, normotensive) and 18.4 +/- 6.2 (T2, hypertensive) in early DN and 7.6 +/- 11.1 (T1) and 20.8 +/- 5.5 (T2) in late DN. The corresponding annual % rates of decline of GFR were 2.0 +/- 0.5 (T1), 1.6 +/- 0.5 (T2, normotensive) and 2.1 +/- 0.3 (T2, hypertensive) in early DN and 9.8 +/- 1.5 (T1) and 9.2 +/- 1.1 (T2) in late DN. AER and GFR responses in each treatment group were closely correlated in late nephropathy (T1, r = -0.67, p = 0.03; T2, r = 0.57, p = 0.02) but not in early nephropathy. In contrast to late DN, the initial decrease in AER with antihypertensive therapy was not shown to predict the subsequent rate of decline of GFR in early DN. It follows that assessment of renoprotection during antihypertensive therapy in early nephropathy should be based not only on albuminuria but also on the GFR response.

Is nonalbuminuric renal insufficiency in type 2 diabetes related to an increase in intrarenal vascular disease?

OBJECTIVE: To investigate the role of intrarenal vascular disease in the pathogenesis of nonalbuminuric renal insufficiency in type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied 325 unselected clinic patients who had sufficient clinical and biochemical information to calculate an estimated glomerular filtration rate (eGFR) using the Modified Diet in Renal Disease six-variable formula, at least two estimations of urinary albumin excretion rates (AER), and a renal duplex scan to estimate the resistance index of the interlobar renal arteries. The resistance index, measured as part of a complications surveillance program, was compared in patients with an eGFR < or >or=60 ml/min per 1.73 m(2) who were further stratified into normo- (AER <20), micro- (20-200), or macroalbuminuria (> 200 microg/min) categories. RESULTS: Patients with an eGFR <60 ml/min per 1.73 m(2) had a higher resistance index of the renal interlobar arteries compared with patients with an eGFR >or=60 ml/min per 1.73 m(2). However, the resistance index was elevated to a similar extent in patients with an eGFR <60 ml/min per 1.73 m(2) regardless of albuminuric status (normo- 0.74 +/- 0.01, micro- 0.73 +/- 0.01, and macroalbuminuria resistance index 0.75 +/- 0.11). Multiple regression analysis revealed that increased age (P < 0.0001), elevated BMI (P = 0.0001), decreased eGFR (P < 0.01), and decreased diastolic blood pressure (P < 0.01), but not an increased AER, were independently associated with an elevated resistance index in patients with impaired renal function. CONCLUSIONS: Subjects with type 2 diabetes and reduced glomerular filtration rate had similar degrees of intrarenal vascular disease, as measured by the intrarenal arterial resistance index, regardless of their AER status. The pathological mechanisms that determine the relationship between impaired renal function and AER status in subjects with type 2 diabetes remain to be elucidated.

Peroxisome proliferator-activated receptor alpha agonist, clofibrate, has profound influence on myocardial fatty acid composition.

The hypolipidemic fibrates have been identified as agonists of the peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a critical role in the regulation of cardiac fatty acid metabolism. Despite the widespread clinical use of fibrates, their role in myocardial oxidative stress and fatty acid composition is less known. In this study, male Sprague-Dawley rats were treated with either vehicle (olive oil, 1 ml/kg) or clofibrate (300 mg/kgday i.p.) for 1-14 days. Lipid peroxidation in heart homogenate was determined by thiobarbituric acid reactive substance (TBARS) assay. Results show that hearts from clofibrate-treated rats are more susceptible to FeSO(4)-induced TBARS production. The antioxidants including catalase and glutathione-related enzymes were marginally affected. We demonstrated that myocardial fatty acid composition was dramatically altered by clofibrate treatment. In hearts from clofibrate-treated rats, the principal n-6 polyunsaturated fatty acids (PUFAs), linoleic acid (C18:2 n-6) and arachidonic acid (C20:4 n-6), was significantly reduced, while the content of the principal n-3 PUFA, docosahexaenoic acid (C22:6 n-3), was markedly increased. The overall effect was to reduce n-6/n-3 ratio and increase the unsaturation extent of myocardial fatty acids. Functional study showed that hearts from clofibrate-treated rats had an improved recovery of post-ischemic contractile function and reduced ischemia/reperfusion (I/R)-induced infarct size. The data shows that clofibrate has a profound impact on cardiac fatty acid composition, which may contribute to its cardioprotective effect.

Nonalbuminuric renal insufficiency in type 2 diabetes.

OBJECTIVE: To determine the prevalence and characteristics of patients with type 2 diabetes who have impaired renal function, defined as a glomerular filtration rate (GFR) <60 ml. min(-1). 1.73 m(-2), and normoalbuminuria. RESEARCH DESIGN AND METHODS: A cross-sectional survey of 301 outpatients attending a single tertiary referral center using the plasma disappearance of isotopic (99m)Tc-diethylene-triamine-penta-acetic acid to measure GFR and at least two measurements of urinary albumin excretion rate (AER) over 24 h to determine albuminuria. RESULTS: A total of 109 patients (36%) had a GFR <60 ml. min(-1). 1.73 m(-2). The overall prevalence of normo-, micro-, and macroalbuminuria was 43 of 109 (39%), 38 of 109 (35%), and 28 of 109 (26%), respectively. Compared with patients with macroalbuminuria, those with normoalbuminuria were more likely to be older and female. After excluding patients whose normoalbuminuric status was possibly related to the initiation of a renin-angiotensin system (RAS) inhibitor before the start of the study, the prevalence of a GFR <60 ml. min(-1). 1.73 m(-2) and normoalbuminuria was 23%. Temporal changes in GFR in a subset of 34 of 109 (32%) unselected patients with impaired renal function were available for comparison over a 3- to 10-year period. The rates of decline in GFR (ml. min(-1). 1.73 m(-2). year(-1)) of -4.6 +/- 1.0, -2.8 +/- 1.0, and -3.0 +/- 07 were not significantly different for normo- (n = 12), micro- (n = 12), and macroalbuminuric (n = 10) patients, respectively. CONCLUSIONS: These results suggest that patients with type 2 diabetes can commonly progress to a significant degree of renal impairment while remaining normoalbuminuric.

Cytokine dialysis: an ex vivo study.

To test the hypothesis that dialysis using a new large pore membrane would achieve effective cytokine removal, blood from six volunteers was incubated with endotoxin (1 mg) and then circulated through a closed circuit with a polyamide membrane (nominal cut-off: 100 kDa). Hemodialysis was conducted at 1 or 9 L/hr of dialysate flow at the start of circulation and after 2 and 4 hours. The peak dialysate/plasma concentration ratios were 0.92 for interleukin (IL)-1beta, 0.67 for IL-6, 0.94 for IL-8, 0.33 for tumor necrosis factor (TNF)-a, and 0.11 for albumin. The dialysate/plasma ratios for all cytokines and albumin were decreased with increased dialysate flow from 1 to 9 L/hr (p < 0.05). Clearances for IL-1beta, IL-6, and IL-8, however, were significantly improved with increased dialysate flow (p < 0.01). There was no increase in TNF-a clearance (not significant) and a decrease in albumin clearance (p < 0.01). The peak clearance at 9 L/hr was 33 ml/min for IL-1beta, 19 for IL-6, 51 for IL-8, 11 for TNF-alpha, and 1.2 for albumin. No adsorption of cytokines was observed. We conclude that cytokine dialysis is achievable through a membrane with a high cut-off point with negligible albumin loss. These findings support the technical feasibility of this new approach to blood purification in sepsis.

Renal structure in normoalbuminuric and albuminuric patients with type 2 diabetes and impaired renal function.

OBJECTIVE: The structural basis of normoalbuminuric renal insufficiency in patients with type 2 diabetes remains to be elucidated. We compared renal biopsy findings in patients with type 2 diabetes and estimated glomerular filtration rate (eGFR) and measured GFR of <60 mL/min/1.73 m2, associated with either normo-, micro-, or macroalbuminuria. RESEARCH DESIGN AND METHODS: In patients with normo- (n = 8) or microalbuminuria (n = 6), renal biopsies were performed according to a research protocol. In patients with macroalbuminuria (n = 17), biopsies were performed according to clinical indication. Findings were categorized according to the Fioretto classification: category 1 (C1), normal/near normal; category 2 (C2), typical diabetic nephropathy (DN) with predominantly glomerular changes; and category 3 (C3), atypical with disproportionately severe interstitial/tubular/vascular damage and with no/mild diabetic glomerular changes. RESULTS: In our study population (mean eGFR 35 mL/min/1.73 m2), typical glomerular changes (C2) of DN were observed in 22 of 23 subjects with micro- or macroalbuminuria compared with 3 of 8 subjects with normoalbuminuria (P = 0.002). By contrast, predominantly interstitial or vascular changes (C3) were seen in only 1 of 23 subjects with micro- or macroalbuminuria compared with 3 of 8 normoalbuminuric subjects (P = 0.08). Mesangial area increased progressively from normal controls to patients with type 2 diabetes and normo-, micro-, and macroalbuminuria. Varying degrees of arteriosclerosis, although not necessarily the predominant pattern, were seen in seven of eight subjects with normoalbuminuria. CONCLUSIONS: Typical renal structural changes of DN were observed in patients with type 2 diabetes and elevated albuminuria. By contrast, in normoalbuminuric renal insufficiency, these changes were seen less frequently, likely reflecting greater contributions from aging, hypertension, and arteriosclerosis.

Less fat reduction per unit weight loss in type 2 diabetic compared with nondiabetic obese individuals completing a very-low-calorie diet program.

The objective was to compare weight loss and change in body composition in obese subjects with and without type 2 diabetes mellitus during a very-low-calorie diet (VLCD) program. Seventy weight-matched subjects with diabetes or normal fasting glucose (controls) participated in a 24-week VLCD study. Primary end points were changes in anthropometry, body composition, and fasting plasma insulin and ß-hydroxybutyrate concentrations. Fifty-one subjects (24 with diabetes) completed the study. No difference in weight loss between the 2 groups at 24 weeks was found by intention-to-treat analysis. Both groups completing the study per protocol had near-identical weight change during the program, with similar weight loss at 24 weeks (diabetes: 8.5 ± 1.3 kg vs control: 9.4 ± 1.2 kg, P = .64). Change in fat mass index correlated with change in body mass index (BMI) in both groups (diabetes: r = 0.878, control: r = 0.920, both P < .001); but change in fat mass index per unit change in BMI was less in the diabetic group compared with controls (0.574 vs 0.905 decrease, P = .003), which persisted after adjusting for age, sex, and baseline BMI (P = .008). Insulin concentrations remained higher and peak ß-hydroxybutyrate concentrations were lower in the diabetic compared with the control group. While following a 24-week VLCD program, obese subjects with and without diabetes achieved comparable weight loss; but the decrease in adiposity per unit weight loss was attenuated in diabetic subjects. Hyperinsulinemia may have inhibited lipolysis in the diabetic group; however, further investigation into other factors is needed.

Urinary proteomics for early diagnosis in diabetic nephropathy.

Diabetic nephropathy (DN) is a progressive kidney disease, a well-known complication of long-standing diabetes. DN is the most frequent reason for dialysis in many Western countries. Early detection may enable development of specific drugs and early initiation of therapy, thereby postponing/preventing the need for renal replacement therapy. We evaluated urinary proteome analysis as a tool for prediction of DN. Capillary electrophoresis-coupled mass spectrometry was used to profile the low-molecular weight proteome in urine. We examined urine samples from a longitudinal cohort of type 1 and 2 diabetic patients (n = 35) using a previously generated chronic kidney disease (CKD) biomarker classifier to assess peptides of collected urines for signs of DN. The application of this classifier to samples of normoalbuminuric subjects up to 5 years prior to development of macroalbuminuria enabled early detection of subsequent progression to macroalbuminuria (area under the curve [AUC] 0.93) compared with urinary albumin routinely used to determine the diagnosis (AUC 0.67). Statistical analysis of each urinary CKD biomarker depicted its regulation with respect to diagnosis of DN over time. Collagen fragments were prominent biomarkers 3-5 years before onset of macroalbuminuria. Before albumin excretion starts to increase, there is a decrease in collagen fragments. Urinary proteomics enables noninvasive assessment of DN risk at an early stage via determination of specific collagen fragments.

Association of ACE2 genetic variants with blood pressure, left ventricular mass, and cardiac function in Caucasians with type 2 diabetes.

BACKGROUND: Cardiovascular disease is common in diabetes, and is associated with activation of the renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE)2 is a recently described member of the RAS, and this study investigated whether ACE2 polymorphisms are associated with hypertension, left ventricular (LV) mass, and cardiac function in type 2 diabetes. METHODS: Variants in ACE2 (rs1978124, rs2074192, rs4240157, rs4646156, rs4646188) were examined in 503 Caucasian subjects with type 2 diabetes. As ACE2 is located on the X chromosome, analyses were performed separately for men and women. Hypertension was defined by a history of hypertension, and/or antihypertensive medications or blood pressure (BP) >130/80 mm Hg. LV mass and systolic function (ejection fraction) were assessed by transthoracic echocardiography. RESULTS: In men, hypertension was more prevalent with the ACE2 rs2074192 C allele (P = 0.023), rs4240157 G allele (P = 0.016) and rs4646188 T allele (P = 0.006). In men, the rs1978124 A allele was associated with a significantly lower ejection fraction compared to the G allele (62.3 ± 13.3 vs. 67.2 ± 10.9%, P = 0.002). This association remained significant after covariate adjustment for age, body mass index, hypertension, antihypertensive treatment, and BP. In women, the prevalence of hypertension was higher (P = 0.009) with the rs4240157 G allele, and the rs1978124 A allele was associated with significantly higher LV mass (P = 0.008). CONCLUSIONS: In Caucasians with type 2 diabetes, genetic variation in ACE2 is associated with hypertension and reduced systolic function in men, and hypertension and increased LV mass in women.

Chronic type 1 diabetes in spontaneously hypertensive rats leads to exacerbated cardiac fibrosis.

INTRODUCTION: Diabetes in human subjects is often associated with hypertension. The aim of this study was to examine the development of cardiac fibrosis following induction of type 1 diabetes in genetically hypertensive rats. METHODS: Diabetes was induced by streptozotocin (STZ) injection in 8-week-old normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) for a duration of 16 or 24 weeks. Aged-matched, nondiabetic WKY and SHRs were used as controls. At termination of treatment, the rats were anaesthetized, hearts arrested in diastole and perfusion fixed. A comprehensive examination of cardiac fibrosis throughout the right and left ventricles was undertaken in picrosirius red-stained sections, using image analysis and by undertaking collagen type I and type III immunohistochemistry. RESULTS: Induction of diabetes in the SHRs led to a marked increase in the levels of interstitial fibrosis in the left ventricle plus septum (LV+S) at both 16 and 24 weeks duration (59% and 43% increase, respectively) and also in the right ventricle after 24 weeks duration of diabetes (35% increase compared to the nondiabetic SHR). Exacerbated perivascular fibrosis was also observed in the LV+S in the diabetic-hypertensive rats at the later time point. These effects of induction of diabetes were not observed in the normotensive strain. CONCLUSIONS/INTERPRETATION: Our findings clearly demonstrate elevations in cardiac fibrosis when type 1 diabetes is combined with hypertension. Our findings thus stress the importance of closely monitoring both blood pressure and glucose levels in type 1 diabetic patients in order to prevent myocardial collagen deposition.

Integrating albuminuria and GFR in the assessment of diabetic nephropathy.

The evaluation of diabetic nephropathy from research and clinical viewpoints depends on the assessment of two continuous variables, albumin excretion rate (AER) and glomerular filtration rate (GFR). These two parameters form the basis of both the European classification of five stages of diabetic nephropathy, assessed according to changes in AER and GFR (hyperfiltration, normoalbuminuria, microalbuminuria, macroalbuminuria and end-stage renal disease), and the National Kidney Foundation classification of five stages of chronic kidney disease based on categories of estimated GFR. Although increases in AER generally precede a decline in GFR, some patients follow a non-albuminuric pathway to renal impairment. In addition, studies indicate that GFR decreases in a linear fashion from normal or above-normal levels. Whether hyperfiltration is part of the pathogenetic process leading to diabetic nephropathy remains unclear. Ideally, both AER and GFR should be assessed at an early stage in patients being evaluated for diabetic nephropathy. New methods such as the use of cystatin-C-based equations for estimating GFR should be considered because current creatinine-based estimates are inaccurate at normal or high GFRs. Serial assessments of both AER and GFR might allow diabetic nephropathy to be diagnosed at early stages of the disease process that are selectively responsive to new interventions. The successful integration of AER categories with the recently defined stages of GFR represents a new challenge in the management of diabetic nephropathy.

Effects of salt supplementation on the albuminuric response to telmisartan with or without hydrochlorothiazide therapy in hypertensive patients with type 2 diabetes are modulated by habitual dietary salt intake.

OBJECTIVE This prospective randomized double-blind placebo-controlled crossover study examined the effects of sodium chloride (NaCl) supplementation on the antialbuminuric action of telmisartan with or without hydrochlorothiazide (HCT) in hypertensive patients with type 2 diabetes, increased albumin excretion rate (AER), and habitual low dietary salt intake (LDS; <100 mmol sodium/24 h on two of three consecutive occasions) or high dietary salt intake (HDS; >200 mmol sodium/24 h on two of three consecutive occasions). RESEARCH DESIGN AND METHODS Following a washout period, subjects (n = 32) received 40 mg/day telmisartan for 4 weeks followed by 40 mg telmisartan plus 12.5 mg/day HCT for 4 weeks. For the last 2 weeks of each treatment period, patients received either 100 mmol/day NaCl or placebo capsules. After a second washout, the regimen was repeated with supplements in reverse order. AER and ambulatory blood pressure were measured at weeks 0, 4, 8, 14, 18, and 22. RESULTS In LDS, NaCl supplementation reduced the anti-albuminuric effect of telmisartan with or without HCT from 42.3% (placebo) to 9.5% (P = 0.004). By contrast, in HDS, NaCl supplementation did not reduce the AER response to telmisartan with or without HCT (placebo 30.9%, NaCl 28.1%, P = 0.7). Changes in AER were independent of changes in blood pressure. CONCLUSIONS The AER response to telmisartan with or without HCT under habitual low salt intake can be blunted by NaCl supplementation. By contrast, when there is already a suppressed renin angiotensin aldosterone system under habitual high dietary salt intake, the additional NaCl does not alter the AER response.