Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization

AbstractThe 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca2+-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations

Antihypertensive Treatment in Diabetic Kidney Disease: The Need for a Patient-Centered Approach

Diabetic kidney disease affects up to forty percent of patients with diabetes during their lifespan. Prevention and treatment of diabetic kidney disease is currently based on optimal glucose and blood pressure control. Renin-angiotensin aldosterone inhibitors are considered the mainstay treatment for hypertension in diabetic patients, especially in the presence of albuminuria. Whether strict blood pressure reduction entails a favorable renal outcome also in non-albuminuric patients is at present unclear. Results of several clinical trials suggest that an overly aggressive blood pressure reduction, especially in the context of profound pharmacologic inhibition of the renin-angiotensin-aldosterone system may result in a paradoxical worsening of renal function. On the basis of this evidence, it is proposed that blood pressure reduction should be tailored in each individual patient according to renal phenotype

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR

AbstractWe are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca2+-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients

Resistant Hypertension, Time-Updated Blood Pressure Values and Renal Outcome in Type 2 Diabetes Mellitus

BACKGROUND: Apparent treatment resistant hypertension (aTRH) is highly prevalent in patients with type 2 diabetes mellitus (T2D) and entails worse cardiovascular prognosis. The impact of aTRH and long-term achievement of recommended blood pressure (BP) values on renal outcome remains largely unknown. We assessed the role of aTRH and BP on the development of chronic kidney disease in patients with T2D and hypertension in real-life clinical practice.METHODS AND RESULTS: Clinical records from a total of 29 923 patients with T2D and hypertension, with normal baseline estimated glomerular filtration rate and regular visits during a 4-year follow-up, were retrieved and analyzed. The association between time-updated BP control (ie, 75% of visits with BP <140/90 mm Hg) and the occurrence of estimated glomerular filtration rate <60 and/or a reduction 6530% from baseline was assessed. At baseline, 17% of patients had aTRH. Over the 4-year follow-up, 19% developed low estimated glomerular filtration rate and 12% an estimated glomerular filtration rate reduction 6530% from baseline. Patients with aTRH showed an increased risk of developing both renal outcomes (adjusted odds ratio, 1.31 and 1.43; P<0.001 respectively), as compared with those with non-aTRH. No association was found between BP control and renal outcomes in non-aTRH, whereas in aTRH, BP control was associated with a 30% (P=0.036) greater risk of developing the renal end points.CONCLUSIONS: ATRH entails a worse renal prognosis in T2D with hypertension. BP control is not associated with a more-favorable renal outcome in aTRH. The relationship between time-updated BP and renal function seems to be J-shaped, with optimal systolic BP values between 120 and 140 mm Hg

Apparent Treatment Resistant Hypertension, Blood Pressure Control and the Progression of Chronic Kidney Disease in Patients with Type 2 Diabetes

Background/Aims: Apparent treatment resistant hypertension (aTRH) is highly prevalent in patients with type 2 diabetes (T2D) and chronic kidney disease (CKD). The impact of aTRH and achievement of recommended blood pressure (BP) values on the rate of glomerular filtration rate (eGFR) loss in CKD patients is poorly known. To assess the role of aTRH and time-updated BP control (BPC) on the progression of CKD in patients with T2D and hypertension (HT) in real life clinical practice. Methods: Clinical records from a total of 2,778 diabetic patients with HT and stage 3 CKD (i.e. baseline eGFR values between 30 and 60 ml/min) and regular visits during a four-year follow-up were analyzed. The association between BPC (i.e. 75% of visits with BP <140/90 mmHg) and eGFR loss (i.e. a >30% reduction from baseline) or worsening of albuminuria status over time was assessed. Results: At baseline 33% of patients had aTRH. Over the 4-year follow-up, 20% had a >30% eGFR reduction. Patients with aTRH had an increased risk of eGFR loss >30% (OR 1.31; P<0.007). In patients with aTRH, BPC was associated with a 79% (P=0.029) greater risk of eGFR reduction despite a 58% (P=0.001) lower risk of albuminuria status worsening. In non-aTRH, no association was found between BPC and renal outcome. Conclusion: In patients with stage 3 CKD the presence of aTRH entails a faster loss of eGFR. More effective prevention of aTRH should be implemented as this condition is associated with a burden of risk not modifiable by tight BP reduction

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

BackgroundAlbuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 3 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 3 CKD in a large cohort of patients affected by T1DM.MethodsA total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 3 CKD (eGFR 30% from baseline was evaluated.ResultsThe mean estimated GFR was 9817mL/min/1.73m(2) and the proportion of patients with albuminuria was 15.3% (n=654) at baseline. About 8% (n=337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR 90ml/min/1.73m(2) and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60mL/min/1.73m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline.Conclusions Albuminuria and eGFR reduction represent independent risk factors for incident stage 3 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Metabolic syndrome, serum uric acid and renal risk in patients with T2D

Background and aims: Metabolic Syndrome (Mets) and increased serum uric acid (SUA), are well known renal risk predictors and often coexist in patients with type 2 diabetes (T2D). Whether they independently contribute to the onset of CKD is at present unclear. Methods and results: Within the AMD Annals database we identified patients with T2D and normal renal function and urine albumin excretion at baseline and regular follow-up visits during a 4-year period. Blood pressure, BMI, HDL, triglycerides, and SUA were available in 14,267 patients. The association between Mets and/or hyperuricemia (HU, top fifth gender specific quintile) and the occurrence of renal outcomes were evaluated. Results: At baseline 59% of patients (n = 8,408) showed Mets and 18% (n = 2,584) HU. Over the 4-year follow-up, 14% (n = 1,990) developed low eGFR (i.e. below 60 mL/min/1.73 m2), and 26% (n = 3,740) albuminuria. After adjustment for confounders, BP 65130/85, low HDL, triglycerides 65150 and HU were independently related to the development of low eGFR (1.57, P<0.001; 1.13, P = 0.056; 1.18, P = 0.008; 1.26, P = 0.001) and of albuminuria (1.35, P<0.001; 1.18, P = 0.001; 1.15, P = 0.002; 1.24, P = 0.001), respectively. The incidence of low eGFR was higher in patients with HU independent of the presence or absence of Mets (21%, OR 1.30, p = 0.009 and 20%, 1.57, p<0.000 respectively), while albuminuria occurred more frequently in those with Mets and HU (32%, OR 1.25, p = 0.005) as compared to the reference group. Conclusions: HU and Mets are independent predictors of CKD and its individual components in patients with T2D

Peripheral artery disease and blood pressure profile abnormalities in hemodialysis patients

Patients undergoing chronic hemodialysis (HD) are at increased risk for peripheral artery disease (PAD). Both ankle-brachial index (ABI) and ambulatory blood pressure monitoring (ABPM) in the interdialytic period have been shown to be strong predictors of all-cause mortality

Diabetic kidney disease in the elderly: Prevalence and clinical correlates

Background: Diabetic kidney disease (DKD) is a major burden in elderly patients with type 2 diabetes (T2DM). Low estimated glomerular filtration rate (eGFR+, &lt; 60 mL/min/1.73 m2) and albuminuria (Alb+) are essential for the diagnosis of DKD, but their association with clinical variables and quality of care may be influenced by ageing. Methods: Here we investigated the association of clinical variables and quality of care measures with eGFR+ and Alb+ in 157,595 T2DM individuals participating to the Italian Association of Clinical Diabetologists (AMD) Annals Initiative, stratified by age. Results: The prevalence of eGFR+ and Alb+ increased with ageing, although this increment was more pronounced for low eGFR. Irrespective of age, both the eGFR+ and Alb + groups had the worst risk factors profile when compared to subjects without renal disease, showing a higher prevalence of out-of target values of HbA1c, BMI, triglycerides, HDL-C, blood pressure and more complex cardiovascular (CVD) and anti-diabetic therapies, including a larger use of insulin In all age groups, these associations differed according to the specific renal outcome examined: male sex and smoking were positively associated with Alb+ and negatively with eGFR+; age and anti-hypertensive therapies were more strongly associated with eGFR+, glucose control with Alb+, whereas BMI, and lipid-related variables with both abnormalities. All these associations were attenuated in the older (&gt; 75 years) as compared to the younger groups (&lt; 65 years; 65-75 years), and they were confirmed by multivariate analysis. Notably, Q-score values &lt; 15, indicating a low quality of care, were strongly associated with Alb+ (OR 8.54; P &lt; 0.001), but not with eGFR+. Conclusions: In T2DM patients, the prevalence of both eGFR and Albuminuria increase with age. DKD is associated with poor cardiovascular risk profile and a lower quality of care, although these associations are influenced by the type of renal abnormality and by ageing. These data indicate that clinical surveillance of DKD should not be unerestimated in old T2DM patients