Leonardo Lucchi

Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end‐stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2‐DE and identified with ESI‐Q‐TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic acid phosphatase precursor, the ribonuclease and the kallikrein‐3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig κ chain C region, Ig κ chain V‐II region Cum, Ig κ‐chain V‐III region SIE, carbonic anhydrase 1, plasma retinol‐binding protein, β‐2‐microglobulin precursor, β‐2‐glycoprotein 1.

Oxidative Metabolism of Polymorphonuclear Leukocytes and Serum Opsonic Activity in Chronic Renal Failure

Luminol-amplified chemiluminescence was used to study the oxidative metabolism of polymorphonuclear leukocytes (PMN), in resting state and in response to opsonized zymosan, in 65 patients with different degrees of chronic renal failure (CRF) or on regular dialysis treatment (RDT). Every patient was compared on the same day with a normal subject. Furthermore, the serum opsonic activity was evaluated, cross-matching zymosan opsonized by serum from CRF-RDT patients and normals with PMN from CRF-RDT patients and normals. PMN resting chemiluminescence showed a progressive increase inversely related to the glomerular filtration rate, and it remained high in patients on RDT. Zymosan-activated chemiluminescence indicated a deficit in phagocytosis for PMN of patients with a glomerular filtration rate lower than 10 ml/min, persisting in RDT patients. The serum opsonic activity was always significantly lower in CRF and in RDT patients than in the control group; this defect was already present in patients with mild renal impairment. Our findings suggest that PMN from CRF or RDT patients have an increased reactive oxygen metabolite production in the resting state that may cause cell and tissue damage; the opsonization impairment and the decreased PMN phagocytic activity contribute to increased vulnerability to infection in these patients.

No Direct Evidence of Increased Lipid Peroxidation in Hemodialysis Patients

Lipid peroxidation, as measured by the thiobarbituric acid test, has been reported to have increased in hemodialysis (HD) patients, even though the test has low specificity in vivo. Conjugated diene fatty acid (CDFA) hydroperoxides are formed during lipid peroxidation, but not all conjugated dienes (CD) detected in humans originate from lipid peroxidation: octadeca-9,11-dienoic acid, a nonhydroperoxide CD derivative of linoleic acid (CDLA), has a dietary origin. We evaluated CDFA hydroperoxides, CDLA and linoleic acid, using high-performance liquid chromatography, in lipids extracted from plasma, adipose tissue and RBC membranes obtained from 25 patients treated with HD, 16 patients treated with hemodiafiltration (HDF) and 29 controls. No differences in the levels of CDFA hydroperoxides and linoleic acid were seen in any of the groups. Concentrations of CDLA were found to be significantly high in the adipose tissue and low in the RBC membranes of HD patients. HDF-treated patients showed the same results as HD patients. No direct evidence of increased lipid peroxidation was found in HD patients. This does not exclude the possibility that lipid peroxidation is increased and escapes direct detection due to the bodys homeostatic control eliminating the increased production of hydroperoxides. Both HD- and HDF-treated patients showed a significant change in CDLA concentrations, either in the adipose tissue, or in the RBC membranes. These dietary CD may be mistaken for markers of lipid peroxidation by conventional methodologies.

Conjugated Diene Fatty Acids in Patients with Chronic Renal Failure: Evidence of Increased Lipid Peroxidation?

Conjugated diene fatty acids (CDFA) were evaluated by second derivative spectrophotometry in the plasma and adipose tissue of 42 chronic renal failure (CFR) patients in conservative treatment, 40 patients treated by hemodialysis (HD) with cuprophane, cellulose acetate or hemophan, 29 treated by hemodiafiltration (HDF) with polysulfone, polyacrylonitrile or polyamide, and 28 healthy controls. Plasma CDFA were also evaluated at the beginning, at 30 min and at the end of the dialytic session. CDFA were unchanged in CRF patients with creatinine clearance (Ccr) > 10 ml/min respect to the controls, CRF patients with Ccr < 10 ml/min showed a higher level of CDFA both in plasma and adipose tissue (p < 0.02). HD patients showed values similar to those of the control group. The lowest level of CDFA was found in HDF patients (p < 0.01 for plasma, p < 0.05 for adipose tissue versus both control and any other group). A significant relationship between plasma and adipose tissue CDFA was found in all groups. In the group of CRF patients with Ccr < 10 ml/min, females exhibited a higher level of CDFA both in plasma and adipose tissue. No significant change was found during dialytic session, independently from the membrane used. CDFA are not only primary products of lipid peroxidation, but also have a dietary origin, primarily from dairy products. Taking into account the reduced dietary intake, the increase in end-stage CRF may be due to an enhanced oxidative stress and/or to abnormalities in CDFA metabolism. Uremic patients, particularly in the predialytic stage, should be considered at risk for increased oxidative stress. HDF treatment better corrects the abnormality compared to conventional HD.

Polymorphonuclear Oxygen Free Radical Production and Complement Activation Induced by Dialysis Membranes as Assayed in an Experimental Model

Activation of polymorphonuclear leukocytes with subsequent production of reactive oxygen metabolites has been reported to occur during hemodialysis related to a membrane bioincompatibility. We used an experimental dialysis model to evaluate, by chemiluminescence, the production of reactive oxygen metabolites and, by C3a, complement activation induced by cuprophan, cellulose acetate, hemophan, polysulfone, polyacrylonitrile, polymethylmethacrylate or polyvinyl chloride blood lines alone. No differences were obtained in the system, at time 30 min compared to initial values, as far as zymosan-activated chemiluminescence is concerned; resting chemiluminescence increased markedly with cellulose acetate (+71%), cuprophan (+49%), polymethylmethacrylate (+22%), hemophan (+21%) but had no variation with polysulfone, polyacrylonitrile and blood line. The time course of C3a levels up to 120 min showed a marked rise with cuprophan and cellulose acetate, a moderate increase with hemophan, polysulfone and blood line, and a decrease with polymethylmethacrylate and polyacrylonitrile. The results obtained documented a different behavior of the production of reactive oxygen metabolites compared to complement activation and support the hypothesis that the production of reactive oxygen metabolites by polymorphonuclear leukocytes is stimulated not only by complement activation but also by a direct dialysis membrane interaction.

Early Initiation of Cinacalcet for the Treatment of Secondary Hyperparathyroidism in Hemodialysis Patients: A Three‐Year Clinical Experience

Despite the availability of standard therapy (vitamin D sterols and phosphate binders) for the treatment of secondary hyperparathyroidism (SHPT) in hemodialyzed (HD) patients, a significant percentage of patients still fail to achieve targets recommended by the Kidney Disease Outcomes Quality Initiative (K/DOQI) of the National Kidney Foundation for parathyroid hormone (PTH), calcium, and phosphorus. The calcimimetic cinacalcet (CN) has been shown to be an effective treatment for SHPT, significantly reducing serum PTH while simultaneously lowering calcium, phosphorus, and calcium-phosphorus product levels, thus increasing the proportion of patients achieving the K/DOQI targets for bone mineral parameters. The aim of this study was to evaluate if early treatment with CN had beneficial effects in HD patients with mild-to-moderate SHPT in whom conventional treatments had failed to achieve NKF-K/DOQI targets for PTH, serum-corrected calcium, and phosphorus while minimizing the risk of paradoxical hypercalcemia and/or hyperphosphatemia. Clinical practice data were collected monthly, starting from 6 months prior to, and up to 36 months after, the start of CN therapy. CN was started at a dose of 30 mg daily or every other day, and titrated thereafter to achieve intact PTH (iPTH) <300 pg/mL. The dose of concomitant vitamin D and phosphate binders were also adjusted in order to achieve K/DOQI targets. Data from 32 patients were collected, 28 of whom had been treated with CN for at least 36 months at the time of data analysis. At baseline, patients had serum iPTH >300 pg/mL (570 ± 295 pg/mL) and/or serum-corrected calcium >9.5 mg/dL. CN induced significant decreases in iPTH, calcium, and calcium-phosphorus product with respect to baseline levels. The percentage of patients within K/DOQI target levels at baseline, 12, 24, and 36 months was 0, 81.2, 83.3, and 86.2% for iPTH; 34.4, 65.6, 86.6, and 89.6% for serum-corrected calcium; 40.6, 56.2, 69.6, and 72.4% for phosphorus; and 37.5, 62.5, 80, and 82.7% for calcium-phosphorus product. The mean dose of CN at the end of the observation period was 38 mg/day. The mean dose of concomitant medication (calcitriol, Al-containing phosphate binders, and sevelamer) decreased from baseline to 36 months. Early treatment with CN in HD patients with SHPT increases the proportion of patients achieving and maintaining K/DOQI targets with a low dose of CN (38 mg/day). These results suggest that the metabolic control obtained with low-dose CN administered early in the course of SHPT can be maintained or increased over time.

Influence of Urinary Calcium Concentration on Erythrocyte Morphology

The effect of the urinary calcium concentration (CaU) on erythrocyte morphology was studied by incubating erythrocytes in urine with prefixed CaUs of 5, 10, 20 and 40 mmol/l by addition of CaCl2. The same experiment was carried out on erythrocytes preincubated with levo-verapamil (l-V) at 10, 100 and 200 mumol/l. Phase contrast microscopy observations were performed at 0, 30, 60, 120, and 240 min by 2 experienced investigators. At 0 min the erythrocytes showed a clear extraglomerular pattern. At 60 min marked morphological and volumetric alterations were evident when the CaU was > or = 10 mmol/l. On the contrary, no change was found when red cells were treated with > or = 100 mumol/l l-V, independent of the CaU. Dysmorphic erythrocyturia has been related to transglomerular passage even if it was sporadically observed in hypercalciuric or lithiasic patients. This work suggests a role for a high CaU in causing the formation of microcytic and warped erythrocytes. In our opinion, in hypercalciuric urine the appearance of dysmorphic or mixed hematuria does not necessarily indicate transglomerular passage.