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Metabolic bone disease in chronic kidney disease and end-stage renal failure represents one of the most severe clinical complication in kidney patients, namely those on maintenance dialysis. Traditionally, bone changes are induced by secondary hyperparathyroidism. The CKD-MBD concept reflects the link between bone and cardiovascular disease in these patients. Studies documented also other bone pathological pathways in renal patients, such as osteoporosis, as in kidney and dialysis patients its risk factors are present as well as in general population. Resulting bone disease in renal disease and failure is far more complex than previously seen. However, the secondary hyperparathyroidism still represents the main pathological pathway.

Kidney international 103(2), 391-402 (2022). doi:10.1016/j.kint.2022.09.017, Published by Elsevier, New York, NY

Background and Aims Thrombotic microangiopathy (TMA) pathogenesis in kidney transplantation is poorly understood. The aim of this study was to evaluate donor and recipients risk factors for posttransplant TMA. Method In this retrospective multicenter national study, we identified both the case and 3M protocol kidney graft biopsies in 5258 recipients with TMA (n=57, 1.1 %) in 1994-2019. To further evaluate the effect of donor kidney on TMA development, the outcomes of the pair kidney grafts from the identical donors (n=43, 6 living donor, 1 pair kidney discarded, 6 insufficient data) as control group was analyzed. Results TMA occurred in 57 patients at 8th (median) post-operative day. Interestingly, neither histological nor laboratory signs of TMA were present in controls but in one case where TMA was evident in both recipients and in donor zero-hour biopsy. TMA associated with acute antibody mediated rejection was found in 11 cases (19%). Basic demographics between TMA and control group were similar, however longer cold ischemia time (p < 0,05, Figure 1) and more frequent delayed graft function (p < 0,05) were observed in TMA group. Patients with TMA experienced lower death-censored 5-year graft survival (56 vs. 84 %) in comparison with control group (p < 0,05, HR 2,904 (95% CI 1,411; 6,127), Figure 2). Conclusion TMA significantly affects the long-term kidney graft survival. Longer cold ischemia time was identified as the only risk factor and thus recipients genetic background is highly suspected in its pathogenesis.

Indoxyl sulfate has been identified as a major factor in the dysregulation of several genes. It is classified as a poorly dialyzable uremic toxin and thus a leading cause in the poor survival rate of dialysis patients. A monocentric, prospective, open cohort study was performed in 43 male patients undergoing chronic renal replacement therapy in a single hemodialysis center. The aim of the study was to determine the influence of acetate- versus citrate-buffered dialysis fluids in hemodialysis (HD) and postdilution hemodiafiltration (HDF) settings on the elimination of indoxyl sulfate. Also, additional factors potentially influencing the serum concentration of indoxyl sulfate were evaluated. For this purpose, the predialysis and postdialysis concentration ratio of indoxyl sulfate and total protein was determined. The difference was of 1.15 (0.61; 2.10), 0.89 (0.53; 1.66), 0.32 (0.07; 0.63), and 0.44 (0.27; 0.77) μmol/g in acetate HD and HDF and citrate HD and HDF, respectively. Acetate HD and HDF were superior when concerning IS elimination when compared to citrate HD and HDF. Moreover, residual diuresis was determined as the only predictor of lower indoxyl sulfate concentration, suggesting that it should be preserved as long as possible. This trial is registered with EU PAS Register of Studies EUPAS23714.

We present a case of severe calciphylaxis in both thighs and calves in a patient with end-stage renal disease and advanced secondary hyperparathyroidism with successful outcome after modified therapeutic approach. The cause of calciphylaxis is multifactorial. In our case, not only severe hyperparathyroidism and mediocalcinosis, but also medication (warfarin, calcium and active vitamin D) was involved. Because the initial conservative therapy was not successful, we indicated parathyroidectomy. However, we were not able to localize parathyroid glands and we contraindicated bilateral neck exploration due to the patientʼs critical status. Therefore, we decided for total thyroidectomy with total parathyroidectomy. Surgery was uncomplicated and histology confirmed that all four parathyroid glands were removed. The expected post-operative hypocalcaemia was asymptomatic and we did not use any calcium supplementation or vitamin D. Thyroid hormone replacement was easy. After surgery, the large and multiple subcutaneous defects started to heal. We achieved complete healing within several months of continuing dedicated care. There is no recurrence after three years. Prompt and radical surgical parathyroidectomy was extremely useful in our patient.

Traditionally, secondary hyperparathyroidism (SHPT) due to low calcitriol synthesis in failing kidneys has been treated with synthetic vitamin D receptor (VDR) activators. Recently, also the importance of low native vitamin D status beyond the issue of SHPT has been recognized in these patients. The aim of this work was to evaluate the effect of cholecalciferol supplementation in haemodialysis patients with low vitamin D serum levels. Another aim was to evaluate dual vitamin D therapy (cholecalciferol supplementation plus paricalcitol) in haemodialysis patients with vitamin D deficiency and concomitant SHPT. Ninety clinically stable maintenance haemodialysis patients were included. Supervised cholecalciferol supplementation was administered due to low vitamin D status. Patients with SHPT were also treated with synthetic VDR activator. Two pre hoc subgroups for statistical analysis were formed: patients treated solely with cholecalciferol (N = 34; 5,000 IU once weekly) and patients treated with a combination of cholecalciferol (identical dose, i.e. 5,000 IU/week) plus paricalcitol (N = 34, median dose 10 μg/week). Follow-up visit was scheduled 15 weeks later. Serum concentrations of calcidiol (25-D), parathyroid hormone (PTH) and beta-cross laps (CTX) were assessed at baseline and at follow-up. Serum calcium, phosphate and alkaline phosphatase (ALP) were monitored monthly. Only non-calcium gastrointestinal phosphate binders were administered. Dialysate calcium was 1.5 mmol/L in all patients, and no oral calcium-containing preparations were prescribed. Depending on data distribution, parametric or nonparametric statistical methods were used for comparison within each group (i.e. baseline vs. follow-up data) as well as between groups. In the whole group of 90 patients, mean baseline 25-D serum level was 20.3 (standard deviation 8.7) nmol/L, and it increased to 66.8 (19) nmol/L (p

The Ubiquitin-proteasome system has recently been shown to be involved in the regulation of cytokine expression. We tested the hypothesis of whether the in vivo administration of proteasome inhibitor MG-132 can modulate cytokine response and mortality in sepsis. Sepsis was induced in mice by caecal ligation and puncture (CLP). Animals were divided into four groups: control, CLP, CLP and 1 microg MG-132/g of b.w. intraperitoneally, and CLP and 10 microg MG-132/g of b.w. Plasma levels of interleukin (IL)-1, tumour necrosis factor-alpha (TNF-alpha, IL-6 and IL-10 were determined by ELISA 6 h after the induction of sepsis. CLP induced significant increase in plasma levels of all measured cytokines. MG-132 treatment resulted in lower increase in IL-1, TNF-alpha and IL-10 levels. IL-6 was not significantly affected. A mortality study revealed prolonged survival in MG-132 treated mice. We conclude that MG-132 treatment decreases inflammatory response and prolongs survival in the CLP model of sepsis.

Proteasome inhibitors are novel potential drugs for therapy of many diseases, and their effects are not fully understood. We investigated direct effects of peptide vinylsulfone inhibitor AdaAhx3L3VS on protein and amino acids metabolism in rat skeletal muscle. Soleus and extensor digitorum longus muscles were incubated in a medium containing 30 micromol/l AdaAhx3L3VS or no inhibitors. Total proteolysis was determined according to the rates of tyrosine release into the medium during incubation. The rates of leucine oxidation and protein synthesis were evaluated during incubation in medium containing L-[1-14C]leucine. Amino acid concentrations in the medium were measured using HPLC. AdaAhx3L3VS decreased tyrosine release into the medium by 21 and 19 %, decreased leucine incorporation into proteins by 22 and 12 %, and increased leucine oxidation by 24 and 19 % in soleus and extensor digitorum longus muscles, respectively. The release of amino acids into the medium was reduced. We conclude that AdaAhx3L3VS significantly decreased proteolysis and protein synthesis and increased leucine oxidation.

The ubiquitin–proteasome system (UPS) is a proteolytic system that is localized in cytosol and nucleus and requires ATP and mostly also the cofactor ubiquitin for proteolysis. Proteins to be degraded by UPS are tagged by polyubiquitin chain in a process precisely regulated by a system of ubiquitinating and deubiquitinating enzymes that assure the specificity and control of proteolysis. The proteasome contains protease active sites that account for the cleavage preferences: chymotryptic, tryptic and peptidylglutamyl (Glickman & Ciechanover 2002). The great importance of the UPS is now evident. It is responsible for the breakdown of a large variety of cell proteins, involved in important biological processes, including cell cycle, cell growth, gene expression, DNA repair, stress response and also programmed cell death (Drexler 1998; Hershko et al. 2000). The activated breakdown of myofibrillar proteins, i.e. actin and myosin, in many serious disorders, including cancer, renal failure, acidosis, infections, burns and sepsis, correlates with enhanced activity of the UPS (Tiao et al. 1994; Mitch & Price 2003). Many proteasome inhibitors have been developed (Kisselev & Goldberg 2001). They are divided into several groups according to the chemical structure and proteasome specificity. Most of them (except the fungal metabolite, lactacystine) are based on short oligopeptidic sequences with an electrophilic structure at the C-terminus. This electrophilic structure can be an aldehyde, a boronate (reversible inhibitors), an epoxyketone or a vinylsulfone (irreversible inhibitors) (Lee & Goldberg 1998). Proteasome inhibitors have been introduced as novel therapeutic agents. The first drug of this class is Bortezomib (Velcade®, Millennium Pharmaceuticals, Cambridge, MA, USA), used for the treatment of multiple myeloma. This and other novel proteasome inhibitors are currently undergoing clinical trials as potential treatment for a growing number of other cancer types (Mack et al. 2003; Davis et al. 2004) and other diseases, such as stroke and inflammatory diseases (Di Napoli & Papa 2003). The mechanism of action of proteasome inhibitors predicts large-scale effects in the organism. In general, proteasome inhibitors seem to have manageable adverse effects, e.g. thrombocytopenia, fatigue and peripheral neuropathy (Richardson et al. 2003). As it is presently believed, the proteasome is responsible for a substantial part of proteolysis in physiological state. Several authors studied the ability of proteasome inhibitors to reduce enhanced protein degradation in various catabolic diseases, including denervation, hyperthyroidism, burn and sepsis (Tawa et al. 1997; Fang et al. 1998; Hobler et al. 1998; Fischer et al. 2000), and their findings support the hypothesis that the ubiquitin–proteasome pathway plays a central role in muscle wasting in these catabolic states. The aim of the present study is to evaluate the direct effects of MG132 and new proteasome inhibitors – ZL3VS and AdaAhx3L3VS – on protein and leucine metabolism in skeletal muscle. In addition to evaluating changes in proteolysis, we studied also changes in protein synthesis and leucine oxidation, as the effects of proteasome inhibitors on these parameters are not clear. The experiments were performed by means of isolated muscles of both intact and septic rats. MG132 belongs to the group of peptide aldehydes. It is a strong inhibitor of the proteasome, but inhibits partly also cathepsins and calpains. ZL3VS is a vinylsulphone inhibitor and AdaAhx3L3VS is a vinylsulphone inhibitor with extended peptide portion, which reveals equal potency to all individual catalytic activities of the proteasome (Kessler et al. 2001).

Acidosis is frequently associated with protein wasting and derangements in amino acid metabolism. As its effect on protein metabolism is significantly modulated by other abnormal metabolic conditions caused by specific illnesses, it is difficult to separate out the effects on protein metabolism solely due to acidosis. The aim of the present study was to evaluate, using a model of isolated perfused rat liver, the direct response of hepatic tissue to acidosis. We have compared hepatic response to perfusion with a solution of pH 7.2 and 7.4 (controls). Parameters of protein and amino acid metabolism were measured using both recirculation and single-pass technique with 4,5-[3H]leucine, [1-14C]leucine and [1-14C]ketoisocaproate (ketoleucine) as tracers and on the basis of difference of amino acid levels in perfusion solution at the beginning and end of perfusion. In liver perfused with a solution of pH 7.2, we observed higher rates of proteolysis, protein synthesis, amino acid utilization and urea production. Furthermore, the liver perfused with a solution of pH 7.2 released a higher amount of proteins to perfusate than the liver perfused with a solution of pH 7.4. Enhanced decarboxylation of ketoisocaproate in liver perfused by a solution of a lower pH indicates increased catabolism of branched-chain amino acids (leucine, valine and isoleucine), decreased reamination of branched-chain keto acids to corresponding essential amino acids and increased ketogenesis from leucine.

Objective. This study was designed to evaluate vitamin D status with separate determination of 25-OH D2and 25-OH D3and its relationship to vitamin D binding protein (VDBP) in patients with chronic kidney disease (CKD) and long-term haemodialysis patients (HD).Methods. 45 CKD patients, 103 HD patients, and 25 controls (C) were included. Plasma vitamin D concentrations were determined using chromatography and VDBP in serum and urine in CKD using enzyme immunoassay.Results. Plasma vitamin D levels were lower in CKD (30.16 ± 16.74 ng/mL) and HD (18.85 ± 15.85 ng/mL) versus C (48.72 ± 18.35 ng/mL),P<0.0001. 25-OH D3was the dominant form of vitamin D. Serum VDBP was higher in CKD (273.2 ± 93.8 ug/mL) versus C (222 ± 87.6 ug/mL) and HD (213.8 ± 70.9 ug/mL),P=0.0003. Vitamin D/VDBP ratio was the highest in C and the lowest in HD; however, there was no correlation between vitamin D and VDBP. Urinary concentration of VDBP in CKD (0.25 ± 0.13 ug/mL) correlated with proteinuria(r=0.43,P=0.003).Conclusions. Plasma levels of vitamin D are decreased in CKD patients and especially in HD patients. 25-OH D3was the major form of vitamin D. Despite urinary losses of VDBP, CKD patients had higher serum VDBP concentrations, indicating compensatory enhanced production. Vitamin D binding protein is not involved in vitamin D deficiency.

Background/Aims: In recent years, one of technical attempts to improve biocompatibility and tolerability of the hemodialysis procedure is the substitution of acetate in dialysis solution with citrate. The aim of our study was to compare two dialysis solutions: traditional bicarbonate dialysis solution containing acetate (3 mmol/L) (solution A); and (solution C) commercially produced citrate-enriched bicarbonate dialysis solution (0.8 mmol/L citrate). Methods: Patients from a single hemodialysis center (N=126) were included in the study. Both conventional low-flux hemodialysis and on-line hemodiafiltration procedures were studied. Both dialysis solutions contained identical calcium (1.5 mmol/L) and magnesium (0.5 mmol/L) concentrations. Results: Parathyroid hormone (iPTH) concentration decreased during procedures with solution A by 64%. On the contrary, when solution C was used, iPTH concentration increased insignificantly by 4%. For solution A, serum calcium and magnesium increased during procedures in patients with predialysis concentrations lower than 2.33 and 0.76 mmol/L, respectively. In procedures with dialysis solution C these concentrations were significantly lower: 2.19 mmol/L for Ca and 0.68 mmol/L for Mg. Conclusion: Our study clearly shows that the substitution of part of acetate with citrate in dialysis solution significantly influences changes of serum calcium, magnesium and parathyroid hormone concentrations during hemodialysis and hemodiafiltration procedures.

Background/Aims: Growth hormone (GH) could have the potential to improve protein metabolism in sepsis but glutamine deficiency has been reported after GH treatment. The aim was to investigate the effects of glutamine deficiency in sepsis with and without GH treatment on protein and amino acid metabolism. Methods: Cecal ligation and puncture (CLP) was used as a model of sepsis. Serious glutamine deficiency was induced by administration of glutamine synthetase inhibitor, methionine sulfoximine (MSO). Young Wistar rats were divided into 5 groups: control; CLP; CLP+MSO; CLP+GH, and CLP+MSO+GH. Parameters of protein metabolism were measured on incubated soleus and extensor digitorum longus muscles: [1-14C]leucine was used to estimate protein synthesis and leucine oxidation, tyrosine release was used to evaluate protein breakdown. Amino acid concentrations in plasma, skeletal muscle and incubation media were measured by HPLC. Results/Conclusions: A reduced muscle glutamine concentration after MSO treatment is not associated with changes in the rates of protein synthesis or breakdown. MSO treatment decreased glutamine release from skeletal muscle and plasma glutamine concentration. Severe glutamine deficiency in GH-treated septic rats resulted in increased release of branched-chain amino acids from skeletal muscle.

Background & aims: Metabolic acidosis is a common finding in critical illness. The aim of the present study was to evaluate acute acidosis as a signal that induces changes in protein metabolism. Methods: In the first study, Wistar rats were infused for 6 h with HCl or saline resulting in blood pH 7.30±0.03 and 7.46±0.02, respectively. The whole body protein metabolism was evaluated using l -[1-14C]leucine. In the second study, soleus and extensor digitorum longus muscles from normal rats were incubated in medium, pH 7.4, 7.3 or 7.0. Protein metabolism was evaluated using l -[1-14C]leucine and tyrosine release. Results: In the in vivo study we observed increased protein turnover–protein synthesis, proteolysis and leucine oxidation and more negative protein balance in rats with acidosis. There was no change in protein synthesis in gastrocnemius muscle. We observed an increase in plasma levels of most amino acids including branched-chain amino acids and a decrease in intracellular amino acid pool in skeletal muscle. In vitro decrease in pH of 0.1 had no effect on protein metabolism, decrease of 0.4 decreased protein turnover and leucine oxidation. Conclusion: Acute metabolic acidosis is a protein wasting condition. Direct effect of acidosis on skeletal muscle is under condition in vivo modified by neurohumoral regulations.

Glutamine deficiency, a common finding in severe illness, has a negative influence on immune status, protein metabolism, and disease outcome. In several studies, a close relationship between glutamine, branched-chain amino acid (BCAA), and protein metabolism was demonstrated. The aim of the present study was to investigate the effect of glutamine deficiency on amino acid and protein metabolism in hepatic tissue using a model of isolated perfused rat liver (IPRL). Parameters of protein metabolism and amino acid metabolism were measured using both recirculation and single pass technique with L-[1-(14)C]leucine and [1-(14)C]ketoisocaproate (KIC) as a tracer. Glutamine concentration in perfusion solution was 0.5 mmol/L in control and 0 mmol/L in the glutamine-deficient group. The net release of glutamine (about 11 micromol/g/h) and higher net uptake of most of the amino acids was observed in the glutamine-deficient group. There was an insignificant effect of lack of glutamine on hepatic protein synthesis, proteolysis, and the release of urea. However, significantly lower release of proteins by the liver perfused with glutamine-deficient solution was observed. The lack of glutamine in perfusion solution caused a significant decrease in leucine oxidation (6.66 +/- 1.04 v 13.67 +/- 2.38, micromol/g dry liver/h, P

Glutamine deficiency, a common finding in severe illness, has a negative influence on immune status, protein metabolism, and disease outcome. In several studies, a close relationship between glutamine, branched-chain amino acid (BCAA), and protein metabolism was demonstrated. The aim of the present study was to investigate the effect of glutamine deficiency on amino acid and protein metabolism in hepatic tissue using a model of isolated perfused rat liver (IPRL). Parameters of protein metabolism and amino acid metabolism were measured using both recirculation and single pass technique with L-[1C]leucine and [1-C]ketoisocaproate (KIC) as a tracer. Glutamine concentration in perfusion solution was 0.5 mmol/L in control and 0 mmol/L in the glutamine-deficient group. The net release of glutamine (about 11 mol/g/h) and higher net uptake of most of the amino acids was observed in the glutamine-deficient group. There was an insignificant effect of lack of glutamine on hepatic protein synthesis, proteolysis, and the release of urea. However, significantly lower release of proteins by the liver perfused with glutamine-deficient solution was observed. The lack of glutamine in perfusion solution caused a significant decrease in leucine oxidation (6.66 1.04 v 13.67 2.38, mol/g dry liver/h, P < .05) and an increase in KIC oxidation (163.7 16.5 v 92.0 12.9 mL/g dry liver/h, P < .05). We conclude that decreased delivery of glutamine to hepatic tissue activates glutamine synthesis, decreases resynthesis of essential BCAA from branched-chain keto acids (BCKA), increases catabolism of BCKA, and has an insignificant effect on protein turnover in hepatic tissue. © 2003 Elsevier Inc. All rights reserved.

Numerous abnormalities of thyroid hormones in end-stage renal disease (ESRD) have been described. Our aim was to analyze the impact of these abnormalities on survival. In 167 hemodialyzed ESRD patients, TSH and thyroid hormone levels (T4, fT4, T3, fT3, rT3) were determined. The patients were then prospectively followed up for up to 5 years and the possible impact of any observed abnormalities on their mortality was studied. Only 16.8 % patients had all six tests within the reference range. The pattern of nonthyroidal illness syndrome was found in 56.3 %. Low T3 was particularly common (44.3 %), and clearly associated with increased 6- and 12-month mortality and decreased overall survival (log rank test, P=0.007). Independent of T3 levels (Spearman correlation, NS), increased rT3 was more frequently observed (9.9 %) than expected from the literature, and was also related to increased mortality and decreased survival (log rank test, P=0.021). Increased rT3 may be more common in ESRD patients than previously described, and together with decreased T3 it may serve as an indicator of poor prognosis in subsequent months.