Advanced Glycation End Products and Bone Metabolism in Patients with Chronic Kidney Disease.

Advanced glycation end products (AGEs) accumulation may be involved in the progression of CKD-bone disorders. We sought to determine the relationship between AGEs measured in the blood, skin, and bone with histomorphometry parameters, bone protein, gene expression, and serum biomarkers of bone metabolism in patients with CKD stages 3 to 5D patients. Serum levels of AGEs were estimated by pentosidine, glycated hemoglobin (A1c), and N-carboxymethyl lysine (CML). The accumulation of AGEs in the skin was estimated from skin autofluorescence (SAF). Bone AGEs accumulation and multiligand receptor for AGEs (RAGEs) expression were evaluated by immunohistochemistry; bone samples were used to evaluate protein and gene expression and histomorphometric analysis. Data are from 86 patients (age: 51 ± 13 years; 60 [70%] on dialysis). Median serum levels of pentosidine, CML, A1c, and SAF were 71.6 pmol/mL, 15.2 ng/mL, 5.4%, and 3.05 arbitrary units, respectively. AGEs covered 3.92% of trabecular bone and 5.42% of the cortical bone surface, whereas RAGEs were expressed in 0.7% and 0.83% of trabecular and cortical bone surfaces, respectively. AGEs accumulation in bone was inversely related to serum receptor activator of NF-κB ligand/parathyroid hormone (PTH) ratio (R = -0.25; p = 0.03), and RAGE expression was negatively related to serum tartrate-resistant acid phosphatase-5b/PTH (R = -0.31; p = 0.01). Patients with higher AGEs accumulation presented decreased bone protein expression (sclerostin [1.96 (0.11-40.3) vs. 89.3 (2.88-401) ng/mg; p = 0.004]; Dickkopf-related protein 1 [0.064 (0.03-0.46) vs. 1.36 (0.39-5.87) ng/mg; p = 0.0001]; FGF-23 [1.07 (0.4-32.6) vs. 44.1 (6-162) ng/mg; p = 0.01]; and osteoprotegerin [0.16 (0.08-2.4) vs. 6.5 (1.1-23.7) ng/mg; p = 0.001]), upregulation of the p53 gene, and downregulation of Dickkopf-1 gene expression. Patients with high serum A1c levels presented greater cortical porosity and Mlt and reduced osteoblast surface/bone surface, eroded surface/bone surface, osteoclast surface/bone surface, mineral apposition rate, and adjusted area. Cortical thickness was negatively correlated with serum A1c (R = -0.28; p = 0.02) and pentosidine levels (R = -0.27; p = 0.02). AGEs accumulation in the bone of CKD patients was related to decreased bone protein expression, gene expression changes, and increased skeletal resistance to PTH; A1c and pentosidine levels were related to decreased cortical thickness; and A1c levels were related to increased cortical porosity and Mlt. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Effects of parathyroidectomy on the biology of bone tissue in patients with chronic kidney disease and secondary hyperparathyroidism.

Secondary hyperparathyroidism is a complication of chronic kidney disease that compromises skeletal integrity. In patients with secondary hyperparathyroidism undergoing parathyroidectomy, parathyroid hormone levels dramatically decrease. The effects of parathyroidectomy on bone tissue are poorly understood, especially regarding the proteins expressed by osteocytes, such as fibroblast growth factor 23, dentin matrix protein 1, matrix extracellular phosphoglycoprotein, sclerostin, receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin, which regulate bone turnover. The objective of this study was to characterize the bone expression of these proteins by immunohistochemistry and correlate these results with those of bone histomorphometry before and after parathyroidectomy. We studied bone biopsies that were obtained from 23 patients before and 12 months after parathyroidectomy. We observed an improvement in bone microarchitecture, but impaired mineralization after parathyroidectomy. We found significant increases in sclerostin and osteoprotegerin expression and a decrease in the RANKL/osteoprotegerin ratio after parathyroidectomy, suggesting that their expression is regulated by parathormone. These proteins correlated with structural and bone formation parameters. We conclude that after parathyroidectomy, significant changes occur in the bone expression of osteocyte proteins and that these proteins potentially regulate bone remodeling.

Comparison of clinical, biochemical and histomorphometric analysis of bone biopsies in dialysis patients with and without fractures.

Chronic kidney disease-mineral bone disorders (CKD-MBD) are associated with increased risk of fracture. Studies report about 3% of fractures in CKD patients, and these occur earlier than in the general population, namely 16 and 13 years earlier for men and women, respectively. Better understanding of the pathophysiology of fractures would probably contribute to new therapeutic approaches. This study aimed to evaluate report of long bone fractures from a bone biopsies bank from patients on hemodialysis and compare clinical and biochemical characteristics, as well as the results of the histomorphometric analysis of trabecular and cortical bone of these patients with a control group (without fractures), paired for age, gender, and time on hemodialysis. Bone proteins (SOST, DMP1 and MEPE) were evaluated by immunohistochemistry. Seventeen patients with fracture and controls were studied. Fracture prevalence was 0.82/1000 patients/year. Serum phosphorus levels were significantly lower in the fracture group. Histomorphometric analysis revealed that all the patients had high turnover disease, and the fracture group had smaller volume and trabecular thickness, greater osteoid surface, smaller eroded surface, smaller mineralizing surface, formation rate and longer mineralization lag time when compared to controls; the DMP1 expression in the cortical bone was smaller and the SOST in the trabecular bone was higher in fractured patients. As conclusion, we found low prevalence of fractures. Both groups had high turnover disease, but the fractured ones presented more impaired bone microarchitecture, as well as lower formation and greater mineralization defect. Bone proteins expression correlated with parameters involved in bone remodeling.

Hypovitaminosis D in patients undergoing kidney transplant: the importance of sunlight exposure.

OBJECTIVES:: Recent studies have shown a high prevalence of hypovitaminosis D, defined as a serum 25-hydroxyvitamin D level less than 30 ng/ml, in both healthy populations and patients with chronic kidney disease. Patients undergoing kidney transplant are at an increased risk of skin cancer and are advised to avoid sunlight exposure. Therefore, these patients might share two major risk factors for hypovitaminosis D: chronic kidney disease and low sunlight exposure. This paper describes the prevalence and clinical characteristics of hypovitaminosis D among patients undergoing kidney transplant. METHODS:: We evaluated 25-hydroxyvitamin D serum levels in a representative sample of patients undergoing kidney transplant. We sought to determine the prevalence of hypovitaminosis D, compare these patients with a control group, and identify factors associated with hypovitaminosis D (e.g., sunlight exposure and dietary habits). RESULTS:: Hypovitaminosis D was found in 79% of patients undergoing kidney transplant, and the major associated factor was low sunlight exposure. These patients had higher creatinine and intact parathyroid hormone serum levels, with 25-hydroxyvitamin D being inversely correlated with intact parathyroid hormone serum levels. Compared with the control group, patients undergoing kidney transplant presented a higher prevalence of 25-hydroxyvitamin D deficiency and lower serum calcium, phosphate and albumin but higher creatinine and intact parathyroid hormone levels. CONCLUSIONS:: Our results confirmed the high prevalence of hypovitaminosis D in patients undergoing kidney transplant. Therapeutic strategies such as moderate sunlight exposure and vitamin D supplementation should be seriously considered for this population.

Hypovitaminosis D in patients undergoing kidney transplant: the importance of sunlight exposure

OBJECTIVES: Recent studies have shown a high prevalence of hypovitaminosis D, defined as a serum 25-hydroxyvitamin D level less than 30 ng/ml, in both healthy populations and patients with chronic kidney disease. Patients undergoing kidney transplant are at an increased risk of skin cancer and are advised to avoid sunlight exposure. Therefore, these patients might share two major risk factors for hypovitaminosis D: chronic kidney disease and low sunlight exposure. This paper describes the prevalence and clinical characteristics of hypovitaminosis D among patients undergoing kidney transplant. METHODS: We evaluated 25-hydroxyvitamin D serum levels in a representative sample of patients undergoing kidney transplant. We sought to determine the prevalence of hypovitaminosis D, compare these patients with a control group, and identify factors associated with hypovitaminosis D (e.g., sunlight exposure and dietary habits). RESULTS: Hypovitaminosis D was found in 79% of patients undergoing kidney transplant, and the major associated factor was low sunlight exposure. These patients had higher creatinine and intact parathyroid hormone serum levels, with 25-hydroxyvitamin D being inversely correlated with intact parathyroid hormone serum levels. Compared with the control group, patients undergoing kidney transplant presented a higher prevalence of 25-hydroxyvitamin D deficiency and lower serum calcium, phosphate and albumin but higher creatinine and intact parathyroid hormone levels. CONCLUSIONS: Our results confirmed the high prevalence of hypovitaminosis D in patients undergoing kidney transplant. Therapeutic strategies such as moderate sunlight exposure and vitamin D supplementation should be seriously considered for this population.

High Dialysate Calcium Concentration May Cause More Sympathetic Stimulus During Hemodialysis.

BACKGROUND/AIMS: Acute activation of sympathetic activation during hemodialysis is essential to maintain blood pressure (BP), albeit long-term overactivity contributes to higher mortality. Low heart rate variability (HRV), a measure of autonomic nervous system activity, and abnormal ankle-brachial index (ABI) are associated with higher mortality in patients on hemodialysis. In this study, we assessed HRV and ABI pre and post dialysis in incident patients on hemodialysis using high (1.75mmol/l) and low (1.25mmol/l) dialysate calcium concentration (DCa). METHODS: HRV was measured as the ratio between low frequency and high frequency power (LF/HF). Thirty patients (age 47±16 years, 67% men) were studied in two consecutive mid-week hemodialysis sessions. RESULTS: Mean BP variation was positive with DCa 1.75 and negative with DCa 1.25 [4.0 (-6.0, 12.2 mmHg) vs. -3.2 (-9.8, 1.3 mmHg); p=0.050]. Reduction of ABI from pre to post HD was related to higher sympathetic activity (p=0.031). The increase in LF/HF ratio was higher with DCa 1.75 (58.3% vs. 41.7% in DCa 1.75 and 1.25, respectively, RR 2.8; p=0.026). CONCLUSION: Although higher DCa is associated with better hemodynamic tolerability during hemodialysis, this occurs at the expense of increased sympathetic activity. Higher sympathetic activity was associated with a decrease of ABI during hemodialysis.

Mineral bone disorder in chronic kidney disease: head-to-head comparison of the 5/6 nephrectomy and adenine models.

BACKGROUND: Experimental models are important to the understanding of the pathophysiology of, as well as the effects of therapy on, certain diseases. In the case of chronic kidney disease-mineral bone disorder, there are currently two models that are used in evaluating the disease: 5/6 nephrectomy (Nx) and adenine-induced renal failure (AIRF). However, the two models have never been compared in studies using animals maintained under similar conditions. Therefore, we compared these two models, focusing on the biochemical, bone histomorphometry, and vascular calcification aspects. METHODS: Wistar rats, initially fed identical diets, were divided into two groups: those undergoing 5/6 Nx (5/6Nx group) and those that were switched to an adenine-enriched diet (AIRF group). After 9 weeks, animals were sacrificed, and we conducted biochemical and bone histomorphometry analyses, as well as assessing vascular calcification. RESULTS: At sacrifice, the mean body weight was higher in the 5/6Nx group than in the AIRF group, as was the mean blood pressure. No differences were seen regarding serum phosphate, ionized calcium, intact parathyroid hormone (PTH), or fibroblast growth factor 23 (FGF23). However, creatinine clearance was lower and fractional excretion of phosphate (FeP) was higher in the AIRF group rats, which also had a more severe form of high-turnover bone disease. Vascular calcification, as evaluated through von Kossa staining, was not observed in any of the animals. CONCLUSIONS: Overt vascular calcification was not seen in either model as applied in this study. Under similar conditions of diet and housing, the AIRF model produces a more severe form of bone disease than does 5/6 Nx. This should be taken into account when the choice is made between these models for use in preclinical studies.

Effects of dietary phosphate on adynamic bone disease in rats with chronic kidney disease--role of sclerostin?

High phosphate intake is known to aggravate renal osteodystrophy along various pathogenetic pathways. Recent studies have raised the possibility that dysregulation of the osteocyte Wnt/ß-catenin signaling pathway is also involved in chronic kidney disease (CKD)-related bone disease. We investigated the role of dietary phosphate and its possible interaction with this pathway in an experimental model of adynamic bone disease (ABD) in association with CKD and hypoparathyroidism. Partial nephrectomy (Nx) and total parathyroidectomy (PTx) were performed in male Wistar rats. Control rats with normal kidney and parathyroid function underwent sham operations. Rats were divided into three groups and underwent pair-feeding for 8 weeks with diets containing either 0.6% or 1.2% phosphate: sham 0.6%, Nx+PTx 0.6%, and Nx+PTx 1.2%. In the two Nx+PTx groups, serum creatinine increased and blood ionized calcium decreased compared with sham control group. They also presented hyperphosphatemia and reduced serum parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) levels. Fractional urinary excretion of phosphate increased in Nx+PTx 1.2% rats despite lower PTH and FGF23 levels than in sham group. These biochemical changes were accompanied by a decrease in bone formation rates. The Nx+PTx 1.2% group had lower bone volume (BV/TV), higher osteoblast and osteocyte apoptosis, and higher SOST and Dickkopf-1 gene expression than the Nx+PTx 0.6% group. Nx+PTx 0.6% rat had very low serum sclerostin levels, and Nx+PTx 1.2% had intermediate sclerostin levels compared with sham group. Finally, there was a negative correlation between BV/TV and serum sclerostin. These results suggest that high dietary phosphate intake decreases bone volume in an experimental model of CKD-ABD, possibly via changes in SOST expression through a PTH-independent mechanism. These findings could have relevance for the clinical setting of CKD-ABD in patients who low turnover bone disease might be attenuated by optimal control of phosphate intake and/or absorption.

Persistence of bone and mineral disorders 2 years after successful kidney transplantation.

BACKGROUND: Studies that have conducted bone biopsies after kidney transplantation are scarce, and the results are conflicting. METHODS: We evaluate the bone histomorphometry, in vitro proliferation, and alkaline phosphatase expression in osteoblasts isolated from bone biopsies from 27 kidney transplant patients. The patients had preserved renal function and were treated with the same immunosuppressive therapy, receiving a minimum dose of corticosteroids. RESULTS: The biochemical analysis revealed that 41% of the patients presented with hypercalcemia, 26% presented with hypophosphatemia, and hypovitaminosis D was detected in 63%. The histomorphometric analysis showed a reduced trabecular number and increased trabecular separation, mineral apposition rate, and mineralization lag time, as well as higher osteoid surface, osteoblastic surface, resorption surface, and osteoclastic surface and a lower mineralizing surface, compared with the controls. Based on the TMV classification, bone turnover was normal in 48%, high in 26%, and low in 26% of patients. Bone mineralization was delayed in 48% of the patients, and 58% of the patients with hypovitaminosis D presented with delayed bone mineralization. Bone volume was low in 37% of the patients. The osteoblasts from patients exhibited a higher degree of proliferation compared with those from controls. CONCLUSION: Eight-two percent of our patients presented with alterations in at least one of the TMV parameters. Persistence of hyperparathyroidism, hypovitaminosis D, and immunosuppressive drugs may have influenced osteoblast function, which would explain many of the bone alterations found in these patients.

Bone plasticity in response to exercise is sex-dependent in rats.

PURPOSE: To characterize the potential sexual dimorphism of bone in response to exercise. METHODS: Young male and female Wistar rats were either submitted to 12 weeks of exercise or remained sedentary. The training load was adjusted at the mid-trial (week 6) by the maximal speed test. A mechanical test was performed to measure the maximal force, resilience, stiffness, and fracture load. The bone structure, formation, and resorption were obtained by histomorphometric analyses. Type I collagen (COL I) mRNA expression and tartrate-resistant acid phosphatase (TRAP) mRNA expression were evaluated by quantitative real-time PCR (qPCR). RESULTS: The male and female trained rats significantly improved their maximum speed during the maximal exercise test (main effect of training; p<0.0001). The male rats were significantly heavier than the females, irrespective of training (main effect of sex; p<0.0001). Similarly, both the weight and length of the femur were greater for the male rats when compared with the females (main effect of sex; p<0.0001 and p<0.0001, respectively). The trabecular volume was positively affected by exercise in male and female rats (main effect of training; p = 0.001), whereas the trabecular thickness, resilience, mineral apposition rate, and bone formation rate increased only in the trained males (within-sex comparison; p<0.05 for all parameters), demonstrating the sexual dimorphism in response to exercise. Accordingly, the number of osteocytes increased significantly only in the trained males (within-sex comparison; p<0.05). Pearson's correlation analyses revealed that the COL I mRNA expression and TRAP mRNA expression were positively and negatively, respectively, related to the parameters of bone remodeling obtained from the histomorphometric analysis (r = 0.59 to 0.85; p<0.05). CONCLUSION: Exercise yielded differential adaptations with respect to bone structure, biomechanical proprieties, and molecular signaling in male and female rats.

Parathyroid hormone and phosphorus overload in uremia: impact on cardiovascular system.

BACKGROUND: Cardiac remodeling in uremia is characterized by left ventricular hypertrophy, interstitial fibrosis and microvascular disease. Cardiovascular disease is the leading cause of death in uremic patients, but coronary events alone are not the prevalent cause, sudden death and heart failure are. We studied the cardiac remodeling in experimental uremia, evaluating the isolated effect of parathyroid hormone (PTH) and phosphorus. METHODS: Wistar rats were submitted to parathyroidectomy (PTx) and 5/6 nephrectomy (Nx); they also received vehicle (V) and PTH at normal (nPTH) or high (hPTH) doses. They were fed with a poor-phosphorus (pP) or rich-phosphorus (rP) diet and were divided into the following groups: 'Sham': G1 (V+normal-phosphorus diet (np)) and 'Nx+PTx': G2 (nPTH+pP), G3 (nPTH+rP), G4 (hPTH+pP) and G5 (hPTH+rP). After 8 weeks, biochemical analysis, myocardium morphometry and arteriolar morphological analysis were performed. In addition, using immunohistochemical analysis, we evaluated angiotensin II, α-actin, transforming growth factor-beta (TGF-ß) and nitrotyrosine, as well as fibroblast growth factor-23 (FGF-23), fibroblast growth factor receptor-1 (FGFR-1) and runt-related transcription factor-2 (Runx-2) expression. RESULTS: Nx animals presented higher serum creatinine levels as well as arterial hypertension. Higher PTH levels were associated with myocardial hypertrophy and fibrosis as well as a higher coronary lesion score. High PTH animals also presented a higher myocardial expression of TGF-ß, angiotensin II, FGF-23 and nitrotyrosine and a lower expression of α-actin. Phosphorus overload was associated with higher serum FGF-23 levels and Runx-2, as well as myocardial hypertrophy. FGFR-1 was positive in the cardiomyocytes of all groups as well as in calcified coronaries of G4 and G5 whereas Runx-2 was positive in G3, G4 and G5. CONCLUSION: In uremia, PTH and phosphorus overload are both independently associated with major changes related to the cardiac remodeling process, emphasizing the need for a better control of these factors in chronic kidney disease.

The bone histology spectrum in experimental renal failure: adverse effects of phosphate and parathyroid hormone disturbances.

Bone disease is a common disorder of bone remodeling and mineral metabolism, which affects patients with chronic kidney disease. Minor changes in the serum level of a given mineral can trigger compensatory mechanisms, making it difficult to evaluate the role of mineral disturbances in isolation. The objective of this study was to determine the isolated effects that phosphate and parathyroid hormone (PTH) have on bone tissue in rats. Male Wistar rats were subjected to parathyroidectomy and 5/6 nephrectomy or were sham-operated. Rats were fed diets in which the phosphate content was low, normal, or high. Some rats received infusion of PTH at a physiological rate, some received infusion of PTH at a supraphysiological rate, and some received infusion of vehicle only. All nephrectomized rats developed moderate renal failure. High phosphate intake decreased bone volume, and this effect was more pronounced in animals with dietary phosphate overload that received PTH infusion at a physiological rate. Phosphate overload induced hyperphosphatemia, hypocalcemia, and changes in bone microarchitecture. PTH at a supraphysiological rate minimized the phosphate-induced osteopenia. These data indicate that the management of uremia requires proper control of dietary phosphate, together with PTH adjustment, in order to ensure adequate bone remodeling.

Apoptosis in kidney and pancreas allograft biopsies.

BACKGROUND: Apoptosis is a particular form of cell death involved in the elimination of somatic cells. In this study, the occurrence of apoptotic cells in kidney and pancreas allograft biopsies was analyzed and correlated with the number of infiltrating macrophages and lymphocytes and granzyme B expression. METHODS: Kidney and pancreas biopsies from patients submitted to simultaneous pancreas-kidney transplantation were classified into three groups: acute rejection, chronic rejection, and transplant cases without evidence of rejection. Formalin-fixed paraffin biopsies were used to identify apoptosis by the terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In normal kidney, only few apoptotic cells were observed. In contrast, in kidney-allograft biopsies, the TUNEL signal was detected in the nuclei of tubular epithelial cells and also in mononuclear cells scattered in the interstitium. In pancreas biopsies, numerous apoptotic cells were detected in acinar cells, in ducts, and occasionally in islets. The number of apoptotic cells in acute pancreas rejection was significantly higher compared with acute rejection of kidney grafts (50+/-14 vs. 21+/-4 cells/mm2; P<0.05). In kidney biopsies, there was a positive correlation between apoptosis and macrophages (r=0.51; P<0.005), and apoptosis versus T lymphocytes (r=0.45; P<0.05). In pancreas biopsies, the number of apoptotic cells correlated only with the number of macrophages (r=0.41; P<0.05). CONCLUSIONS: Apoptosis occurs in kidney and pancreas allograft biopsies, markedly in acute rejection in pancreas biopsies. Although apoptosis may reflect a mechanism of down-regulation of the allograft immune response by eliminating infiltrating cells, the elimination of graft cells may result in graft damage, particularly in pancreas transplantation.