Adipose Tissue Denervation Blunted the Decrease in Bone Formation Promoted by Obesity in Rats.

The impact of obesity upon bone metabolism is controversial since both beneficial or harmful effects have been reported. Bone remodeling is modulated by the central nervous system through cytokines, hormones and neuromodulators. The present study aimed to evaluate the effects evoked by bilateral retroperitoneal white adipose tissue (rWAT) denervation (Dnx) upon bone mineral metabolism and remodeling in an experimental model of obesity in rats. Male Wistar rats were fed during 18 weeks with high-fat diet (HFD) or standard diet (SD) as controls, and rWAT Dnx or Sham surgery was performed at the 14th week. Biochemical and hormonal parameters, bone histomorphometry, rWAT and hypothalamus protein and gene expression were analyzed. The HFD group presented decreased bone formation parameters, increased serum and bone leptin and FGF23, increased serum and hypothalamic neuropeptide Y (NPY) and decreased serum 1,25-dihydroxyvitamin D3 and PTH. After rWAT Dnx, bone markers and histomorphometry showed restoration of bone formation, and serum and hypothalamic NPY decreased, without alteration in leptin levels. The present study shows that the denervation of rWAT improved bone formation in obese rats mediated by a preferential reduction in neurohormonal actions of NPY, emphasizing the relevance of the adipose tissue-brain-bone axis in the control of bone metabolism in obesity.

Urinary Calcium Is Associated with Serum Sclerostin among Stone Formers.

BACKGROUND: Sclerostin plays an important role in bone metabolism and adipose tissue. Animal studies suggest that sclerostin influences urinary calcium (UCa), but this relationship has not been evaluated in stone formers (SFs). We aimed to investigate the association of UCa with serum sclerostin, bone mineral density (BMD), and body composition among SFs. METHODS: Clinical and laboratorial data were retrieved from medical records. Determinants of UCa were studied using linear regression. RESULTS: A total of 107 SFs (35.8 ± 9.3 years, 54% male) with eGFR 99.8 ± 14.5 mL/min/1.73 were studied. Subjects were split by sex and grouped into tertiles of UCa levels. Men in the highest UCa tertile had higher body mass index (BMI) and serum sclerostin, lower lean mass, and a trend towards higher fat mass. Women in the highest tertile had higher BMI and a trend towards higher serum sclerostin. Hypertension and metabolic syndrome, but not lower BMD, were more prevalent in the highest UCa tertile for both sexes. Sclerostin was positively correlated with fat mass and inversely correlated with lean mass among men, but not among women. BMD corrected for BMI at lumbar spine was inversely associated with UCa in a univariate analysis, but only serum sclerostin, hypertension, and NaCl intake were independent determinants of UCa in the multivariate model. CONCLUSION: The present findings disclose that in addition to hypertension and salt intake, serum sclerostin is associated with urinary calcium in stone formers, suggesting that in addition to the hormones traditionally thought to alter calcium reabsorption in the kidney, sclerostin may play a significant additional role, warranting further investigation.

Bone density, microarchitecture and estimated strength in stone formers: a cross-sectional HR-pQCT study.

BACKGROUND: Low areal bone mineral density (BMD), increased fracture risk and altered bone remodeling have been described among stone formers (SFs), but the magnitude of these findings differs by age, sex, menopausal status and urinary calcium (uCa). This study aimed to investigate volumetric BMD (vBMD), bone microarchitecture and biomechanical properties by high-resolution peripheral quantitative computed tomography (HR-pQCT) and finite element analysis (FEA) in young SFs, irrespective of calciuria, further distinguishing trabecular from cortical compartments. METHODS: HR-pQCT/FEA was performed at the distal tibia (DT) and distal radius (DR) in 106 SFs (57 males and 49 premenopausal females; median age 37 years) and compared with 106 non-SFs (NSFs) retrieved from an existing database, matched for age, sex and body mass index (BMI). Biochemical/hormonal serum and urinary parameters were obtained from SFs. RESULTS: SFs exhibited significantly lower trabecular number (TbN) and higher trabecular separation (TbSp) than NSFs at both anatomical sites and lower cortical porosity in the DR. In a subgroup analysis separated by sex, female SFs presented significantly lower TbvBMD, relative bone volume fraction (BV/TV) and TbN and higher TbSp than NSFs at both sites, while male SFs showed significantly lower stiffness and failure load. Multivariate analysis showed TbN to be independently associated with sex and BMI at both sites and with uCa at the DR. CONCLUSIONS: The present findings suggest that bone disease represents an early event among SFs, associated at least in part with calcium excretion and mainly characterized by trabecular bone microarchitecture impairment, especially among women, but with reduced bone strength parameters in men.

Vascular Calcification Is Associated with Fetuin-A and Cortical Bone Porosity in Stone Formers.

Background: Nephrolithiasis has been associated with bone loss and vascular calcification (VC), reflecting abnormal extraosseous calcium deposition. Fetuin-A (Fet-A) acts as a potent inhibitor of ectopic mineralization. The aim of the present study was to evaluate the prevalence of VC in stone formers (SF) and non-stone formers (NSF) and to investigate potential determinants of VC among SF, including circulating levels of Fet-A and bone microarchitecture parameters. Methods: Abdominal aortic calcification (AAC) was assessed using available computed tomography in SF and in age-, sex-, and BMI-matched NSF (potential living kidney donors). Serum Fet-A was measured in stored blood samples from SF. Bone microarchitecture parameters were obtained as a post hoc analysis of a cross-sectional cohort from young SF evaluated by high-resolution peripheral quantitative computed tomography (HR-pQCT). Results: A total of 62 SF (38.0 [28.0−45.3] years old) and 80 NSF (40.0 [37.0−45.8] years old) were included. There was no significant difference in AAC scores between SF and NSF. However, when dividing SF according to mean AAC score, below <5.8% (n = 33) or above ≥5.8% (n = 29), SF with higher AAC presented significantly higher BMI and tibial cortical porosity (Ct.Po) and significantly lower serum HDL, klotho, Fet-A, and eGFR. Urinary calcium did not differ between groups, but fractional excretion of phosphate was higher in the former. Upon multivariate regression, BMI, serum Fet-A, and tibial Ct.Po remained independently associated with AAC. Conclusions: This study suggests an association between reduced circulating Fet-A levels and increased bone Ct.Po with VC in SF.

Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used "housekeeping" genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10-12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/-:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/-, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.

Interplay between gut microbiota, bone health and vascular calcification in chronic kidney disease.

Deregulations in gut microbiota may play a role in vascular and bone disease in chronic kidney disease (CKD). As glomerular filtration rate declines, the colon becomes more important as a site of excretion of urea and uric acid, and an increased bacterial proteolytic fermentation alters the gut microbial balance. A diet with limited amounts of fibre, as well as certain medications (eg phosphate binders, iron supplementation, antibiotics) further contribute to changes in gut microbiota composition among CKD patients. At the same time, both vascular calcification and bone disease are common in patients with advanced kidney disease. This narrative review describes emerging evidence on gut dysbiosis, vascular calcification, bone demineralization and their interrelationship termed the 'gut-bone-vascular axis' in progressive CKD. The role of diet, gut microbial metabolites (ie indoxyl sulphate, p-cresyl sulphate, trimethylamine N-oxide (TMAO) and short-chain fatty acids (SCFA)), vitamin K deficiency, inflammatory cytokines and their impact on both bone health and vascular calcification are discussed. This framework may open up novel preventive and therapeutic approaches targeting the microbiome in an attempt to improve cardiovascular and bone health in CKD.

Comparison of the Effects of Mesenchymal Stem Cells with Their Extracellular Vesicles on the Treatment of Kidney Damage Induced by Chronic Renal Artery Stenosis.

BACKGROUND: Chronic renal artery stenosis is considered one of the most common causes of renovascular hypertension (RH). Chronic hypoxia can lead to irreversible damage to renal tissue and to a progressive deterioration of renal function. We have previously shown that bone marrow-derived mesenchymal stem cells (BMSCs) improved renal parenchyma and function in a model of RH (2 kidneys, 1 clip model (2K-1C) in rats. Microvesicles (MVs) and exosomes (EXs) released by MSCs have been shown to induce effects similar to those induced by whole cells but with fewer side effects. In this study, we compared the effects of adipose-derived MSCs (ASCs) with those of the MVs and EXs released by ASCs on tissue inflammation and renal function in 2 K-1C rats. RESULTS: Flow cytometry analysis showed that even after 15 days, ASCs were still detected in both kidneys. The expression of a stem cell homing marker (SDF1-α) was increased in ASC-treated animals in both the stenotic and contralateral kidneys. Interestingly, SDF1-α expression was also increased in MV- and EX-treated animals. A hypoxia marker (HIF1-α) was upregulated in the stenotic kidney, and treatments with ASCs, MVs, and EXs were effective in reducing the expression of this marker. Stenotic animals showed a progressive increase in systolic blood pressure (SBP), while animals treated with ASCs, MVs, and EXs showed a stabilization of SBP, and this stabilization was similar among the different treatments. Stenotic animals developed significant proteinuria, which was reduced by ASCs and MVs but not by EXs. The increased expression of Col I and TGFß in both kidneys was reduced by all the treatments, and these treatments also effectively increased the expression of the anti-inflammatory cytokine IL-10 in both kidneys; however, only ASCs were able to reduce the overexpression of the proinflammatory cytokine IL-1ß in both kidneys of 2K-1C animals. CONCLUSION: The results of this study demonstrated that the EVs released by ASCs produced beneficial results but with lower efficacy than whole cells. ASCs produced stronger effects in this model of renal chronic hypoxia, and the use of EVs instead of whole cells should be evaluated depending on the parameter to be corrected.

Dietary Recommendations for Bariatric Patients to Prevent Kidney Stone Formation.

Bariatric surgery (BS) is one of the most common and efficient surgical procedures for sustained weight loss but is associated with long-term complications such as nutritional deficiencies, biliary lithiasis, disturbances in bone and mineral metabolism and an increased risk of nephrolithiasis, attributed to urinary metabolic changes resultant from low urinary volume, hypocitraturia and hyperoxaluria. The underlying mechanisms responsible for hyperoxaluria, the most common among all metabolic disturbances, may comprise increased intestinal oxalate absorption consequent to decreased calcium intake or increased dietary oxalate, changes in the gut microbiota, fat malabsorption and altered intestinal oxalate transport. In the current review, the authors present a mechanistic overview of changes found after BS and propose dietary recommendations to prevent the risk of urinary stone formation, focusing on the role of dietary oxalate, calcium, citrate, potassium, protein, fat, sodium, probiotics, vitamins D, C, B6 and the consumption of fluids.

Expression of vitamin D receptor, CYP27B1 and CYP24A1 hydroxylases and 1,25-dihydroxyvitamin D3 levels in stone formers.

The expression of vitamin D receptor (VDR) and 1,25-dihydroxyvitamin D3 [1,25(OH)D] levels exceed the values of controls in some but not all hypercalciuric stone formers (HSF). We aimed to evaluate serum 1,25(OH)D levels, the expression of VDR, CYP27B1, and CYP24A1 hydroxylases in HSF in comparison with normocalciuric stone formers (NSF) and healthy subjects (HS). Blood samples, 24-h urine collections and a 3-day dietary record were obtained from 30 participants from each of the groups. The expression of VDR, CYP27B1, and CYP24A1 was measured by flow cytometry. HSF presented significantly higher urinary volume, sodium, magnesium, oxalate, uric acid, and phosphorus than NSF and HS. Calcium intake was lower in HSF versus NSF and HS (442 ± 41 vs 594 ± 42 and 559 ± 41 mg/day, respectively, p = 0.027). Ionized calcium was significantly lower in HSF than NSF (1.29 ± 0.0 vs 1.31 ± 0.0 mmol/L, p < 0.01). Serum 1,25(OH)D was significantly higher in HSF and NSF than HS (22.5 ± 1.2; 22.2 ± 1.2 vs 17.4 ± 1.2 pg/ml, p = 0.007) but serum 25(OH)D, PTH, klotho and plasma FGF-23 did not differ between groups. VDR expression was higher in HSF and NSF than HS (80.8 ± 3.2; 78.7 ± 3.3 vs 68.6 ± 3.2%, p = 0.023). Although CYP27B1 and CYP24A1 expressions were similar among all groups, the ratio of 1,25(OH)D/CYP24A1 was higher in HSF and NSF than in HS (1.43 ± 0.25 and 0.56 ± 0.10 vs 0.34 ± 0.06, p = 0.00). Stone formers, regardless of urinary calcium excretion, had higher VDR expression and 1,25(OH)D levels than HS, even in ranges considered normal. Higher 1,25(OH)D/CYP24A1 ratio suggested a lower degradation of 1,25(OH)D by CYP24A1 in HSF and NSF.

Caffeine Accelerates Cystic Kidney Disease in a Pkd1-Deficient Mouse Model.

BACKGROUND/AIMS: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst formation and growth, leading to end-stage renal disease. A higher kidney volume is predictive of a more accelerated decline in renal function. This study aimed to examine the effects of caffeine, a phosphodiesterase inhibitor, on the progression of cystic kidney disease in a mouse model orthologous to human disease (Pkd1cond/cond:Nestincre). METHODS: Caffeine was administered to male cystic (CyCaf) and noncystic (NCCaf) mice (Pkd1cond/cond) from conception and at the postweaning period through 12 weeks of life (3 mg/d), while control animals consumed water (CyCtrl and NCCtrl). Renal ultrasonography was performed at 10 weeks of life to calculate total kidney volume and cystic index. At the end of the protocol, blood and urine samples were collected for biochemical analysis, and animals were euthanized. Kidneys were harvested to obtain renal tissue for determinations of adenosine 3´5´-cyclic monophosphate (cAMP) by an enzymatic immunoassay kit and phosphorylated extracellular signal-regulated kinase (p-ERK) by Western blotting. Renal fibrosis (picrosirius staining), renal cell proliferation (ki-67 immunohistochemistry) and apoptotic rates (TUNEL analysis) were also determined. RESULTS: At 12 weeks, CyCaf mice exhibited higher serum urea nitrogen, renal cystic index, total kidney volume, kidney cell proliferation, apoptosis and fibrosis compared with CyCtrl mice. Serum cystatin C was significantly higher in CyCaf than in NCCaf and NCCtrl mice. CyCaf mice had higher total kidney weight than all other groups but not higher heart and liver weight. The levels of cAMP and p-ERK did not differ among the groups. CONCLUSION: Caffeine consumption from conception through 12 weeks led to increased cystic index and total kidney volume and worsened renal function in Pkd1-deficient cystic mice, suggesting that high consumption of caffeine may contribute to a faster progression of renal disease in ADPKD.

Behavioral, electrophysiological and neuropathological characteristics of the occurrence of hypertension in pregnant rats.

Pre-eclampsia (PE) affects approximately 2 to 8% of pregnant women, causing blood pressure above 140 × 90 mmHg and proteinuria, normally after the 20th gestation week. If unsuccessfully treated, PE can lead to self-limited seizures (Eclampsia) that could eventually result in death of the mother and her fetus. The present study reports an experimental model of preeclampsia hypertension in pregnant (HP) and non-pregnant (H) Wistar rats by partially clamping one of their renal arteries. Pregnant (P) and non-pregnant (C) controls were provided. Differently from controls (C and P), H and HP animals presented a steady rise in BP two weeks after renal artery clamping. Injection of pentylenetetrazol (PTZ) induced behavioral and electroencephalographic seizures in all groups, which were increased in number, duration, amplitude and power accompanied by decreased latency in HP animals (p < 0.05). Consistent results were obtained in in vitro experimentation. Immunohistochemistry of hippocampus tissue in HP animals showed decreased density of neurons nuclei in CA1, CA3 and Hilus and increased density of astrocytes in CA1, CA3 and gyrus (p < 0.05). The present findings show that the clamping of one renal arteries to 0.15 mm and PTZ administration were able to induce signs similar to human PE in pregnant Wistar rats.

Fish Oil Supplementation Reduces Inflammation but Does Not Restore Renal Function and Klotho Expression in an Adenine-Induced CKD Model.

BACKGROUND: Chronic kidney disease and inflammation promote loss of Klotho expression. Given the well-established anti-inflammatory effects of omega-3 fatty acids, we aimed to investigate the effect of fish oil supplementation in a model of CKD. METHODS: Male C57BL/6 mice received supplementation with an adenine-enriched diet (AD, n = 5) or standard diet (CTL, n = 5) for 10 days. Two other experimental groups were kept under the adenine diet for 10 days. Following adenine withdrawal on the 11th day, the animals returned to a standard diet supplemented with fish oil (Post AD-Fish oil, n = 9) or not (Post AD-CTL, n = 9) for an additional period of 7 days. RESULTS: Adenine mice exhibited significantly higher mean serum urea, creatinine, and renal expression of the pro-inflammatory markers Interleukin-6 (IL-6), C-X-C motif chemokine 10 (CXCL10), and Interleukin-1ß (IL-1ß), in addition to prominent renal fibrosis and reduced renal Klotho gene expression compared to the control. Post AD-Fish oil animals demonstrated a significant reduction of IL-6, C-X-C motif chemokine 9 (CXCL9), and IL-1ß compared to Post AD-CTL animals. However, serum creatinine, renal fibrosis, and Klotho were not significantly different in the fish oil-treated group. Furthermore, renal histomorphological changes such as tubular dilatation and interstitial infiltration persisted despite treatment. CONCLUSIONS: Fish oil supplementation reduced renal pro-inflammatory markers but was not able to restore renal function nor Klotho expression in an adenine-induced CKD model.

miR-26a modulates HGF and STAT3 effects on the kidney repair process in a glycerol-induced AKI model in rats.

Acute kidney injury is mostly reversible, and hepatocyte growth factor (HGF) has a relevant role in the tissue repair. MicroRNA (miR)-26a is an endogenous modulator of HGF. The role of miR-26a in the kidney repair process was evaluated in Wistar rats submitted to an acute kidney injury model of rhabdomyolysis induced by glycerol (6 mL/kg). Animals were evaluated 3, 12, 48, 96, and 120 hours after glycerol injection. Serum creatinine (SCr) and gene expression of HGF, c-met, signal transducer and activator of transcription 3 (STAT3), and miR-26a were estimated. Also, tubular NK52E cells were transfected with anti-miR26a and stimulated with Fe3+ for 24 hours to mimic the effects of myoglobin in vitro. SCr was highest after 48 hours. After 96 hours, SCr started to decrease, characterizing the recovery phase, with normalization after 120 hours. HGF expression increased during the onset phase (3 hours), with a low relationship with miR-26a. In contrast, in the recovery phase, the increase in miR-26a was coincident with HGF messenger RNA suppression, suggesting that in the recovery phase, miR-26a may have a role in HGF modulation. Fe3+ induced cellular death after 3 hours and proliferation after 24 hours. There was no correlation between miR-26a and STAT3 during the death phase; however, during the proliferation phase, an increase in STAT3 was paralleled with a decrease in miR-26a. miR-26a silencing induced increases in cell viability and the phosphorylated form of STAT3 protein expression in cells receiving Fe3+ . In conclusion, miR-26a may have a key role in modulating HGF levels after its proliferative effects have been triggered.

Hyperoxaluria in a Model of Mini-Gastric Bypass Surgery in Rats.

BACKGROUND: Bariatric surgery is associated with hyperoxaluria hence predisposing to nephrolithiasis. The present study aimed to investigate the underlying mechanisms contributing to increased urinary oxalate in a mini-gastric bypass (MGB) surgery model in rats under different dietary conditions. The expression of intestinal oxalate transporters was also evaluated. METHODS: Male rats underwent MGB (n = 21) or Sham procedure (n = 21) and after recovery were fed a standard or high-fat diet with or without oxalate for 8 weeks. Stool and urine were collected before surgery (baseline) and at the end of protocol (final), when intestinal fragments were harvested for expression of Slc26a3 and Slc26a6 oxalate transporters. RESULTS: MGB groups fed with fat, irrespective of oxalate supplementation, presented steatorrhea. In MGB animals fed with fat and oxalate (Fat + Ox), final values of urinary oxalate and calcium oxalate supersaturation risk were markedly and significantly increased versus baseline or Sham animals under the same diet, as well as MGB groups under other diets. Slc26a3 was decreased in biliopancreatic limbs of MGB rats, probably reflecting a physiological adaptation to the restriction of food passage. Slc26a6 was not altered in any harvested intestinal fragment. CONCLUSIONS: A high-fat and oxalate diet induced hyperoxaluria and elevation in calcium oxalate supersaturation risk in a MGB rat model. The presence of fat malabsorption and increased dietary oxalate absorption, but not modifications of Slc26a3 and Slc26a6 oxalate transporters, accounted for these findings, suggesting that bariatric patients may benefit from a low-fat and low-oxalate diet.

Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance.

Metabolic syndrome is a cluster of metabolic risk factors such as obesity, diabetes and cardiovascular diseases. Mitochondria is the main site of ATP production and its dysfunction leads to decreased oxidative phosphorylation, resulting in lipid accumulation and insulin resistance. Our group has demonstrated that kinins can modulate glucose and lipid metabolism as well as skeletal muscle mass. By using B2 receptor knockout mice (B2R-/-) we investigated whether kinin action affects weight gain and physical performance of the animals. Our results show that B2R-/- mice are resistant to high fat diet-induced obesity, have higher glucose tolerance as well as increased mitochondrial mass. These features are accompanied by higher energy expenditure and a lower feed efficiency associated with an increase in the proportion of type I fibers and intermediary fibers characterized by higher mitochondrial content and increased expression of genes related to oxidative metabolism. Additionally, the increased percentage of oxidative skeletal muscle fibers and mitochondrial apparatus in B2R-/- mice is coupled with a higher aerobic exercise performance. Taken together, our data give support to the involvement of kinins in skeletal muscle fiber type distribution and muscle metabolism, which ultimately protects against fat-induced obesity and improves aerobic exercise performance.

Transplantation of bone marrow mesenchymal stem cells decreases oxidative stress, apoptosis, and hippocampal damage in brain of a spontaneous stroke model.

Stroke is the most common cause of motor disabilities and is a major cause of mortality worldwide. Adult stem cells have been shown to be effective against neuronal degeneration through mechanisms that include both the recovery of neurotransmitter activity and a decrease in apoptosis and oxidative stress. We chose the lineage stroke-prone spontaneously hypertensive rat (SHRSP) as a model for stem cell therapy. SHRSP rats can develop such severe hypertension that they generally suffer a stroke at approximately 1 year of age. The aim of this study was to evaluate whether mesenchymal stem cells (MSCs) decrease apoptotic death and oxidative stress in existing SHRSP brain tissue. The results of qRT-PCR assays showed higher levels of the antiapoptotic Bcl-2 gene in the MSC-treated animals, compared with untreated. Our study also showed that superoxide, apoptotic cells, and by-products of lipid peroxidation decreased in MSC-treated SHRSP to levels similar those found in the animal controls, Wistar Kyoto rats. In addition, we saw a repair of morphological damage at the hippocampal region after MSC transplantation. These data suggest that MSCs have neuroprotective and antioxidant potential in stroke-prone spontaneously hypertensive rats.