Comparative Phosphoproteomic Profiling in Prefrontal Cortex of Prenatal Stressed Offspring Rats

Background: Many investigations indicate that prenatal stress caused depressive-like disturbances in offspring rats. The underlying pathogenic mechanisms have not yet been fully unravelled. The PFC has been shown to play a role in susceptibility to stress during fetal, thus we focus our attention on differential protein phosphorylation in this region of PS-S(Susceptibility to PS) offspring rats.Method: SPT was used to screen for susceptibility to PS. The validity of prenatally stressed model was verified by other common depression-like behaviors. We used MS-based TMT quantitative proteomics in combination with phosphopeptide enrichment method to compare phosphoproteomic profiling in prefrontal cortex of of PS-S and CON offspring rats. Results: Totally, 3418 phosphoproteins, 8404 phosphopeptides and 12175 phosphosites were identified in this analysis. According to the screening criteria, 902 phosphopeptides increased and 609 decreased in the PFC of PS-S group compared to the control rats. GO enrichment analysis indicated that the main enriched terms in CC category were ‘synapse part’, ‘myelin sheath’, ‘synapse’, ‘neuron part’ and ‘axon’. The phosphorproteins enriched in MF and BP category were mainly related to cytoskeleton and projection morphogenesis associated proteins. KEGG pathway enrichment analyses identified 30 significant KEGG pathways, the top five pathways included salivary secretion, Endocrine and other factor-regulated calcium reabsorption, Pancreatic secretion and Insulin secretion. Motifs such as......_S_P...RR, ......_S_PE...., ......_S_PV...., ......_S_P.H...and ..S..._S_PT....were the top five motifs enriched in phosphorylated sites.Conclusion: PS may induce depressive-like behaviors in offspring rats through regulating the phosphorylation of protein mainly related to synapse, myelin sheath, neuron and cytoskeleton. And the phosphorylation of related proteins may act as key pathogenic hits. Data are available via ProteomeXchange with identifier PXD026563.

Research on the Mechanism of Chuanxiong in Treating Diabetic Nephropathy by Network Pharmacology and Molecular Docking Technique

Background Chuanxiong hort. It has been used in the clinic, but its main active components and drug mechanism are unknown. Objective To explore the mechanism of diabetic nephropathy (DN) treated with chuanxiong hort. Methods the active components and targets of chuanxiong Hort. were collected by TCPSP database, the DN gene expression data were collected by NCBI database, DN related genes were obtained by differential analysis. Furthermore, String and Cytoscape tools were used to construct the regulatory network of chuanxiong and the main active components for DN treatment. At the same time, PPI network interaction analysis was performed for core genes and GO. KEGG analysis was performed, prediction of molecular docking using Auto Dock Tools for critical genes. Results eight active components of chuanxiong Hort. were screened out, and one was recorded repeatedly. They were Angelica lactone A, ligusticum chuanxiong naphthofurolactone, Myricetin, 1-palmitic acid-2-linoleic acid-3-olein and ferulic acid ester of conifer. In addition, the active components interact with 15 disease targets, namely PGR, NR3C1, NCOA1, NCOA2, NR3C2, PTGS1, F2, F7, ESR1, AR, PPARG, ESR2, DPP4, HSP90AA1 and PRKACA. Besides, GO analysis of target genes, involved in the regulation of DN by chuanxiong Hort., were the regulation of cellular response to steroid hormone stimulation, the binding of nuclear receptors to steroid hormone receptors and the principle of cAMP-dependent protein kinase complexes. What's more, results of KEGG analysis showed that estrogen signalling pathway, endocrine and other factors-regulated calcium reabsorption and adipocyte adipogenesis played a role in the regulation of DN. Further molecular docking showed that NCOA1 and NCOA2 could interact with Angelica lactone A, Myricetin, Chrysophanol, chuanxiong naphthalize and Chrysophanol. Conclusion the active components of chuanxiong Hort., including Angelica Lactone A, Myricetin, Chrysophanol, chuanxiong naphthafunolide and Chrysophanol, can affect the regulation of estrogen signalling pathway, endocrine and other factors regulating calcium reabsorption and adipogenesis of adipocytes through the regulation of steroid hormone stimulation and regulation of cAMP-dependent protein kinase complex, and then play a therapeutic role on DN.

Primary Hyperparathyroidism in Homozygous Sickle Cell Patients: A Hemolysis-Mediated Hypocalciuric Hypercalcemia Phenotype?

Primary hyperparathyroidism (pHPT) has been reported to have a higher prevalence in sickle cell disease (SCD) patients, including a high rate of recurrence following surgery. However, most patients are asymptomatic at the time of diagnosis, with surprisingly infrequent hypercalciuria, raising the issue of renal calcium handling in SCD patients. We conducted a retrospective study including (1) 64 hypercalcemic pHPT non-SCD patients; (2) 177 SCD patients, divided into two groups of 12 hypercalcemic pHPT and 165 non-pHPT; (3) eight patients with a diagnosis of familial hypocalciuric hypercalcemia (FHH). Demographic and biological parameters at the time of diagnosis were collected and compared between the different groups. Determinants of fasting fractional excretion of calcium (FeCa2+) were also analyzed in non-pHPT SCD patients. Compared to non-SCD pHPT patients, our data show a similar ionized calcium and PTH concentration, with a lower plasmatic calcitriol concentration and a lower daily urinary calcium excretion in pHPT SCD patients (p < 0.0001 in both cases). Fasting FeCa2+ is also surprisingly low in pHPT SCD patients, and thus inadequate to be considered hypercalcemia, recalling the FHH phenotype. FeCa2+ is also low in the non-pHPT SCD control group, and negatively associated with PTH and hemolytic biomarkers such as LDH and low hemoglobin. Our data suggest that the pHPT biochemical phenotype in SCD patients resembles the FHH phenotype, and the fasting FeCa2+ association with chronic hemolysis biomarkers strengthens the view of a potential pharmacological link between hemolytic by-products and calcium reabsorption, potentially through a decreased calcium-sensing receptor (CaSR) activity.

Acute decrease of urine calcium by amiloride in healthy volunteers under high sodium diet

Background Amiloride is a competitive blocker of the epithelial sodium channel (ENaC) in the renal collecting duct. It is a less potent diuretic than thiazides or loop diuretics, but is often used in association for its potassium-sparing profile. Whether amiloride has hypocalciuric effect similar to thiazides remains unclear. Animal studies and experiments on cell lines suggested that amiloride increases calcium reabsorption in the distal nephron, but human studies are scarce. Methods We performed a post hoc analysis of a study with 48 healthy males (age, 23.2 ± 3.9 years) who were assigned to a high sodium (Na)/low potassium (K) diet for 7 days before receiving 20 mg of amiloride p.o. Urinary excretions of electrolytes were measured at 3 and 6 hours afterward; we calculated the relative changes in urinary excretion rates after amiloride administration. Results The high Na/low K diet led to an expected suppression of plasma renin and aldosterone. Amiloride showed a mild natriuretic effect associated with a decreased kaliuresis. Urinary calcium excretion dropped substantially (by 80%) 3 hours after amiloride administration and remained low at the 6th hour. At the same time, fractional excretion of lithium decreased by a third, reflecting an increased proximal tubular reabsorption. Conclusion During a high Na/low K diet, amiloride had a strong acute hypocalciuric effect, most probably mediated by increased proximal calcium reabsorption, even though distal effect cannot be excluded. Further studies should establish if chronic amiloride or combined amiloride/thiazide treatment may decrease calciuria more efficiently and be useful in preventing kidney stones.

Soluble α-klotho anchors TRPV5 to the distal tubular cell membrane independent of FGFR1 by binding TRPV5 and galectin-1 simultaneously

Hypercalciuria is one of early manifestations of diabetic nephropathy (DN). This is partially due to a decrease in the expression of renal transient receptor potential vanilloid type 5 (TRPV5), which is responsible for renal calcium reabsorption. Soluble klotho was previously determined to increase TRPV5 by cleaving sialic acid, causing TRPV5 to bind to membrane protein galectin-1. However, a recent study showed that soluble klotho binds to α2-3-sialyllactose - where sialic acid is located - on TRPV5, rather than cleave it. Here, we report that soluble klotho tethers TRPV5 on the membrane by binding both TRPV5 and galectin-1, thereby protecting membrane TRPV5 from diabetes-induced endocytosis. We injected recombinant soluble α-klotho protein (rKL) into db/db and db/m mice for 8 weeks and collected urine and the kidney. We administered rKL, AZD4547 (FGFR1 inhibitor), and OTX008 (Galectin 1 inhibitor) to cultured mouse distal tubular cells, with or without 30 mM high glucose (HG) exposure. db/db mice showed increased renal calcium excretion and decreased renal TRPV5 expression. rKL treatment reversed this change. In vitro, TRPV5 expression in distal tubular cells decreased under HG conditions, and rKL successfully upregulated TRPV5 with or without FGF23. Also, immunofluorescence showed co-localization of klotho, TRPV5, and galectin-1 in distal tubule cells, suggesting that klotho binds to both TRPV5 and galectin 1. Moreover, when both FGFR1 and galectin-1 were inhibited, rKL failed to increase TRPV5 under HG conditions. Our results indicate that soluble klotho prevents TRPV5 from degradation and subsequent diabetes-induced endocytosis by anchoring TRPV5 through binding with both TRPV5 and galectin-1.

Contribution of Bone Tissue to Regulation of Calcium and Phosphate Metabolism. Role of FGF23 and Klotho Protein

Bone tissue actively contributes to the regulation of systemic homoeostasis, and particularly the maintenance of calcium-phosphate balance. The parathyroid hormone-vitamin D feedback axis is balanced by the recently discovered bone-FGF23-kidney hormonal axis. An active complex consisting of FGF23, a receptor and Klotho protein blocks phosphate reabsorption in the proximal tubules, increasing urine phosphate levels and decreasing blood phosphate levels. Mutations of the gene mediating FGF23 transcription lead to a number of diseases, examples including autosomal dominant hypophosphataemic rickets. Klotho protein is a cofactor for FGF23 displaying cardio-, vaso- and nephroprotective activity. It increases calcium reabsorption in the kidneys and inhibits phosphate reabsorption. It also exerts antioxidative and anti-insulin effects and inhibits tissue calcification and apoptosis. As an inhibitor of bone resorption, osteoprotegerin becomes an important contributor to bone remodelling, while RANK/RANKL signalling inhibition is used in the treatment of postmenopausal osteoporosis. Osteocalcin plays an important role in energy metabolism in the human body. Sclerostin exerts a strong catabolic effect on bone tissue. Newly identified contributors to the regulation of calcium and phosphate homoeostasis suggest that bone tissue plays a complex role in the systemic metabolism.

Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (PCa). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with β-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and PCa was measured. PCa in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of PCa in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

Structure-based characterization of novel TRPV5 inhibitors

Transient receptor potential vanilloid 5 (TRPV5) is a highly calcium selective ion channel that acts as the rate-limiting step of calcium reabsorption in the kidney. The lack of potent, specific modulators of TRPV5 has limited the ability to probe the contribution of TRPV5 in disease phenotypes such as hypercalcemia and nephrolithiasis. Here, we performed structure-based virtual screening (SBVS) at a previously identified TRPV5 inhibitor binding site coupled with electrophysiology screening and identified three novel inhibitors of TRPV5, one of which exhibits high affinity, and specificity for TRPV5 over other TRP channels, including its close homologue TRPV6. Cryo-electron microscopy of TRPV5 in the presence of the specific inhibitor and its parent compound revealed novel binding sites for this channel. Structural and functional analysis have allowed us to suggest a mechanism of action for the selective inhibition of TRPV5 and lay the groundwork for rational design of new classes of TRPV5 modulators.