Study On The Role Of Type ? Sodium-dependent Phosphate Transporter In Phosphate Metabolism And The Regulatory

Pi has a variety of physiological functions,and its metabolic balance can ensure the normal level of multiple physiological functions of the body.Pi metabolic imbalance can be directly caused by a variety of heriditery metabolic diseases,and is also one of the important complications of multi-system clinical diseases.The transmembrane transport of Pi from apical plasma membrane of proximal tubule is the rate-limiting step and the main site of metabolism regulation.This ratelimiting step is mediated by the Type ? sodium-dependent phosphate cotransporter(Npt2).Currently found Npt2a and Npt2c mainly associated with kidney Pi transport,they specificity expressed in renal proximal convoluted tubules epithelial cells of brush border.The power generated by sodium ion concentration difference retrieved Pi from the apical plasma membrane.Therefore,Npt2 is a key factor of maintaining the balance of Pi metabolism.As the regulatory of inorganic phosphate mechanism has remained elusive until now,there is non-specific treatment for diseases related to disorders of phosphate metabolism.Traditionally,1,25 dihydroxyvitamin D3(1,25(OH)2D)and parathyroid hormone(PTH)have been considered to be the most important factors in regulating Pi metabolism.Fibroblast growth factor 23(FGF23),the central regulator of Pi metabolism,exerts regulatory function in Npt2,which mediate about 80%reabsorption process,located in renal proximal tubule(RPT).Furthermore,FGF23 is also the independent risk factor for the occurrence,development and prognosis of phosphate metabolism-related diseases.The previous studies confirmed.that severe phosphate disorders were caused by mutations of Npt2 gene,it is noteworthy that Npt2a played the key role.On the basis of previous studies,this study established the gene knockout mouse model,and induce changes in phosphate and FGF23 levels through special diets,and further detected corresponding changes in phosphorus metabolism,biochemical indicators and key factors in the pathway to reveal the pathogenesis and development mechanism of diseases related to disorders of phosphate metabolism as well as the role of FGF23 in the regulation molecular mechanism of phosphate metabolism.This study consists of two parts:In the first part,new Npt2 mutations leading to HHRH(Hereditary Hypophosphatemia Rickets with Hypercalciuria)were detected through gene sequencing and analysis of patients.Furthermore,to know more about the novel mutations and new symptoms of the HHRH,such as renal mineralization and kidney stones.In the second part,by establishing a gene knockout animal model,then different dietary interventions to detect level changes in phosphate metabolism and regulatory factors and renal mineralization.The core factors of phosphate metabolism regulation were further identified,meanwhile,the potential risk factors and predictors were explored.In conclusion,this study may clarify the relevant mechanisms and key factors of phosphorus metabolism and regulation.Search for novel pathogenic mutations,optimize existing treatments and seek new treatment stretegy for clinically manifested diseases related to phosphorus metabolism disorders.ObjectivesCompound heterozygous and homozygous(comp/hom)mutations in solute carrier family 34,member 3(SLC34A3),the gene encoding the sodium(Na+)-dependent phosphate cotransporter 2c(NPT2c),cause hereditary hypophosphatemic rickets with hypercalciuria(HHRH),a disorder characterized by renal phosphate wasting resulting in hypophosphatemia,correspondingly elevated 1,25(OH)2 vitamin D levels,hypercalciuria,and rickets/osteomalacia.In this study,genetic sequencing and analysis were performed on 5 previously unreported HHRH kindreds to search for new pathogenic genetic mutations.Additional studies were to determine whether these biochemical parameters are independent ofgenotype and can guide therapy to prevent nephrocalcinosis,nephrolithiasis,and potentially,CKD.Subjects and Methods1.Subjects:5 previously undiagnosed HHRH kindreds were selected from the outpatient and inpatient Department of Endocrinology of Massachusetts General Hospital from January 2013 to December 2015.(Because of the confidentiality agreements,patients and other related information cannot be disclosed)2.Methods:Clinical evaluation were assessed for all the patients,family members and control persons.Laboratory assays:25-Hydroxy vitamin D levels?1,25(OH)2D levels?serum PTH levels and FGF23 level were performed at laboratories used by the different investigators(normal ranges are provided in parentheses after each value).Evaluation of renal mineralization on imaging studies(ultrasound or computed tomography).Results1.Five new HHRH kindreds were referred to us for genetic evaluation.HHRH patients and the family members SLC34A3/Npt2c nucleotide sequenced and analyzed.A novel homozygous missense mutation?a novel in-frame deletion mutation and a compound heterozygous novel deletion mutation.2.We first evaluated the prevalence of renal mineralization(or kidney stone)in the comp/homcarriers of SLC34A3/NPT2c mutations.This was significantly increased compared with the available genotyped relatives;15 of 43(35%)comp/hom carriers of SLC34A3/NPT2c mutations presented with renal stones(P=0.05),of whom 6(14%)individuals also had evidence for renal calcium/phosphate deposition.Furthermore,renal ultrasound studies showed renal calcium/phosphate deposition in 13 of 43(30%)com/hom individuals.3.A combined analysis of the initial biochemical findings in our new HHRH families showed reduced mean serum P,TRP,and intact PTH in carriers of SLC34A3/NPT2cmutations,whereas serum Ca,1,25(OH)2D,and urinary calcium excretion(uCa/uCrea)ratio were increased.Differences were significant based on oneway ANOVA for these analyze when comparing wildtype,heterozygous,and comp/homcarriers.Despite the small number of healthy siblings,serum P was also significantly reduced in heterozygous carriers compared with healthy siblings and heterozygous means for 1,25(OH)2D and TRP were above and below the normal range,respectively.As all the analyses combined,1,25(OH)2D?TRP and FGF23 were strong predictor of renal mineralization even kidney stone.Conclusions1.In this study,five new HHRH patients and the family members SLC34A3/Npt2c nucleotide sequenced and analyzed,3 novel mutations were found.2.The study showed that renal mineralization and/or renal stones may be an important,often unrecognized initial symptom in carriers of comp/hom SLC34A3/NPT2c mutations.Meanwhile,it is a great warning of HHRH treatment potential risks.3.Serum 1,25(OH)2D,phosphate,and FGF23 may be predictors of renal mineralization and kidney stones of HHRH patients.Furthermore,these biochemical parameters can guide therapy to prevent renal mineralization,kidney stones and potentially CKD.4.Degree of renal mineralization of Npt2a-/-mice directly correlates with blood phosphate and FGF23 levels and urine calcium excretion.A combined univariate linear regression analysis of all Npt2a-/-mice showed a significant direct correlation of the urine calcium/urine creatinine ratio(U-Ca/U-cre)and of the calcium excretion index(CEI)with%calcified area.5.Multivariate linear regression analysis suggests that plasma phosphate,serum FGF23,urine calcium,urine phosphate independent predictors of renal mineralization.ObjectivesReabsorption of phosphate from the urine in the renal proximal tubules is the ratelimiting step of metabolism and regulation.The previous studies confirmed that mutations in the sodium phosphate co-transporters(Npt2a and Npt2c)cause HHRH.The study established Npt2a-/-mice model and the wild type mice(WT)as control.Intervened all the mice with different content of calcium phosphorus diet.Then observed the degree of renal mineralization.Meanwhile,all the biochemical parameters related with phosphorus metabolism and regulation further evaluation.Additional of calcium and phosphate to their diet further increase renal mineralization in Npt2a-/mice but not WT mice.We use different statistical methods analyzed all the biochemical parameters related phosphate metabolism to search for the real Npt2a-/-mice independent risk factor related to renal mineralization.Our observations in Npt2a-/mice,if confirmed in humans,may be relevant for the optimization of existing and the development of novel therapies to prevent renal calcium phosphate deposits.Subjects and Methods1.Subjects:Male and female C57BL/6 mice were obtained from Charles River Laboratory,MA.Male and female Npt2a-/-mice were purchased from the Jackson Laboratory.At 8 weeks of age all the mice were randomized to special diets using egg whites as protein source for 10 to 30 weeks:HPC(high phosphate and high calcium)diet(TD.96348)contained 20%Lactose,2.0%Ca,1.25%Pi;HP(high phosphate)diet(TD.85349)contained 0.6%Ca,1.2%Pi and CO(control diet)diet(TD.09803)contained 0.6%Ca,0.3%Pi.2.Methods:Mice were euthanized in deep anesthesia with isoflurane by removal of vital organs and blood.Laboratory examinations?kidney histology?imaging studies(ultrasound or computed tomography)were assessed for all mice.Urine calcium(Ca)?blood urea nitrogen(S-BUN)?urine creatinine(U-crea)?serum 1,25dihydroxyvitaminD(1,25(OH)2D)?urine oxalate(U-oxalate)?Urine citrate?plasma parathyroid hormone(PTH)and fibroblast growth factor 23(FGF23)were detected by kit.The urine anion gap was calculated using the formula urine Na(mmol/l)+urine K(mmol/l)±urine Cl(mmol/l).SI correction factors are for Ca(mg/dl)*0.25=Ca(mmol/L),P(mg/dl)₀.32=P(mmol/L),creatinine(mg/dl)₈8.4=creatinine(umol/L).Fractional excretion indexes were calculated using the formula PEI=urine Pi/(urine creatinine_plasma Pi)or CEI=urine Ca/(urine creatinine_serum Ca),respectively.Results1.Npt2a-/-mice form renal mineral deposits on HP diet,renal mineralization was present in both intraluminal and interstitial compartments.2.Compared to WT mice serum Pi,plasma PTH and FGF23 were decreased in Npt2a-/-mice on the CO diet,while serum 1,25(OH)2D and urine calcium were increased.HP diet increased phosphaturia in Npt2a-/-mice and HPC diet increased calciuria in WT and Npt2a-/-mice.The urine calcium phosphorus product was increased in Npt2a-/-mice on all three diets but not in WT mice,albeit significantly only on HPC diet.Lack of increase of the excretion of phosphate on HPC diet when compared to HP diet may be due to decreased intestinal phosphate absorption as CaHPO4 salt and suppression of PTH by this diet's calcium content.Serum BUN levels were in the normal range for all groups.3.Npt2a-/-mice fed HPC diet(n=12)showed 0.58±0.08%calcified area,while Npt2a/-mice fed CO diet(n=21)showed 0.27±0.18%calcified area(p<0.0001 vs.HPC diet).Mineralized area was reduced in Npt2a-/-mice fed a HP diet(0.23±0.08%calcified area,n=23)when compared to HPC diet,but was similar when compared to Npt2a-/-mice fed CO diet.No mineralization was observed in WT mice on HPC or HP diet,but mineralization was seen in two of ten WT mice on CO diet,albeit less than in Npt2a-/-mice on the same diet.Mineralization size was similar on all three diets.4.Degree of renal mineralization of Npt2a-/-mice directly correlates with blood phosphate and FGF23 levels and urine calcium excretion.A combined univariate linear regression analysis of all Npt2a-/-mice showed a significant direct correlation of the urine calcium/urine creatinine ratio(U-Ca/U-cre)and of the calcium excretion index(CEI)with%calcified area.5.Multivariate linear regression analysis suggests that plasma phosphate,serum FGF23,urine calcium,urine phosphate independent predictors of renal mineral deposits.Conclusions1.We HP/HC diet induced Npt2a-/-mice renal mineralization was present in both intraluminal and interstitial compartments caused by calcium and phosphate urinary excretion increased,meanwhile,same deposit unhappened in WT mice.It can be concluded that Npt2a-/-mice renal mineralization was not only related to genetic mutations,it may directly correlated with serum phosphate,serum FGF23 level and calcium urinary excretion index.2.The phosphate reabsorption in renal proximal tubule mediated by type ? sodiumdependent Pi cotransporter(Npt2a/Npt2c),the process regulated by many hormones and factors.FGF23,the central regulator of Pi metabolism,is positive correlation of mineralization degree.Furthermore,plasm FGF23 is an independent predictor of renal mineral deposits.3.Oral phosphate supplements are currently used to treat HHRH patients.However,there is concern that this therapy might contribute to the formation of renal mineralization.Our observations in Npt2a-/-mice,if confirmed in humans,may be relevant for the optimization of existing and the development of novel therapies to prevent renal mineralization and kidney stone in HHRH patients.