Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I(PH1),the most common and lifethreatening type of primary hyperoxaluria.The comp...Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I(PH1),the most common and lifethreatening type of primary hyperoxaluria.The compact Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)from the Prevotella and Francisella 1(Cpf1)protein simplifies multiplex gene editing and allows for all-in-one adeno-associated virus(AAV)delivery.We hypothesized that the multiplex capabilities of the Cpf1system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1(Hao1)and lactate dehydrogenase A(Ldha)genes.Study cohorts included treated PH1 rats(Agxt Q84X rats injected with AAV-AsCpf1 at 7 days of age),phosphate-buffered saline(PBS)-injected PH1 rats,untreated PH1 rats,and age-matched wild-type(WT)rats.The most efficient and specific CRISPR RNA(crRNA)pairs targeting the rat Hao1and Ldha genes were initially screened ex vivo.In vivo experiments demonstrated efficient genome editing of the Hao1 and Ldha genes,primarily resulting in small deletions.This resulted in decreased transcription and translational expression of Hao1 and Ldha.Treatment significantly reduced urine oxalate levels,reduced kidney damage,and alleviated nephrocalcinosis in rats with PH1.No liver toxicity,ex-liver genome editing,or obvious offtarget effects were detected.We demonstrated the AAVAsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1,serving as a proof-ofconcept for the development of multiplex genome editingbased gene therapy.展开更多
BACKGROUND Primary hyperoxaluria type 1(PH1)is a rare autosomal recessive disease stemming from a deficiency in liver-specific alanine-glyoxylate aminotransferase,resulting in increased endogenous oxalate deposition a...BACKGROUND Primary hyperoxaluria type 1(PH1)is a rare autosomal recessive disease stemming from a deficiency in liver-specific alanine-glyoxylate aminotransferase,resulting in increased endogenous oxalate deposition and end-stage renal disease.Organ transplantation is the only effective treatment.However,its approach and timing remain controversial.CASE SUMMARY We retrospectively analyzed 5 patients diagnosed with PH1 from the Liver Transplant Center of the Beijing Friendship Hospital from March 2017 to December 2020.Our cohort included 4 males and 1 female.The median age at onset was 4.0 years(range:1.0-5.0),age at diagnosis was 12.2 years(range:6.7-23.5),age at liver transplantation(LT)was 12.2 years(range:7.0-25.1),and the follow-up time was 26.3 mo(range:12.8-40.1).All patients had delayed diagnosis,and 3patients had progressed to end-stage renal disease by the time they were diagnosed.Two patients received preemptive LT;their estimated glomerular filtration rate was maintained at>120 mL/min/1.73 m2,indicating a better prognosis.Three patients received sequential liver and kidney transplantation.After transplantation,serum and urinary oxalate decreased,and liver function recovered.At the last follow-up,the estimated glomerular filtration rates of the latter 3 patients were 179,52 and 21 mL/min/1.73 m2.CONCLUSION Different transplantation strategies should be adopted for patients based on their renal function stage.Preemptive-LT offers a good therapeutic approach for PH1.展开更多
Primary hyperoxaluria type 1(PH1)is a rare but devastating autosomal recessive inherited disease caused by mutations in gene AGXT.Pathogenic mutations of AGXT were mostly reported in Caucasian but infrequently in Asia...Primary hyperoxaluria type 1(PH1)is a rare but devastating autosomal recessive inherited disease caused by mutations in gene AGXT.Pathogenic mutations of AGXT were mostly reported in Caucasian but infrequently in Asian,especially in Chinese.To update the genotypes of PH1 in the Chinese population,we collected and identified 7 Chinese probands with PH1 from 2013 to 2017 in our center,five of whom had delayed diagnosis and failed in kidney transplantation.Samples of peripheral blood DNA from the 7 patients and their family members were collected and sequencing analysis was performed to test the mutations of gene AGXT.Western blotting and enzyme activity analysis were conducted to evaluate the function of the mutations.Furthermore,a systematic review from 1998 to 2017 was performed to observe the genetic characteristics between Chinese and Caucasian. The results showed that a total of 12 mutations were identified in the 7 pedigrees.To the best of ourknowledge,2 novel variants of A GXT,p.Gly41 Trp and p.Leu33Met,were first reported.Bioinformatics and functional analysis showed that only 7 mutations led to a reduced expression of alanine-glyoxylate amino transferase (AGT)at a protein level.The systematic review revealed significant population heterogeneity in PH1.In conclusion,new genetic subtypes and genetic characteristics of PH1 are updated in the Chinese population. Furthermore,a genotype-phenotype correlation is found in PH1.展开更多
Objective:Hyperoxaluria leads to calcium oxalate crystal formation and subsequent urolithiasis.This study aims to analyse the effect of treatment compliance in hyperoxaluria,firstly by analysis of patients with non-pr...Objective:Hyperoxaluria leads to calcium oxalate crystal formation and subsequent urolithiasis.This study aims to analyse the effect of treatment compliance in hyperoxaluria,firstly by analysis of patients with non-primary hyperoxaluria and secondly via systematic review in patients with any hyperoxaluria.Methods:In a retrospective cohort study,adults with non-primary hyperoxaluria managed with dietary counselling in 2013 were enrolled.Twenty-four-hour(24 h)urine collections initially and at 6 months were obtained.Compliance was assessed by self-reported dietary compliance and 24 h urinary volume>2 L.Patients were followed for 24 months.Primary outcomes were urinary oxalate and calcium 24 h load at 6 months,and urolithiasis-related procedural rates at 24 months.A Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA)-compatible systematic review of compliance among hyperoxaluric patients was performed.Results:In the cohort study,of 19 eligible patients(4 female)with median age 52 years,10(53%)were considered compliant.Compared with the non-compliant group,these patients had significantly increased subsequent 24 h urinary volume(2250 mL vs.1600 mL;p=0.008)and lower procedural rates(10%vs.56%;p=0.033).Subsequent 24 h urinary oxalate load was nonsignificantly lower in compliant patients.Systematic review regarding compliance in hyperoxaluric patients revealed five studies.Only one utilised dietary counselling or analysed compliant vs.non-compliant patients,finding no difference.None examined the effect of compliance on procedural rates.展开更多
Hyperoxaluria is characterized by an increased urinary excretion of oxalate. Primary and secondary hyperoxaluria are two distinct clinical expressions of hyperoxaluria. Primary hyperoxaluria is an inherited error of m...Hyperoxaluria is characterized by an increased urinary excretion of oxalate. Primary and secondary hyperoxaluria are two distinct clinical expressions of hyperoxaluria. Primary hyperoxaluria is an inherited error of metabolism due to defective enzyme activity. In contrast, secondary hyperoxaluria is caused by increased dietary ingestion of oxalate, precursors of oxalate or alteration in intestinal microflora. The disease spectrum extends from recurrent kidney stones, nephrocalcinosis and urinary tract infections to chronic kidney disease and end stage renal disease. When calcium oxalate burden exceeds the renal excretory ability, calcium oxalate starts to deposit in various organ systems in a process called systemic oxalosis. Increased urinary oxalate levels help to make the diagnosis while plasma oxalate levels are likely to be more accurate when patients develop chronic kidney disease. Definitive diagnosis of primary hyperoxaluria is achieved by genetic studies and if genetic studies prove inconclusive, liver biopsy is undertaken to establish diagnosis. Diagnostic clues pointing towards secondary hyperoxaluria are a supportive dietary history and tests to detect increased intestinal absorption of oxalate. Conservative treatment for both types of hyperoxaluria includes vigorous hydration and crystallization inhibitors to decrease calcium oxalate precipitation. Pyridoxine is also found to be helpful in approximately 30% patients with primary hyperoxaluria type 1. Liver-kidney and isolated kidney transplantation are the treatment of choice in primary hyperoxaluria type 1 and type 2 respectively. Data is scarce on role of transplantation in primary hyperoxaluria type 3 where there are no reports of end stage renal disease so far. There are ongoing investigations into newer modalities of diagnosis and treatment of hyperoxaluria. Clinical differentiation between primary and secondary hyperoxaluria and further between the types of primary hyperoxaluria is very important because of implications in treatment and diagnosis. Hyperoxaluria continues to be a challenging disease and a high index of clinical suspicion is often the first step on the path to accurate diagnosis and management.展开更多
Hyperoxaluria can cause not only nephrolithiasis and nephrocalcinosis,but also renal parenchymal disease histologically characterized by deposition of calcium oxalate crystals throughout the renal parenchyma,profound ...Hyperoxaluria can cause not only nephrolithiasis and nephrocalcinosis,but also renal parenchymal disease histologically characterized by deposition of calcium oxalate crystals throughout the renal parenchyma,profound tubular damage and interstitial inflammation and fibrosis.Hyperoxaluric nephropathy presents clinically as acute or chronic renal failure that may progress to endstage renal disease(ESRD).This sequence of events,well recognized in the past in primary and enteric hyperoxalurias,has also been documented in a few cases of dietary hyperoxaluria.Estimates of oxalate intake in patients with chronic dietary hyperoxaluria who developed chronic kidney disease or ESRD were comparable to the reported average oxalate content of the diets of certain populations worldwide,thus raising the question whether dietary hyperoxaluria is a primary cause of ESRD in these regions.Studies addressing this question have the potential of improving population health and should be undertaken,alongside ongoing studies which are yielding fresh insights into the mechanisms of intestinal absorption and renal excretion of oxalate,and into the mechanisms of development of oxalate-induced renal parenchymal disease.Novel preventive and therapeutic strategies for treating all types of hyperoxaluria are expected to develop from these studies.展开更多
Enzyme therapeutics have great potential for the treatment of systemic disorders such as urolithiasis and nephrocalcinosis, which are caused by the excessive accumulation of oxalate. However, exogenous enzymes have sh...Enzyme therapeutics have great potential for the treatment of systemic disorders such as urolithiasis and nephrocalcinosis, which are caused by the excessive accumulation of oxalate. However, exogenous enzymes have short half-lives in vivo and elicit high immunogenicity, which largely limit the therapeutic outcomes. Herein, we report a delivery strategy whereby therapeutic enzymes are encapsulated within a thin zwitterionic polymer shell to form enzyme nanocapsules. The strategy is exemplified by the encapsulation of oxalate oxidase （OxO） for the treatment of hyperoxaluria, because as-synthesized OxO nanocapsules have a prolonged blood circulation half-life and elicit reduced immunogenicity. Our design of enzyme nanocapsules that enable the systemic delivery of therapeutic enzymes can be extended to various biomedical applications.展开更多
Background Idiopathic hyperoxaluria （IH） may be caused by increased endogenous formation or exogenous absorption of oxalic acid.Characterization of the molecular pathogenesis of IH has been hampered by the lack of a...Background Idiopathic hyperoxaluria （IH） may be caused by increased endogenous formation or exogenous absorption of oxalic acid.Characterization of the molecular pathogenesis of IH has been hampered by the lack of an ideal animal model.We therefore established a stabile rat IH model in order to analyze variation in gene expression profile in the jejunum and to investigate the association between IH pathogenesis and exogenous absorption of oxalic acid.Methods A rat model of IH was established and three female rats with IH were assigned to the study group,while three normal rats served as controls.Total RNA was isolated from the jejunum of rats in the two groups and mRNA was purified,reversely transcribed,labeled with Cy5 or Cy3 and hybridized to 27K Rat Genome Array.Differences in gene expression profile between the 2 groups were analyzed by bioinformatics methods.Results Comparative analysis revealed that the expression of 517 genes was up-regulated and that of 203 genes was down-regulated by at least two-fold in the jejunum of rats with idiopathic hyperoxaluria.These genes are related to many functions including cell signal transduction,DNA binding and transcription,ATP binding,ion binding and transport,cell receptors,immunity,cyclins,cytoskeleton structure,and metabolic proteins.Kyoto encyclopedia of genes and genomes （KEGG） signaling pathway analysis revealed that the variations of 239 pathway functional changes are statistically significant （P 〈0.05）.Conclusions cDNA microarray can be used effectively to screen differentially expressed genes in the jejunum of rats with idiopathic hyperoxaluria. These differentially expressed genes may underlie idiopathic hyperoxaluria pathophysiology and provide a platform for further studying molecular pathogenetic mechanisms.展开更多
基金partially supported by the Science and Technology Commission of Shanghai Municipality (22YF1426900,20140900200)National Natural Science Foundation of China (32001057)。
文摘Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I(PH1),the most common and lifethreatening type of primary hyperoxaluria.The compact Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)from the Prevotella and Francisella 1(Cpf1)protein simplifies multiplex gene editing and allows for all-in-one adeno-associated virus(AAV)delivery.We hypothesized that the multiplex capabilities of the Cpf1system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1(Hao1)and lactate dehydrogenase A(Ldha)genes.Study cohorts included treated PH1 rats(Agxt Q84X rats injected with AAV-AsCpf1 at 7 days of age),phosphate-buffered saline(PBS)-injected PH1 rats,untreated PH1 rats,and age-matched wild-type(WT)rats.The most efficient and specific CRISPR RNA(crRNA)pairs targeting the rat Hao1and Ldha genes were initially screened ex vivo.In vivo experiments demonstrated efficient genome editing of the Hao1 and Ldha genes,primarily resulting in small deletions.This resulted in decreased transcription and translational expression of Hao1 and Ldha.Treatment significantly reduced urine oxalate levels,reduced kidney damage,and alleviated nephrocalcinosis in rats with PH1.No liver toxicity,ex-liver genome editing,or obvious offtarget effects were detected.We demonstrated the AAVAsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1,serving as a proof-ofconcept for the development of multiplex genome editingbased gene therapy.
文摘BACKGROUND Primary hyperoxaluria type 1(PH1)is a rare autosomal recessive disease stemming from a deficiency in liver-specific alanine-glyoxylate aminotransferase,resulting in increased endogenous oxalate deposition and end-stage renal disease.Organ transplantation is the only effective treatment.However,its approach and timing remain controversial.CASE SUMMARY We retrospectively analyzed 5 patients diagnosed with PH1 from the Liver Transplant Center of the Beijing Friendship Hospital from March 2017 to December 2020.Our cohort included 4 males and 1 female.The median age at onset was 4.0 years(range:1.0-5.0),age at diagnosis was 12.2 years(range:6.7-23.5),age at liver transplantation(LT)was 12.2 years(range:7.0-25.1),and the follow-up time was 26.3 mo(range:12.8-40.1).All patients had delayed diagnosis,and 3patients had progressed to end-stage renal disease by the time they were diagnosed.Two patients received preemptive LT;their estimated glomerular filtration rate was maintained at>120 mL/min/1.73 m2,indicating a better prognosis.Three patients received sequential liver and kidney transplantation.After transplantation,serum and urinary oxalate decreased,and liver function recovered.At the last follow-up,the estimated glomerular filtration rates of the latter 3 patients were 179,52 and 21 mL/min/1.73 m2.CONCLUSION Different transplantation strategies should be adopted for patients based on their renal function stage.Preemptive-LT offers a good therapeutic approach for PH1.
基金This work was supported by the grants from the Special Project of Ministry of Health (No.201302009)and the National Natural Science Foundation of China (No. 81700300).
文摘Primary hyperoxaluria type 1(PH1)is a rare but devastating autosomal recessive inherited disease caused by mutations in gene AGXT.Pathogenic mutations of AGXT were mostly reported in Caucasian but infrequently in Asian,especially in Chinese.To update the genotypes of PH1 in the Chinese population,we collected and identified 7 Chinese probands with PH1 from 2013 to 2017 in our center,five of whom had delayed diagnosis and failed in kidney transplantation.Samples of peripheral blood DNA from the 7 patients and their family members were collected and sequencing analysis was performed to test the mutations of gene AGXT.Western blotting and enzyme activity analysis were conducted to evaluate the function of the mutations.Furthermore,a systematic review from 1998 to 2017 was performed to observe the genetic characteristics between Chinese and Caucasian. The results showed that a total of 12 mutations were identified in the 7 pedigrees.To the best of ourknowledge,2 novel variants of A GXT,p.Gly41 Trp and p.Leu33Met,were first reported.Bioinformatics and functional analysis showed that only 7 mutations led to a reduced expression of alanine-glyoxylate amino transferase (AGT)at a protein level.The systematic review revealed significant population heterogeneity in PH1.In conclusion,new genetic subtypes and genetic characteristics of PH1 are updated in the Chinese population. Furthermore,a genotype-phenotype correlation is found in PH1.
文摘Objective:Hyperoxaluria leads to calcium oxalate crystal formation and subsequent urolithiasis.This study aims to analyse the effect of treatment compliance in hyperoxaluria,firstly by analysis of patients with non-primary hyperoxaluria and secondly via systematic review in patients with any hyperoxaluria.Methods:In a retrospective cohort study,adults with non-primary hyperoxaluria managed with dietary counselling in 2013 were enrolled.Twenty-four-hour(24 h)urine collections initially and at 6 months were obtained.Compliance was assessed by self-reported dietary compliance and 24 h urinary volume>2 L.Patients were followed for 24 months.Primary outcomes were urinary oxalate and calcium 24 h load at 6 months,and urolithiasis-related procedural rates at 24 months.A Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA)-compatible systematic review of compliance among hyperoxaluric patients was performed.Results:In the cohort study,of 19 eligible patients(4 female)with median age 52 years,10(53%)were considered compliant.Compared with the non-compliant group,these patients had significantly increased subsequent 24 h urinary volume(2250 mL vs.1600 mL;p=0.008)and lower procedural rates(10%vs.56%;p=0.033).Subsequent 24 h urinary oxalate load was nonsignificantly lower in compliant patients.Systematic review regarding compliance in hyperoxaluric patients revealed five studies.Only one utilised dietary counselling or analysed compliant vs.non-compliant patients,finding no difference.None examined the effect of compliance on procedural rates.
文摘Hyperoxaluria is characterized by an increased urinary excretion of oxalate. Primary and secondary hyperoxaluria are two distinct clinical expressions of hyperoxaluria. Primary hyperoxaluria is an inherited error of metabolism due to defective enzyme activity. In contrast, secondary hyperoxaluria is caused by increased dietary ingestion of oxalate, precursors of oxalate or alteration in intestinal microflora. The disease spectrum extends from recurrent kidney stones, nephrocalcinosis and urinary tract infections to chronic kidney disease and end stage renal disease. When calcium oxalate burden exceeds the renal excretory ability, calcium oxalate starts to deposit in various organ systems in a process called systemic oxalosis. Increased urinary oxalate levels help to make the diagnosis while plasma oxalate levels are likely to be more accurate when patients develop chronic kidney disease. Definitive diagnosis of primary hyperoxaluria is achieved by genetic studies and if genetic studies prove inconclusive, liver biopsy is undertaken to establish diagnosis. Diagnostic clues pointing towards secondary hyperoxaluria are a supportive dietary history and tests to detect increased intestinal absorption of oxalate. Conservative treatment for both types of hyperoxaluria includes vigorous hydration and crystallization inhibitors to decrease calcium oxalate precipitation. Pyridoxine is also found to be helpful in approximately 30% patients with primary hyperoxaluria type 1. Liver-kidney and isolated kidney transplantation are the treatment of choice in primary hyperoxaluria type 1 and type 2 respectively. Data is scarce on role of transplantation in primary hyperoxaluria type 3 where there are no reports of end stage renal disease so far. There are ongoing investigations into newer modalities of diagnosis and treatment of hyperoxaluria. Clinical differentiation between primary and secondary hyperoxaluria and further between the types of primary hyperoxaluria is very important because of implications in treatment and diagnosis. Hyperoxaluria continues to be a challenging disease and a high index of clinical suspicion is often the first step on the path to accurate diagnosis and management.
文摘Hyperoxaluria can cause not only nephrolithiasis and nephrocalcinosis,but also renal parenchymal disease histologically characterized by deposition of calcium oxalate crystals throughout the renal parenchyma,profound tubular damage and interstitial inflammation and fibrosis.Hyperoxaluric nephropathy presents clinically as acute or chronic renal failure that may progress to endstage renal disease(ESRD).This sequence of events,well recognized in the past in primary and enteric hyperoxalurias,has also been documented in a few cases of dietary hyperoxaluria.Estimates of oxalate intake in patients with chronic dietary hyperoxaluria who developed chronic kidney disease or ESRD were comparable to the reported average oxalate content of the diets of certain populations worldwide,thus raising the question whether dietary hyperoxaluria is a primary cause of ESRD in these regions.Studies addressing this question have the potential of improving population health and should be undertaken,alongside ongoing studies which are yielding fresh insights into the mechanisms of intestinal absorption and renal excretion of oxalate,and into the mechanisms of development of oxalate-induced renal parenchymal disease.Novel preventive and therapeutic strategies for treating all types of hyperoxaluria are expected to develop from these studies.
文摘Enzyme therapeutics have great potential for the treatment of systemic disorders such as urolithiasis and nephrocalcinosis, which are caused by the excessive accumulation of oxalate. However, exogenous enzymes have short half-lives in vivo and elicit high immunogenicity, which largely limit the therapeutic outcomes. Herein, we report a delivery strategy whereby therapeutic enzymes are encapsulated within a thin zwitterionic polymer shell to form enzyme nanocapsules. The strategy is exemplified by the encapsulation of oxalate oxidase （OxO） for the treatment of hyperoxaluria, because as-synthesized OxO nanocapsules have a prolonged blood circulation half-life and elicit reduced immunogenicity. Our design of enzyme nanocapsules that enable the systemic delivery of therapeutic enzymes can be extended to various biomedical applications.
文摘Background Idiopathic hyperoxaluria （IH） may be caused by increased endogenous formation or exogenous absorption of oxalic acid.Characterization of the molecular pathogenesis of IH has been hampered by the lack of an ideal animal model.We therefore established a stabile rat IH model in order to analyze variation in gene expression profile in the jejunum and to investigate the association between IH pathogenesis and exogenous absorption of oxalic acid.Methods A rat model of IH was established and three female rats with IH were assigned to the study group,while three normal rats served as controls.Total RNA was isolated from the jejunum of rats in the two groups and mRNA was purified,reversely transcribed,labeled with Cy5 or Cy3 and hybridized to 27K Rat Genome Array.Differences in gene expression profile between the 2 groups were analyzed by bioinformatics methods.Results Comparative analysis revealed that the expression of 517 genes was up-regulated and that of 203 genes was down-regulated by at least two-fold in the jejunum of rats with idiopathic hyperoxaluria.These genes are related to many functions including cell signal transduction,DNA binding and transcription,ATP binding,ion binding and transport,cell receptors,immunity,cyclins,cytoskeleton structure,and metabolic proteins.Kyoto encyclopedia of genes and genomes （KEGG） signaling pathway analysis revealed that the variations of 239 pathway functional changes are statistically significant （P 〈0.05）.Conclusions cDNA microarray can be used effectively to screen differentially expressed genes in the jejunum of rats with idiopathic hyperoxaluria. These differentially expressed genes may underlie idiopathic hyperoxaluria pathophysiology and provide a platform for further studying molecular pathogenetic mechanisms.
