Chronic renal failure and shortened lifespan in COL4A3+/- mice: an animal model for thin basement membrane nephropathy

A heterozygous mutation in autosomal Alport genes COL4A3 and COL4A4 can be found in 20 to 50% of individuals with familial benign hematuria and diffuse glomerular basement membrane thinning (thin basement membrane nephropathy [TBMN]). Approximately 1% of humans are heterozygous carriers of mutations in the autosomal Alport genes and at risk for developing renal failure as a result of TBMN. The incidence and pathogenesis of renal failure in heterozygous COL4A3/4 mutation carriers is still unclear and was examined further in this study using COL4A3 knockout mice. In heterozygous COL4A3(+/-) mice lifespan, hematuria and renal function (serum urea and proteinuria) were monitored during a period of 3 yr, and renal tissue was examined by light and electron microscopy, immunohistochemistry, and Western blot. Lifespan of COL4A3(+/-) mice was found to be significantly shorter than in healthy controls (21.7 versus 30.3 mo). Persistent glomerular hematuria was detected starting in week 9; proteinuria of > 0.1 g/L started after 3 mo of life and increased to > 3 g/L after 24 mo. The glomerular basement membrane was significantly thinned (167 versus 200 nm in wild type) in 30-wk-old mice, coinciding with focal glomerulosclerosis, tubulointerstitial fibrosis, and increased levels of TGF-beta and connective tissue growth factor. The renal phenotype in COL4A3(+/-) mice resembled the clinical and histopathologic phenotype of human cases of TBMN with concomitant progression to chronic renal failure. Therefore, the COL4A3(+/-) mouse model will help in the understanding of the pathogenesis of TBMN in humans and in the evaluation of potential therapies.     

A Monocyte chemoattractant protein-1 (MCP-1) polymorphism and outcome after renal transplantation

Among the factors modulating transplant rejection and cardiovascular disease, chemokines and their respective receptors deserve special attention. In this respect, increased expression of MCP-1 and the corresponding receptor CCR2 have been demonstrated in renal transplant rejection and coronary artery disease. The impact of the MCP-1-2518G and CCR2-64I genotypes on renal allograft function was investigated in 232 patients who underwent transplantation over an 11-yr period. Genomic DNA was genotyped using PCR with sequence-specific primers followed by restriction fragment length polymorphism analysis. Eighteen (7.8%) patients were homozygous for the MCP-1-2518G mutation. The G/G allele of MCP-1 -2518 behaved as a determinant for long-term allograft survival and resulted in reduction of the mean graft survival, as compared with the heterozygous (A/G) or wild-type (A/A) allele (67 +/- 14 versus 95 +/- 4 mo; Log rank P = 0.0052). The 64I mutation of CCR2 had no effect on kidney graft failure (93 +/- 6 and 91 +/- 5 mo, respectively; P = 0.81). None of the investigated polymorphisms showed a significant shift in gene frequency in acute rejection and rejection-free groups. In conjunction with these findings, peripheral blood mononuclear cells from kidney transplant recipients carrying the G-allele were characterized by a 2.5-fold higher MCP-1 secretion (P < 0.05). In conclusion, recipients of renal transplants homozygous for the -2518 G mutation of the MCP-1 gene are at risk for premature kidney graft failure. This variant of MCP-1 may be a future predictor for long-term kidney graft failure.     

Role of aquaporin water channels in kidney function studied using transgenic mice

Several aquaporin (AQP) water transporting proteins are expressed in mammalian kidney: AQP1 in plasma membranes of proximal tubule, thin descending limb of Henle, and descending vasa recta; AQP2 in collecting duct luminal membrane; AQP3 and AQP4 in collecting duct basolateral membrane; AQP6 in intercalated cells; and AQP7 in the S3 segment of proximal tubule. To define the role of aquaporins in renal physiology, we have generated and characterized transgenic null mice deficient in AQP1, AQP3, and AQP4, individually and in combinations, as well as AQP2 mutant mice, in which the T126M mutation causing human nephrogenic diabetes insipidus was introduced. AQP1-deficient mice are polyuric and unable to concentrate their urine in response to water deprivation or vasopressin administration. AQP1 deletion greatly reduces osmotic water permeability in proximal tubule, thin descending limb of Henle, and descending vasa recta, resulting in defective proximal tubule fluid absorption and medullary countercurrent exchange. Mice lacking AQP3 have low basolateral membrane water permeability in cortical collecting duct and excrete large quantities of dilute urine. Mice lacking AQP4 have low water permeability in inner medullary collecting duct, but manifest only a mild defect in maximum urinary concentrating ability. These data, taken together with phenotype analyses of brain, lung, and gastrointestinal organs, support the paradigm that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. The renal phenotype data in aquaporin knockout mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents. Received: March 19, 2001 / Accepted: March 22, 2001     

Prevalence of alpha1-antitrypsin phenotypes in patients with IgA nephropathy

BACKGROUND: alpha1-antitrypsin (AAT) is the main protease inhibitor in the blood. Several different AAT phenotypes exist. The most common variant is the MM phenotype, which is also associated with normal AAT levels. The less common phenotypes with Z and S variants are associated with low AAT levels. AAT deficiency is a risk factor for pulmonary emphysema, liver impairment and some immune-mediated diseases, some of which are also associated with IgA nephropathy (IgAN). In fact, liver impairment resulting from AAT deficiency may directly contribute to renal abnormalities resembling IgAN. PATIENTS AND METHODS: We investigated AAT phenotype and AAT levels in 100 IgAN patients who did not have end-stage liver disease. Fifteen patients in our sample had secondary IgAN. We also tested for the presence of renal deposition of AAT in patients heterozygous for AAT variants as well as in a randomly chosen group of patients with MM phenotype. We checked for any association between AAT phenotype and the progression of IgAN as well as the prevalence of diseases associated with IgAN (i.e. secondary IgAN). RESULTS: Twelve patients in our sample were heterozygous for AAT variants. Phenotypes were MZ in 5 patients, MS in 3, MF in 1, ML in 2 and ME in 1 patient. AAT levels were lower in these 12 patients than in those homozygous for the M variant (1.17+/-0.46 vs. 1.44+/-0.34 g/l, p < 0.05). We found renal deposition of AAT in 2 heterozygous patients and in 1 of the 12 patients which were randomly chosen. End-stage renal (ESRF) failure developed in 3 of the 12 heterozygous patients and in 6 of the 88 homozygous patients (p = 0.07) during the follow-up. The prevalence of heterozygosity was significantly higher in patients with secondary IgAN than in those with primary IgAN ((5/15 vs. 7/85; p < 0.02). CONCLUSIONS: AAT phenotype is not associated with the risk of primary IgA nephropathy, but might have an impact on disease outcome as well as on the risk of secondary IgAN.     

Renal phenotype of UT-A urea transporter knockout mice

The urea transporters UT-A1 and UT-A3 mediate rapid transepithelial urea transport across the inner medullary collecting duct (IMCD). In a previous study, using a new mouse model in which both UT-A1 and UT-A3 were genetically deleted from the IMCD (UT-A1/3(-/-) mice), we investigated the role of these transporters in the function of the renal inner medulla. Here the authors report a new series of studies investigating more generally the renal phenotype of UT-A1/3(-/-) mice. Pathologic screening of 33 tissues revealed abnormalities in both the testis (increased size) and kidney (decreased size and vascular congestion) of UT-A1/3(-/-) mice. Total urinary nitrate and nitrite (NOx) excretion rates in UT-A1/3(-/-) mice were more than double those in wild-type mice. Total renal blood flow was not different between UT-A1/3(-/-) and wild-type mice but underwent a greater percentage decrease in response to NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME) infusion. Whole kidney GFR (FITC-inulin clearance) was not different in UT-A1/3(-/-) mice compared with controls and underwent a similar increase in response to a greater dietary protein intake. Fractional urea excretion was markedly elevated in UT-A1/3(-/-) mice on a 40% protein diet, reaching 102.4 +/- 8.8% of the filtered load, suggesting that there may be active urea secretion somewhere along the renal tubule. Although there was a marked urinary concentrating defect in UT-A1/3(-/-) mice, there was no decrease in aquaporin 2 or aquaporin 3 expression. Furthermore, although urea accumulation in the inner medulla was markedly attenuated, there was no decrease in sodium ion concentration in tissue from outer medulla or two levels of the inner medulla. These results support our conclusion that the urinary concentrating defect in UT-A1/3(-/-) mice is caused by a failure of urea transport from the IMCD lumen to the inner medullary interstitium, resulting in osmotic diuresis.     

Ochronotic rheumatism in Algeria: clinical, radiological, biological and molecular studies--a case study of 14 patients in 11 families

OBJECTIVES: To confirm alkaptonuria and ochronotic arthropathy diagnosis by mutation screening of the homogentisate 1,2-dioxygenase (HGD) gene. Try to establish a genotype-phenotype correlation in the five subjects with a molecular study on HGD gene. METHODS: We report 14 alkaptonuria cases (10 men and four women) in 11 Algerian families. Consanguineous matings were evidenced in only three families (F = 1/16). Molecular analysis was performed by sequencing genomic DNA in order to identify the mutations of the HGD gene. RESULTS: Alkaptonuria was always confirmed by urinary homogentisic acid determination. Four different mutations of the HGD gene were found: an homozygous missense mutation, Serine189Isoleucine in two sisters with a mild phenotype; an homozygous splice site mutation (IVS1-1G > A) in a man with a severe phenotype (death at 61 years old from renal failure); a silent mutation, Alanine470Alanine at the heterozygous state in a man with a mild phenotype; a 'G' deletion at the position c.819 which causes a frameshift after Gly217(Gly217fs) that runs into a stop codon at c. 850. This mutation is novel and was found in heterozygosis in a woman with a mild phenotype. CONCLUSIONS: The two homozygous mutations were associated, respectively, with a severe and a mild phenotype but no genotype-phenotype correlation could be found.     

The Uromodulin C744G mutation causes MCKD2 and FJHN in children and adults and may be due to a possible founder effect

Autosomal dominant medullary cystic kidney disease type 2 (MCKD2) is a tubulo-in terstitial nephropathy that causes renal salt wasting, hyperuricemia, gout, and end-stage renal failure in the fifth decade of life. This disorder was described to have an age of onset between the age of 20-30 years or even later. Mutations in the Uromodulin (UMOD) gene were published in patients with familial juvenile hyperuricemic nephropathy (FJHN) and MCKD2. Clinical data and blood samples of 16 affected individuals from 11 different kindreds were collected. Mutational analysis of the UMOD gene was performed by exon polymerase chain reaction (PCR) and direct sequencing. We found the heterozygous C744G (Cys248Trp) mutation, which was originally published by our group, in an additional four kindreds from Europe and Turkey. Age of onset ranged from 3 years to 39 years. The phenotype showed a variety of symptoms such as urinary concentration defect, vesicoureteral reflux, urinary tract infections, hyperuricemia, hypertension, proteinuria, and renal hypoplasia. Haplotype analysis showed cosegragation with the phenotype in all eight affected individuals indicating that the C744G mutation may be due to a founder effect. Moreover, we describe a novel T229G (Cys77Gly) mutation in two affecteds of one kindred. Three of the affected individuals were younger than 10 years at the onset of MCKD2/FJHN. Symptoms include recurrent urinary tract infections compatible with the published phenotype of the Umod knockout mouse model. This emphasizes that MCKD2 is not just a disease of the young adult but is also relevant for children.     

常染色体隐性遗传性远端肾小管酸中毒患儿ATP6V0A4和ATP6V1B1基因突变分析

目的 分析常染色体隐性遗传性远端肾小管酸中毒(rdRTA)患儿ATP6V0A4和ATP6V1B1基因的突变,进行基因型和表型的相关性研究.方法 PCR扩增基因组DNA,直接测序分析来自3个家系3例患儿的ATP6V0A4和ATP6V1B1基因的突变位点,选取不相关的100例健康人作为对照.结果 1例患儿携带ATP6V0A4基因的1个新的纯合无义突变(p.R194X);1例患儿携带ATP6V1B1基因1个新的杂合无义突变(p.R114X)和1个已经报道过的杂合突变p.I386fsX441;第3例患儿未发现以上2个基因的突变.结论 对中国rdRTA患者基因突变分析有利于了解该类疾病的基因型和表型的相关性,增强临床医生对该类疾病的认识和治疗.     

Novel COL4A4 splice defect and in-frame deletion in a large consanguine family as a genetic link between benign familial haematuria and autosomal Alport syndrome.

BACKGROUND: Alport syndrome (AS) is a common hereditary cause for end-stage renal failure due to a defect in type IV collagen genes. The molecular pathogenesis of benign familial haematuria (BFH) is not fully understood. Evidence from linkage analyses and mutation studies point to a role of the COL4A3/COL4A4 genes. The present study describes molecular changes of the COL4A4 gene that cause both diseases: autosomal recessive AS and BFH in a consanguine family with a 400-year-old history of haematuria. METHODS: RNA and DNA were isolated and analysed by RT-PCR, PCR, DNA and cDNA sequencing, and Southern blotting. Evaluation of family members comprised creatinine clearence, urine analysis, audiometry and past medical history. RESULTS: Forefathers of this family moved to a German village in the 17th century. Sporadic episodes of macrohaematuria have been reported ever since. Numerous family members with haematuria including the parents of the index family were heterozygous for a splice defect eliminating exon 25 from the alpha4(IV) cDNA. The daughter (15 years old, creatinine clearence 27 ml/min, proteinuria 5 g/day, hearing loss) was homozygous for the mutation, while the son (22 years old, creatinine clearance 68 ml/min, proteinuria 11 g/day, hearing loss, splitted and thickened glomerular basement membrane) was heterozygous. Further analysis showed a second mutation, an 18 bp in-frame deletion in exon 25, for which numerous family members were heterozygous, and both children were homozygous. CONCLUSIONS: The COL4A4 splice defect causes BFH-phenotype in heterozygous, and AS in homozygous state. The clinical spectrum of heterozygous individuals reaches from macrohaematuria, intermittent microhaematuria to isolated deafness. The 18 bp in-frame deletion aggravates the phenotype in the compound heterozygous son. These results give further evidence that BFH and autosomal AS are in fact both type IV collagen diseases.     

Role of parathyroid hormone-related peptide and Indian hedgehog in skeletal development

Parathyroid hormone-related peptide (PTHrP), which frequently causes the humoral hypercalcemia of malignancy syndrome, is an autocrine/paracrine regulator of chondrocyte proliferation and differentiation that acts through the PTH/PTHrP receptor (PTH1R). PTHrP is generated in response to Indian hedgehog (Ihh), which mediates its actions through the membrane receptor patched, but interacts also with hedgehog-interacting protein (Hip). Mice lacking PTHrP show accelerated chondrocyte differentiation, and thus premature ossification of those bones that are formed through an endochondral process, and similar but more-severe abnormalities are observed in PTH1R-ablated animals. The mirror image of these skeletal findings, i.e., a severe delay in chondrocyte differentiation and endochondral ossification, is observed in transgenic mice that overexpress PTHrP under the control of the α1(II) procollagen promoter. Severe abnormalities in chondrocyte proliferation and differentiation are also observed in two genetic disorders in humans that are most likely caused by mutations in the PTH1R. Heterozygous PTH1R mutations that lead to constitutively activity were identified in Jansen metaphyseal chondrodysplasia, and homozygous or compound heterozygous mutations that lead to less-active or completely inactive receptors were identified in patients with Blomstrand lethal chondrodysplasia. Based on the growth plate abnormalities observed in these human disorders and in mice with abnormal expression of either PTHrP or the PTH1R, it appears plausible that impaired expression of PTHrP and/or its receptor contributes to the growth abnormalities in children with end-stage renal disease. In fact, mild-to-moderate renal failure leads in animals to a reduction in PTH1R expression in growth plates and impaired growth, but it remains uncertain whether this contributes to altered chondrocyte growth and differentiation. Received: 18 March 1999 / Revised: 21 December 1999 / Accepted: 29 December 1999     

Pkd1 inactivation induced in adulthood produces focal cystic disease

Autosomal dominant polycystic kidney disease, the most common monogenetic disorder, is characterized by gradual replacement of normal renal parenchyma by fluid-filled cysts. Mutations in either PKD1 or PKD2 cause autosomal dominant polycystic kidney disease. Pkd1(-/-) or Pkd2(-/-) mice develop rapid renal cystic disease and exhibit embryonic lethality; this supports the "two-hit" hypothesis, which proposes that a germline mutation in PKD1 (or PKD2) followed by a second somatic mutation later in life is responsible for the phenotype. Here, for investigation of the loss of Pkd1 at specific times of development, an inducible Pkd1-knockout mouse model was generated. Inactivation of Pkd1 in 5-wk-old mice resulted in formation of only focal renal cysts 6 to 9 wk later but in a severe polycystic phenotype nearly 1 yr later. Cysts derived from either collecting tubules or distal tubules but not from proximal tubules, which correlated with sites of Cre-mediated recombination. Inactivation of Pkd1 in 1-wk-old mice, however, resulted in massive cyst disease 6 wk later, despite a similar pattern of Cre-mediated recombination between 1- and 5-wk-old kidneys. Moreover, a germline heterozygous Pkd1 mutation facilitated cyst formation when a somatic Pkd1 mutation was induced. A marked increase in proliferating cell nuclear antigen expression was observed in cyst-lining epithelia and in normal-looking tubules adjacent to but not in those distant from cysts. These data suggest that Pkd1 inactivation is not sufficient to initiate the cell proliferation necessary for cyst formation; a paracrine mechanism may account for focal cell proliferation and regional disease progression. We propose that an additional genetic or nongenetic "third hit" may be required for rapid development of cysts in polycystic kidney disease.     

Vascular endothelial growth factor a signaling in the podocyte-endothelial compartment is required for mesangial cell migration and survival

The glomerular filtration barrier separates the blood from the urinary space and consists of two major cell types: podocytes and fenestrated endothelial cells. Mesangial cells sit between the capillary loops and provide structural support. Proliferation and loss of mesangial cells both are central findings in a number of renal diseases, including diabetic nephropathy and mesangiolysis, respectively. Using cell-specific gene targeting, it was shown previously that vascular endothelial growth factor A (VEGF-A) production by podocytes is required for glomerular endothelial cell migration, differentiation, and survival. For further investigation of the effect of gene dose and VEGF-A knockdown within the glomerulus, mice that carry one hypomorphic VEGF-A allele and one podocyte-specific null VEGF-A allele (VEGFhypo/loxP,Neph-Cre+/-) were generated; in these mice, the "allelic dose" of VEGF-A is intermediate between glomerular-specific heterozygous and null states. VEGFhypo/loxP,Neph-Cre+/- mice die at 3 wk of age from renal failure. Although endothelial cell defects are observed, striking loss of mesangial cells occurs postnatally. In addition, differentiated mesangial cells cannot be found in glomeruli of podocyte-specific null VEGF-A mice (VEGFloxP/loxP,Cre+/-). Together, these results demonstrate a key role for VEGF-A production in the podocyte for mesangial cell survival and differentiation.     

Genetic modifiers of the insulin resistance phenotype in mice

Insulin resistance can result from genetic interactions among susceptibility alleles. To identify genetic loci predisposing to insulin resistance, we used crosses between different strains of mice with a targeted null allele of the insulin receptor gene. On the genetic background of B6 mice, the insulin receptor gene mutation causes mild hyperinsulinemia. In contrast, on the genetic background of 129/Sv mice, the same mutation causes severe hyperinsulinemia, suggesting that the 129/Sv strain harbors alleles that interact with the insulin receptor mutation and predispose to insulin resistance. As a first step to identify these alleles, we generated an F2 intercross between insulin receptor heterozygous mutant mice on B6 and 129/Sv backgrounds (B6IR x 129IR) and performed a genome-wide scan with polymorphic markers at a 20-cM resolution. We report the identification of loci on chromosomes 2 (logarithm of odds [LOD] 5.58) and 10 (LOD 5.58) that show significant evidence for linkage to plasma insulin levels as a quantitative trait. These findings indicate that targeted mutations in knockout mice can be used to unravel the complex genetic interactions underlying insulin resistance.     

Generation of the first autosomal dominant osteopetrosis type II (ADO2) disease models

Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl⁻/1H⁺ antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.     

Feline polycystic kidney disease mutation identified in PKD1

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder in humans that causes the formation of fluid-filled renal cysts, often leading to renal failure. PKD1 mutations cause 85% of ADPKD. Feline PKD is autosomal dominant and has clinical presentations similar to humans. PKD affects approximately 38% of Persian cats worldwide, which is approximately 6% of cats, making it the most prominent inherited feline disease. Previous analyses have shown significant linkage between the PKD phenotype and microsatellite markers linked to the feline homolog for PKD1. In this report, the feline PKD1 gene was scanned for causative mutations and a C>A transversion was identified at c.10063 (human ref NM_000296) in exon 29, resulting in a stop mutation at position 3284, which suggests a loss of approximately 25% of the C-terminus of the protein. The same mutation has not been identified in humans, although similar regions of the protein are truncated. The C>A transversion has been identified in the heterozygous state in 48 affected cats examined, including 41 Persians, a Siamese, and several other breeds that have been known to outcross with Persians. In addition, the mutation is segregating concordantly in all available PKD families. No unaffected cats have been identified with the mutation. No homozygous cats have been identified, supporting the suggestion that the mutation is embryonic lethal. These data suggest that the stop mutation causes feline PKD, providing a test to identify cats that will develop PKD and demonstrating that the domestic cat is an ideal model for human PKD.     

Ureteric bud apoptosis and renal hypoplasia in transgenic PAX2-Bax fetal mice mimics the renal-coloboma syndrome

In humans, PAX2 haploinsufficiency causes renal-coloboma syndrome (RCS) involving eye abnormalities, renal hypoplasia, and renal failure in early life. The authors previously showed that heterozygous mutant Pax2 mice have smaller kidneys with fewer nephrons, associated with elevated apoptosis in the ureteric bud (UB). However, PAX2 may have a variety of developmental functions such as effects on cell fate and differentiation. To determine whether apoptosis alone is sufficient to cause a UB branching deficit, the authors targeted a pro-apoptotic gene (Baxalpha) to the embryonic kidney under the control of human PAX2 regulatory elements. The exogenous PAX2 promoter directed Baxalpha gene expression specifically to the developing kidney UB, eye, and mid/hindbrain. At E15.5 PAX2Promoter-Baxalpha fetal mice exhibited renal hypoplasia, elevated UB apoptosis, and retinal defects, mimicking the phenotype observed in RCS. The kidneys of E15.5 PAX2Promoter-Baxalpha fetal mice were 55% smaller than those of wild-type fetal mice, and they contained 70% of the normal level of UB branching. The data indicate that loss of Pax2 anti-apoptotic activity is sufficient to account for the reduced UB branching observed in RCS and suggest that elevated UB apoptosis may be a key process responsible for renal hypoplasia. The authors propose a morphogenic unit model in which cell survival influences the rate of UB branching and determines final nephron endowment.     

Mutation analysis of 5 children with primary distal renal tubular acidosis

Objective To analyze the mutations of causal genes in 5 children with primary distal renal tubular acidosis (dRTA), and explore their association of genotype and phenotype, so as to raise the awareness of the disease. Methods The whole exome sequencing was used to identify mutations in these 5 children from 5 families. Results A total of 4 different mutations of ATP6V0A4 gene were found in 2 dRTA children, including a novel heterozygous intron mutation (c.639+1G＞A), a reported heterozygous nonsense variant (c.580C＞T, p.Arg194*) and 2 novel heterozygous duplications (c.1504dupT, p.Tyr502Leufs*22; c.2351dupT, p.Phe785Ilefs*28). Two novel heterozygous missense mutations of ATP6V1B1 gene (c.409C＞T, p.Pro137Ser; c.904C＞T, p.Arg302Trp) were identified in the third child, and a heterozygous missense mutation of SLC4A1 gene (c.1765C＞A, p.Arg589Ser) previously reported was found in the fourth child. No mutation of the dRTA-related causal genes was found in the fifth child. Furthermore, the mutations of causal genes in each of the first three children were compound heterozygous, which were consistent with the autosomal recessive inheritance pattern, and the variant from the fourth child was de novo. Conclusions The present study has found 7 mutations, including 5 novel variants, which enriches the human gene mutation database (HGMD) and contributes to a better understanding of the disease mechanisms.     

Insulin effect during embryogenesis determines fetal growth: a possible molecular link between birth weight and susceptibility to type 2 diabetes

Low birth weight has been reported to be associated with impaired insulin secretion and insulin resistance. It has been proposed that this association results from fetal programming in response to the intrauterine environment (the thrifty phenotype hypothesis). To elucidate the relationship between birth weight and genetically determined defects in insulin secretion, we measured the birth weights of neonates derived from crosses of male pancreatic beta-cell type glucokinase knockout (Gck+/-) mice and female wild-type (WT) or Gck+/- mice. In 135 offspring, birth weights were lower in the presence of a fetal heterozygous mutation and higher in the presence of a maternal heterozygous mutation. Moreover, Gck-/- neonates had significantly smaller birth weights than WT or Gck+/- neonates (means +/- SE 1.49+/-0.03 [n = 30] vs. 1.63+/-0.03 [n = 30] or 1.63+/-0.02 [n = 50] g, respectively; P<0.01). Thus, Gck mutations in beta-cells may impair insulin response to glucose and alter intrauterine growth as well as glucose metabolism after birth. This study has confirmed the results of a previous report that human subjects carrying mutations in Gck had reduced birth weights and has provided direct evidence for a link between insulin and fetal growth. Moreover, birth weights were reduced in insulin receptor substrate-1 knockout mice despite normal insulin levels. Taken together, these results suggest that a genetically programmed insulin effect during embryogenesis determines fetal growth and provides a possible molecular link between birth weight and susceptibility to type 2 diabetes.     

Deletion of the kinase domain in death-associated protein kinase attenuates tubular cell apoptosis in renal ischemia-reperfusion injury

Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase localized to renal tubular epithelial cells. To elucidate the contribution of DAPK activity to apoptosis in renal ischemia-reperfusion (IR) injury, wild-type (WT) mice and DAPK-mutant mice, which express a DAPK deletion mutant that lacks a portion of the kinase domain, were subjected to renal pedicle clamping and reperfusion. After IR, DAPK activity was elevated in WT kidneys but not in mutant kidneys (1785.7 +/- 54.1 pmol/min/mg versus 160.7 +/- 60.6 pmol/min/mg). Furthermore, there were more TUNEL-positive nuclei and activated caspase 3-positive cells in WT kidneys than in mutant kidneys after IR (24.0 +/- 5.9 nuclei or 9.4 +/- 0.6 cells per high-power field [HPF] versus 6.3 +/- 2.2 nuclei or 4.4 +/- 0.7 cells/HPF at 40 h after ischemia). In addition, the increase in p53-positive tubule cells after IR was greater in WT kidney than in mutant kidneys (9.9 +/- 1.4 cells/HPF versus 0.8 +/- 0.4 cells/HPF), which is consistent with the theory that DAPK activity stabilizes p53 protein. Finally, serum creatinine levels after IR were higher in WT mice than in mutant mice (2.54 +/- 0.34 mg/dl versus 0.87 +/- 0.24 mg/dl at 40 h after ischemia). Thus, these results indicate that deletion of the kinase domain from DAPK molecule can attenuate tubular cell apoptosis and renal dysfunction after IR injury.     

Compensatory up-regulation of angiotensin II subtype 1 receptors in alpha ENaC knockout heterozygous mice

BACKGROUND: In mice, a partial loss of function of the epithelial sodium channel (ENaC), which regulates sodium excretion in the distal nephron, causes pseudohypoaldosteronism, a salt-wasting syndrome. The purpose of the present experiments was to examine how alpha ENaC knockout heterozygous (+/-) mice, which have only one allele of the gene encoding for the alpha subunit of ENaC, control their blood pressure (BP) and sodium balance. METHODS: BP, urinary electrolyte excretion, plasma renin activity, and urinary adosterone were measured in wild-type (+/+) and heterozygous (+/-) mice on a low, regular, or high sodium diet. In addition, the BP response to angiotensin II (Ang II) and to Ang II receptor blockade, and the number and affinity of Ang II subtype 1 (AT1) receptors in renal tissue were analyzed in both mouse strains on the three diets. RESULTS: In comparison with wild-type mice (+/+), alpha ENaC heterozygous mutant mice (+/-) showed an intact capacity to maintain BP and sodium balance when studied on different sodium diets. However, no change in plasma renin activity was found in response to changes in sodium intake in alpha ENaC +/- mice. On a normal salt diet, heterozygous mice had an increased vascular responsiveness to exogenous Ang II (P < 0.01). Moreover, on a normal and low sodium intake, these mice exhibited an increase in the number of AT1 receptors in renal tissues; their BP lowered markedly during the Ang II receptor blockade (P < 0.01) and there was a clear tendency for an increase in urinary aldosterone excretion. CONCLUSIONS: alpha ENaC heterozygous mice have developed an unusual mechanism of compensation leading to an activation of the renin-angiotensin system, that is, the up-regulation of AT1 receptors. This up-regulation may be due to an increase in aldosterone production.