Congenital adrenal hypoplasia, myopathy, and glycerol kinase deficiency: Molecular genetic evidence for deletions

Glycerol kinase deficiency (GKD) is an X-linked recessive trait that occurs in association with congenital adrenal hypoplasia (AH) and developmental delay with or without congenital dystrophic myopathy. Several such patients have recently been reported to have cytological deletions of chromosome region Xp21 and/or of DNA markers that map near the locus for Duchenne muscular dystrophy (DMD) in band Xp21. We have examined the initial family reported in the literature and, using prometaphase chromosome studies and Southern blot analysis with 13 different DNA probes derived from band Xp21, have found no deletions within this region of the X chromosome. When DNA samples from six other unrelated affected males were analyzed, four of them were found to have different-size deletions within Xp21. Thus, the form of GKD associated with AH and dystrophic myopathy exhibits significant genetic heterogeneity at the DNA level. No deletions were detected in two patients with isolated GK deficiency. Comparison of our molecular studies of unrelated patients with deletions of DNA segments allows us to define the region of Xp21 (between probes J-Bir and L1.4) that most likely contains the genes for GKD and AH. This location is distal to the DMD locus. The patients with progressive muscular dystrophy tended to have larger deletions that include markers known to derive from the DMD locus, while GKD/AH/dystrophic-myopathy patients without current evidence of deletion seemed to have a milder, nonprogressive form of congenital myopathy.     

Glycerol kinase deficiency: evidence for complexity in a single gene disorder

Glycerol kinase deficiency (GKD) occurs as part of an Xp21 contiguous gene syndrome or as isolated GKD. The isolated form can be either symptomatic with episodic metabolic and central nervous system (CNS) decompensation or asymptomatic with hyperglycerolemia and glyceroluria only. To better understand the pathogenesis of isolated GKD, we sought individuals with point mutations in the GK coding region and measured their GK enzyme activities. We identified six individuals with missense mutations: four (N288D, A305V, M428T, and Q438R) among males who were asymptomatic and two (D198G, R405Q) in individuals who were symptomatic. GK activity measured in lymphoblastoid cell lines or fibroblasts was similar for the symptomatic and the asymptomatic individuals. Mapping of the individuals' missense mutations to the three-dimensional structure of Escherichia coli GK revealed that the symptomatic individuals' mutations are in the same region as a subset of the mutations among the asymptomatic individuals, adjacent to the active-site cleft. We conclude that, like many other disorders, GK genotype does not predict GKD phenotype. We hypothesize that the phenotype of an individual with GKD is a complex trait influenced by additional, independently inherited genes.     

Gene therapy for murine glycerol kinase deficiency: importance of murine ortholog

A glycerol kinase (Gyk) knock-out (KO) mouse model permits improved understanding of glycerol kinase (GK) deficiency (GKD) pathogenesis, however, early death of affected mice limits its utility. The purpose of this work was to delay death of affected males to investigate thoroughly their phenotypes. An adenoviral vector carrying the human (Adeno-XGK) or mouse (Adeno-XGyk) GK gene was injected into KO mice within 24 h of birth. Adeno-XGK did not change KO mouse survival time despite liver GK activity greater than 100% of wild type. However, Adeno-XGyk improved KO mouse survival time greater than two-fold. These investigations demonstrate that gene replacement therapy for Gyk KO mice is more efficacious using murine Gyk than human GK. These studies expand our understanding of GKD pathogenesis in the murine model, and show that while murine GKD is more severe than in humans, GKD mice have similar metabolic disturbances to affected humans with hypoglycemia and acidemia.     

6-Keto-prostaglandin F1 alpha is not added to urine during passage through the rat urinary bladder in vivo

Studies were conducted to determine whether prostaglandins are added to the urine during its passage through the rat urinary bladder in vivo. Control rats and rats with chronic streptozotocin-induced diabetes were anesthetized with Inactin, 100 mg/kg i.p., and urine was collected simultaneously from both kidneys. Urine from the left kidney was collected directly from the renal pelvis via a ureteral cannula, while urine from the right kidney was collected via a cannula in the urinary bladder. Prostaglandins in the urine were measured by radioimmunoassay. No difference in urinary concentration or rate of excretion of 6-keto-PGF1 alpha or PGE2 was seen between ureteral urine and bladder urine from either normal or diabetic rats. The results of this study indicate that in vivo there is no intralumenal addition of either 6-keto-PGF1 alpha or PGE2 to the urine by the ureteral bladder of rats.     

Deletion mapping of Aland Island eye disease to Xp21 between DXS67 (B24) and Duchenne muscular dystrophy.

Aland Island Eye Disease (AIED) is an X-linked form of ocular hypopigmentation--also known as Forsius-Eriksson, or type 2, ocular albinism--in which affected males demonstrate subnormal visual acuity, protanomalous red-green colorblindness, axial myopia, astigmatism, hypoplasia of the fovea, and hypopigmentation of the fundus. A patient has previously been described who, in addition to AIED, manifested a contiguous gene syndrome which included congenital adrenal hypoplasia (AHC), glycerol kinase deficiency (GKD), and Duchenne muscular dystrophy (DMD). In the present paper report we report the molecular genetic analysis of his deletion. Initially, multiplex polymerase-chain-reaction amplification was used to screen for a DMD-locus deletion which was then further characterized, using DMD cDNA and genomic probes, via Southern blot analysis. The deletion includes the region encompassed by probes C7 (DXS28) and DMD cDNA 8. Probes B24 (DXS67) and DMD cDNA 5b-7 show normal hybridization patterns and appear to flank the deletion, while the DMD cDNA 8 detects a junction fragment. Molecular genetic techniques have mapped the deletion in this patient to the subbands Xp21.3-21.2, between DXS67 and DMD.     

Single-cell RNA-Seq analysis identified kidney progenitor cells from human urine

Dear Editor, Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra.Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases.Among them, renal tubular cells and podocytes have been identified and 2D or 3D cultured from human urine specimens (Oliveira Arcolino et al., 2015;Schutgens et al.,2019).Particularly, kidney stem cell/progenitor cells were successfully recovered from pediatric patient urine and then cultured for kidney regenerative purpose by the Romagnani group.However, they also showed that such cells cannot be recovered from healthy individuals (Lazzeri et al., 2015).It remains unknown whether similar types of progenitor cells can be found in different individuals, either healthy or diseased.     

An evolutionary switch in tissue-specific gene expression. Abundant expression of alpha 1-antitrypsin in the kidney of a wild mouse species

alpha 1-Antitrypsin (AT), one of the major proteinase inhibitors in mammalian serum, is generally considered to be synthesized exclusively in the liver. We have found that a wild-derived Mus species, Mus caroli, expresses AT mRNA in kidney at levels approaching that in liver; no other mouse, inbred or wild-derived, exhibits this striking property. Liver and kidney mRNAs from M. caroli encode very similar AT polypeptides that are distinct from that encoded by Mus musculus liver mRNA. In vivo, liver AT is secreted into the bloodstream, while kidney AT, which is processed differently from the liver protein, is excreted into the urine. Analysis of RNA from a hybrid between M. musculus and M. caroli indicates that a cis-acting genetic element may be responsible for the difference in AT expression. Restriction enzyme digestion patterns of AT genomic sequences in M. caroli DNA are considerably different from those in M. musculus; in addition, these sequences are undermethylated in liver DNA from M. musculus and in liver and kidney DNA from M. caroli, reflecting the respective patterns of expression. Further studies of the altered tissue specificity of AT expression that is apparent in these two related species should lead to new insights into the nature and evolution of genetic determinants of tissue-specific phenotypes.     

Characterization of patients with glycerol kinase deficiency utilizing cDNA probes for the Duchenne muscular dystrophy locus

Summary Genomic DNA from five previously unreported patients with glycerol kinase deficiency (GKD), dystrophic myopathy, and adrenal insufficiency were studied with genomic probes and cDNA probes for the Duchenne muscular dystrophy (DMD) locus. These individuals, together with those reported by ourselves and others, show that patients with a contigous gene syndrome involving the DMD, GK, and adrenal hypoplasia congenita (AHC) loci have a broader distribution of microdeletion breakpoints than those observed among patients with classical DMD. This study demonstrates the use of the DMD cDNA probes to delineate the centromeric deletion breakpoints for patients with Xp21 microdeletions extending beyond the DMD locus. It also shows the practical diagnostic application of the DMD cDNA probes when the diagnosis of GKD is entertained in a patient with known DMD and only DNA is available for study.     

6-keto-prostaglandin F1α is not added to urine during passage through the rat urinary bladder

Studies were conducted to determine whether prostaglandins are added to the urine during its passage through the rat urinary blader . Control rats and rats with chronic streptozotocin-induced diabetes were anesthetized with Inactin, 100 mg/kg i.p., and urine was collected simultaneously from both kidneys. Urine from the left kidney was collected directly from the renal pelvis via a ureteral cannula, while urine from the right kidney was collected via a cannula in the urinary bladder. Prostaglandins in the urine were measured by radioimmunoassay. No difference in urinary concentration or rate of excretion of 6-keto-PGF_1α or PGE₂ was seen between ureteral urine and bladder urine from either normal or diabetic rats. The results of this study indicate that there is no intralumenal addition of either 6-keto-PGF_1α or PGE₂ to the urine by the ureteral bladder of rats.     

Ischemic Acute Kidney Injury Perturbs Homeostasis of Serine Enantiomers in the Body Fluid in Mice: Early Detection of Renal Dysfunction Using the Ratio of Serine Enantiomers

The imbalance of blood and urine amino acids in renal failure has been studied mostly without chiral separation. Although a few reports have shown the presence of D-serine, an enantiomer of L-serine, in the serum of patients with severe renal failure, it has remained uncertain how serine enantiomers are deranged in the development of renal failure. In the present study, we have monitored serine enantiomers using a two-dimensional HPLC system in the serum and urine of mice after renal ischemia-reperfusion injury (IRI), known as a mouse model of acute kidney injury. In the serum, the level of D-serine gradually increased after renal IRI in parallel with that of creatinine, whereas the L-serine level decreased sharply in the early phase after IRI. The increase of D-serine was suppressed in part by genetic inactivation of a D-serine-degrading enzyme, D-amino acid oxidase (DAO), but not by disruption of its synthetic enzyme, serine racemase, in mice. Renal DAO activity was detected exclusively in proximal tubules, and IRI reduced the number of DAO-positive tubules. On the other hand, in the urine, D-serine was excreted at a rate nearly triple that of L-serine in mice with sham operations, indicating that little D-serine was reabsorbed while most L-serine was reabsorbed in physiological conditions. IRI significantly reduced the ratio of urinary D−/L-serine from 2.82±0.18 to 1.10±0.26 in the early phase and kept the ratio lower than 0.5 thereafter. The urinary D−/L-serine ratio can detect renal ischemia earlier than kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) in the urine, and more sensitively than creatinine, cystatin C, or the ratio of D−/L-serine in the serum. Our findings provide a novel understanding of the imbalance of amino acids in renal failure and offer a potential new biomarker for an early detection of acute kidney injury.     

Concanavalin A-captured glycoproteins in healthy human urine

Both the urinary proteome and its glycoproteome can reflect human health status, and more directly, functions of kidney and urinary tracts. Because the high abundance protein albumin is not N-glycosylated, the urine N-glycoprotein enrichment procedure could deplete it, and urine proteome could thus provide a more detailed protein profile in addition to glycosylation information especially when albuminuria occurs in some kidney diseases. In terms of describing the details of urinary proteins, the urine glycoproteome is even a better choice than the proteome itself. Pooled urine samples from healthy volunteers were collected and acetone-precipitated for proteins. N-Linked glycoproteins enriched with concanavalin A affinity purification were separated and analyzed by SDS-PAGE-reverse phase LC/MS/MS or two-dimensional LC/MS/MS. A total of 225 urinary proteins were identified based on two-hit criteria with reliability over 97% for each peptide. Among these proteins, 94 were identified in previous urine proteome works, 150 were annotated as glycoproteins in Swiss-Prot, and 43 were predicted as glycoproteins by NetNGlyc 1.0. A number of known biomarkers and disease-related glycoproteins were identified. Because changes in protein quantity or the glycosylation status can lead to changes in the concanavalin A-captured glycoprotein profile, specific urine glycoproteome patterns might be observed for specific pathological conditions as multiplex urinary biomarkers. Knowledge of the urine glycoproteome is important in understanding kidney and body function.     

Using an Isolated Rat Kidney Model to Identify Kidney Origin Proteins in Urine

The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases.     

Gamma-glutamyltransferase release by the post ischemic kidney: multiple forms and cellular pH

The release of gamma-glutamyltransferase from renal tubule cells was studied in situ following 30 minutes of ischemia. The ischemic kidney enzyme level fell 33 percent after 15 minutes of reflow of which only 1.2 percent was recovered in the urine; none was released into the renal vein. At this time the overwhelming majority of the enzyme appears bound to membranes in both the kidney and the urine. In the subsequent 15 minutes renal levels continue to decline while urinary excretion accounts for 5 percent of that disappearing from the kidney. Interestingly the form of the enzyme present in kidney and urine shifts to a soluble form coinciding with cellular alkalosis, urinary alkalinization and a rise in ATP levels. Alkalinization of renal homogenates result in a 2-fold increase in the soluble enzyme form. The results are consonant with the immediate loss of brush border enzyme via uptake into the cell or release into the urine with the former pathway predominating; subsequent appearance of the soluble enzyme appears to reflect intracellular alkaline proteinase activity and exocytosis. The form in which the enzyme is excreted may provide a useful clinical index: membranous reflecting cellular necrosis and soluble reflecting cellular recovery.     

NGAL-Siderocalin in kidney disease

Kidney damage induces the expression of a myriad of proteins in the serum and in the urine. The function of these proteins in the sequence of damage and repair is now being studied in genetic models and by novel imaging techniques. One of the most intensely expressed proteins is lipocalin2, also called NGAL or Siderocalin. While this protein has been best studied by clinical scientists, only a few labs study its underlying metabolism and function in tissue damage. Structure-function studies, imaging studies and clinical studies have revealed that NGAL-Siderocalin is an endogenous antimicrobial with iron scavenging activity. This review discusses the "iron problem" of kidney damage, the tight linkage between kidney damage and NGAL-Siderocalin expression and the potential roles that NGAL-Siderocalin may serve in the defense of the urogenital system. This article is part of a Special Issue entitled: Cell Biology of Metals.     

联合标记物检测在重症感染中合并急性肾损伤的诊断价值

目的:探讨尿中性粒细胞明胶酶相关载脂蛋白(neutrophil gelatinase-associated lipocalin,NGAL)、尿肾损伤分子-1(kidneyinjury molecule-1,Kim-1)、尿N-乙酰-β-D-氨基葡萄糖苷酶(N-acecyl-β-D-glucosaminidase,NAG)、尿微量白蛋白(mALB)在重症感染中合并急性肾损伤的敏感性及临床价值。方法:回顾分析60例在新疆自治区人民医院ICU住院的重症感染合并急性肾损伤(AKI)患者的尿NGAL、Kim-1、NAG及mALB的变化情况。健康体检者20例为对照组。尿NGAL、Kim-1、mALB测定采用酶联免疫法(ELISA)检测,尿NAG测定采用对硝基苯酚(PNP)比色法检测,并以ROC曲线分析其敏感性。结果:AKI组患者尿液中的NGAL、Kim-1、NAG、mALB的测定浓度明显高于对照组,差异具有统计学意义(P<0.001),通过ROC曲线、诊断试验结果显示:尿NGAL、Kim-1曲线下面积分别为0.986、0.956,95%可信区间分别是0.968～1.004、0.910～1.001,较尿NAG、mALB更具有敏感性(P<0.001)。结论:尿NGAL、尿Kim-1的浓度检测对重症感染合并急性肾损伤的诊断更具有敏感性,与NAG、mALB联合检测有助于急性肾损伤的早期监测,对预防急性肾损伤的发生、发展具有重要的临床价值。     

Detection of immunoreactive napsin A in human urine

Human napsin A is an aspartic proteinase highly expressed in kidney and lung. To elucidate whether napsin A is excreted in the urine we have performed an immunochemical study using anti-napsin A polyclonal antibody. As a result an immunoreactive band at approx. 38 kDa was detected which corresponds to the molecular mass of recombinant active human napsin A. A deglycosylation study showed that excreted napsin A is N-glycosylated on apparently all of the three potential glycosylation sites. Immunoreactive napsin A was also observed in urine from patients with a transplanted kidney whose kidney function appeared half to fully normal. On the other hand, no or very low immunostaining was detected in samples from patients with diseased kidneys. The urinary excretion pattern correlates well with the enzymatic activity of napsin A. These data show that human napsin A is excreted as functional proteinase in the urine. Furthermore, immunochemical studies suggest a relation between urinary excretion of napsin A and renal function. More specifically, lack of urinary excretion of napsin A could potentially serve as a tool for the detection of kidney dysfunction.     

Modification of genital kidney nephrons for sperm transport in a plethodontid salamander,Eurycea longicauda

Salamanders possess kidneys with two distinct regions: a caudal pelvic portion and cranial genital portion. Nephrons of the pelvic region are responsible for urine formation and transport. Nephrons of the genital region transport sperm from testes to Wolffian ducts; however, nephrons of the genital region possess all the same functional regions found in pelvic kidney nephrons that are involved with urine formation and transport (renal corpuscles, proximal tubules, distal tubules, and collecting ducts). Morphological similarities between pelvic and genital regions stimulated past researchers to hypothesize that nephrons of genital kidneys possess dual function; that is, sperm transport and urine formation/transport. Considering size of glomeruli is directly related to the total amount of blood plasma filtered into the Bowman's space, we tested the hypothesis that nephrons of genital kidneys have reduced urine formation function by comparing glomerular size between nephrons of pelvic and genital kidney regions in Eurycea longicauda with general histological techniques. Light microscopy analysis revealed that glomeruli of pelvic kidneys were significantly larger than those measured from genital kidneys. Transmission electron microscopy analysis also revealed modifications in genital kidney nephrons when compared to pelvic kidney nephrons that suggested a decrease in urine formation function in genital kidneys. Such modifications included a decrease in basal and lateral plasma membrane folding in genital kidney proximal and distal tubules compared to that of pelvic kidney proximal and distal tubules. Genital kidney proximal tubules were also ciliated, which was not observed in pelvic kidney proximal tubules. In conclusion, although structurally similar at the histological level, it appears that nephrons of genital kidneys have decreased urine formation function based on glomerular size comparison and nephron ultrastructure.     

Genetic basis for lithium toxicity and its relationship with tissue distribution: a diallel cross analysis of six strains of mice

Lithium toxicity measured as the time to death following an intraperitoneal injection of a lethal dose (900 mg/kg) of lithium chloride was determined in 8-week-old males from six genetic strains of mice (Balb/c, C3H/S, C57BL/6J, DBA/2, 129/ReJ, and S.W.) and their F1 hybrids. These genotypes were also evaluated for the Li+ distribution in the body 1 h after an i.p. injection of a 200 mg/kg dose of LiCl. Genotype differences in toxicity and lithium concentration in eight tissues, excreta, and urine were all statistically significant. Significant rank correlation with toxicity was found for Li+ levels in three of the tissues (kidney, lung, and blood), excreta, and urine. Diallel cross analysis was used to investigate the genetic properties of the mouse strains in responding to the toxic effects of lithium and in regulating its levels in these tissues, excreta, and urine. Our results suggest that genetically influenced membrane transport which regulates the elimination of the ion via urine and feces is an essential component in determining lithium toxicity. In general, there was a high degree of intra- and inter-genic interaction among genetic determinants of toxicity and its distribution in a number of tissues. The order of dominance of the strains as determined by (Vr, Wr) graph was different for each of the systems analysed. It is realized that the Li+ distribution in the different parts of the body is not the only factor affecting lithium toxicity.