Performance of the ACMG-AMP criteria in a large familial renal glucosuria cohort with identified SLC5A2 sequence variants.

Familial Renal Glucosuria (FRG) is a co-dominantly inherited trait characterized by orthoglycaemic glucosuria. From 2003 to 2015 we have reported several cohorts validating SLC5A2 (16p11.2), encoding SGLT2 (Na+/glucose cotransporter family member 2), as the gene responsible for FRG. The aim of this work was to validate the variants identified in our extended FRG cohort of published, as well more recent unreported cases, according to the ACMG-AMP 2015 criteria. Forty-six variants were evaluated, including 16 novel alleles first described in this study. All are rare, ultra-rare or absent from population databases and most are missense changes. According to the ACMG-AMP standards, only 74% of the variants were classified as P/LP. The lack of descriptions of unrelated patients with similar variants or failing to test additional affected family members, averted a conclusion for pathogenicity in the alleles that scored VUS, highlighting the importance of both family testing and variant reporting. Finally, the cryo-EM structure of the hSGLT2-MAP17 complex in the empagliflozin-bound state improved the ACMG-AMP pathogenicity score by identifying critical/functional protein domains.

Identification of ten novel SLC5A2 mutations and determination of the renal threshold for glucose excretion in Chinese patients with familial renal glucosuria.

BACKGROUND: Familial renal glucosuria (FRG) is a rare renal tubular disorder characterized by isolated persistent glucosuria without both abnormal glucose metabolism and any signs of proximal tubular dysfunction. SLC5A2 gene mutations are responsible for most FRG cases. METHODS: Quantitative test for 24-hour urine glucose and RTG were determined in 9 families (totaling 25 subjects). All coding regions, including intron-exon boundaries, were analyzed with PCR followed by direct sequence analysis. RESULTS: Ten novel mutations were identified (c.331 T > C, p.W111R; c.374T>C, p.M125T; c.394C>T, p.R132C; c.612G>C, p.Q204H; c.829C>T, p.P277S; c.880G>A, p.D294N; c.1129G>A, p.G377S; c.1194C>A, p.F398L; c.1540C > T, p.P514S and c.1573C>T, p.H525Y). c.886(-10_-31)del that is specific to Chinese population was found in 5 out of 9 families, with a mutation rate of 28% (5/18). The compound heterozygotes presented with much lower RTG values (1.28 ±â€¯0.10 mmol/L), compared with the carriers of heterozygous variants (5.14 ±â€¯0.77 mmol/L) (p<0.01); c.886(-10_-31)del heterozygotes had significant lower RTG values than others (4.43 ±â€¯0.37 vs 5.7 ±â€¯0.51 mmol/L; p<0.01). CONCLUSIONS: Ten novel SLC5A2 mutations are found and c.886(-10-31)del may be a hot spot mutation in Chinese population. Compound heterozygotes had much lower RTG values than simple heterozygotes. Mixed-meal tolerance test is a simple method for determining RTG in FRG patients.

Hypothalamic POMC Deficiency Improves Glucose Tolerance Despite Insulin Resistance by Increasing Glycosuria.

Hypothalamic proopiomelanocortin (POMC) is essential for the physiological regulation of energy balance; however, its role in glucose homeostasis remains less clear. We show that hypothalamic arcuate nucleus (Arc)POMC-deficient mice, which develop severe obesity and insulin resistance, unexpectedly exhibit improved glucose tolerance and remain protected from hyperglycemia. To explain these paradoxical phenotypes, we hypothesized that an insulin-independent pathway is responsible for the enhanced glucose tolerance. Indeed, the mutant mice demonstrated increased glucose effectiveness and exaggerated glycosuria relative to wild-type littermate controls at comparable blood glucose concentrations. Central administration of the melanocortin receptor agonist melanotan II in mutant mice reversed alterations in glucose tolerance and glycosuria, whereas, conversely, administration of the antagonist Agouti-related peptide (Agrp) to wild-type mice enhanced glucose tolerance. The glycosuria of ArcPOMC-deficient mice was due to decreased levels of renal GLUT 2 (rGLUT2) but not sodium-glucose cotransporter 2 and was associated with reduced renal catecholamine content. Epinephrine treatment abolished the genotype differences in glucose tolerance and rGLUT2 levels, suggesting that reduced renal sympathetic nervous system (SNS) activity is the underlying mechanism for the observed glycosuria and improved glucose tolerance in ArcPOMC-deficient mice. Therefore, the ArcPOMC-SNS-rGLUT2 axis is potentially an insulin-independent therapeutic target to control diabetes.

Determination of the renal threshold for glucose excretion in Familial Renal Glucosuria.

BACKGROUND/AIMS: Familial Renal Glucosuria (FRG) is characterized by the presence of persistent isolated glucosuria in the absence of hyperglycemia. Mutations in SLC5A2, the gene coding for the sodium-glucose co-transporter 2 (SGLT2), are responsible for FRG. Phenotype/genotype correlations in FRG have mostly relied on the quantification of Urinary Glucose Excretion (UGE), which is dependent on both the filtered glucose load and the renal glucose reabsorptive capacity. In the current work, the renal threshold for glucose excretion (RTG) was determined in an FRG cohort, with the purpose of characterizing the impact of SGLT2 mutations on renal glucose transport. METHODS: From January to December of 2013, eight FRG individuals with identified SLC5A2 mutations were enrolled. Patients were given a Mixed-Meal Tolerance Test during which blood glucose and UGE were measured over a 4 h period and the data was used to calculate RTG, according to a recently validated protocol. RESULTS: In patients with homozygous mutations, RTG values were very low, with a mean (SD) of 0.95 (1.17) mmol/l, compared to commonly reported values of approximately 10-11.1 mmol/l in healthy subjects. In subjects with heterozygous mutations, mean (SD) RTG values were 4.91 (1.23) mmol/l, which are approximately one-half of the values in subjects without mutations. CONCLUSIONS: In FRG, mutations in SLC5A2 lead to reductions in RTG and increases in UGE. Because determination of RTG is not influenced by the filtered glucose load, the calculated RTG values provide a more refined measure of the impact of mutations on renal glucose transport than can be obtained from UGE alone.

Novel SLC5A2 variants contribute to renal glucosuria in Chinese families: abnormal expression and dysfunction of variant SLC5A2.

Familial renal glucosuria (FRG) is characterized by persistent glucosuria despite normal serum glucose and the absence of overt tubular dysfunction. Variants in solute carrier family 5 (sodium-glucose cotransporter), member 2 (SLC5A2) have been reported in FRG patients. However, the functional and expression-related consequences of such variants have been scarcely investigated. In the current study, we studied five FRG families and identified six missense mutations, including four novel variants (c.1051T>C/.(C351R), c.1400T>C/p.(V467A), c.1420G>C/p.(A474P), c.1691G>A/p.(R564Q); RNA not analyzed) and two variants that had been previously reported (c.294C>A/p.(F98L), c.736C>T/p.(P246S); RNA not analyzed). The probands were either heterozygous or compound heterozygous for SLC5A2 variants and had glucosuria of 5.9%-19.6 g/day. Human 293 cells were transfected with plasmid constructs to study the expression and function of SLC5A2 variants in vitro. Western blotting revealed that the expression levels of SLC5A2-351R-GFP, SLC5A2-467A-GFP, SLC5A2-474P-GFP, and SLC5A2-564Q-GFP were significantly decreased compared with wild-type SLC5A2-GFP (37%-55%). Confocal microscopy revealed that three variants (c.1400T>C, c.1420G>C, c.1691G>A) resulted in a loss of the punctate membrane pattern typical of wild-type SLC5A2. All variants had a significantly lower transport capacity in than the wild-type control. The current study provides a starting point to further investigate the molecular mechanism of SLC5A2 in FRG families and provides functional clues for antidiabetes drugs.

Familial renal glycosuria and modifications of glucose renal excretion.

Under physiological conditions, the kidneys contribute to glucose homoeostasis by producing glucose by gluconeogenesis and preventing glucose loss in urine. The glucose filtered by the glomeruli is completely reabsorbed in the renal proximal tubule. Renal gluconeogenesis produces 25% of the circulating glucose in the postabsorptive state, while the amount of glucose reabsorbed by the kidneys largely exceeds the quantity synthesized by kidney gluconeogenesis. Sodium-glucose cotransporter type 2 (SGLT-2) and glucose transporter 2 (GLUT2) carry out more than 90% of renal glucose uptake. In diabetes, both gluconeogenesis and renal glucose reabsorption are increased. The augmentation of glucose uptake in diabetes is due to the overexpression of renal glucose transporters SGLT-2 and GLUT2 in response to the increase in expression of transcription activator hepatic nuclear factor 1-alpha (HNF1α). The rise in glucose uptake contributes to hyperglycaemia and induces glomerular hyperfiltration by increasing sodium and water reabsorption in the proximal tubule that, in turn, modifies urine flux at the macula densa. SGLT-2 inhibitors improve glycaemic control and prevent renal hyperfiltration in diabetes. Loss of SGLT-2 transporter function is a benign state characterized by glycosuria. In contrast, mutations of other glucose transporters expressed in the kidney are responsible for severe disorders.

Inhibidores SGLT-2: de la corteza del manzano y la glucosuria familiar al tratamiento de la diabetes mellitus tipo 2 / Sodium-glucose co-transporter-2 inhibitors: from the bark of apple trees and familial renal glycosuria to the treatment of type 2 diabetes mellitus

El arsenal terapéutico para el tratamiento de la hiperglucemia en la diabetes mellitus tipo 2 aún es insuficiente. Actualmente asistimos a la introducción de una nueva vía en el tratamiento hipoglucemiante a través de la inducción de glucosuria para disminuir la disponibilidad del sustrato metabólico, esto es, la glucosa. Los inhibidores del cotransportador de sodio/glucosa tipo 2 han mostrado en los ensayos clínicos una eficacia comparable a otros fármacos hipoglucemiantes orales. En este artículo se discuten los aspectos básicos generales de este nuevo concepto de tratamiento, y de la eficacia y seguridad de este nuevo grupo terapéutico (AU)

The therapeutic armamentarium for the treatment of hyperglycemia in type 2 diabetes mellitus is still inadequate. We are currently witnessing the introduction of a new mode of hypoglycemic treatment through induction of glycosuria to decrease the availability of the metabolic substrate, i.e. glucose. Clinical trials have shown that sodium-glucose co-transporter-2 (SGLT2) inhibitors are as efficacious as other oral hypoglycemic drugs. This article discusses the basic features of this new treatment concept and the efficacy and safety of this new drug group (AU)

Hydrogen sulfide deficiency and diabetic renal remodeling: role of matrix metalloproteinase-9.

Matrix metalloproteinase-9 (MMP-9) causes adverse remodeling, whereas hydrogen sulfide (H2S) rescues organs in vascular diseases. The involvement of MMP-9 and H2S in diabetic renovascular remodeling is, however, not well characterized. We determined whether MMP-9 regulates H2S generation and whether H2S modulates connexin through N-methyl-d-aspartate receptor (NMDA-R)-mediated pathway in the diabetic kidney. Wild-type (WT, C57BL/6J), diabetic (Akita, C57BL/6J-Ins2(Akita)), MMP-9(-/-) (M9KO), double knockout (DKO) of Akita/MMP-9(-/-) mice and in vitro cell culture were used in our study. Hyperglycemic Akita mice exhibited increased level of MMP-9 and decreased production of H2S. H2S-synthesizing enzymes cystathionine-ß-synthase and cystathionine-γ-lyase were also diminished. In addition, increased expressions of NMDA-R1 and connexin-40 and -43 were observed in diabetic kidney. As expected, MMP-9 mRNA was not detected in M9KO kidneys. However, very thin protein expression and activity were detected. No other changes were noticed in M9KO kidney. In DKO mice, all the above molecules showed a trend toward baseline despite hyperglycemia. In vitro, glomerular endothelial cells treated with high glucose showed induction of MMP-9, attenuated H2S production, NMDA-R1 induction, and dysregulated conexin-40 and -43 expressions. Silencing MMP-9 by siRNA or inhibition of NMDA-R1 by MK801 or H2S treatment preserved connexin-40 and -43. We conclude that in diabetic renovascular remodeling MMP-9 plays a major role and that H2S has therapeutic potential to prevent adverse diabetic renal remodeling.

The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter.

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition.

[Sodium-glucose co-transporter-2 inhibitors: from the bark of apple trees and familial renal glycosuria to the treatment of type 2 diabetes mellitus]. / Inhibidores SGLT-2: de la corteza del manzano y la glucosuria familiar al tratamiento de la diabetes mellitus tipo 2.

The therapeutic armamentarium for the treatment of hyperglycemia in type 2 diabetes mellitus is still inadequate. We are currently witnessing the introduction of a new mode of hypoglycemic treatment through induction of glycosuria to decrease the availability of the metabolic substrate, i.e. glucose. Clinical trials have shown that sodium-glucose co-transporter-2 (SGLT2) inhibitors are as efficacious as other oral hypoglycemic drugs. This article discusses the basic features of this new treatment concept and the efficacy and safety of this new drug group.

Familial renal glucosuria: a clinicogenetic study of 23 additional cases.

BACKGROUND: Familial renal glucosuria (FRG) is an inherited renal tubular disorder characterized by persistent isolated glucosuria in the absence of hyperglycemia that is caused by mutations in the sodium-glucose cotransporter SGLT2 coding gene, SLC5A2. OBJECTIVE: We conducted molecular and phenotype analyses of a cohort of 23 unrelated Korean children with FRG. METHODS: Mutational analysis of the SLC5A2 gene was conducted in this multicenter study organized by the Korean Society of Pediatric Nephrology. RESULTS: A total of 21 different SLC5A2 mutations were detected, including 19 novel mutations. All patients had at least one mutated allele; ten patients had homozygous or compound heterozygous mutations and 13 patients had a single heterozygous mutation. Most mutations were private. Patients with two mutations were diagnosed earlier with larger amounts of urinary glucose excretion than patients with single mutations. Pedigree analysis data were consistent with the inheritance of a codominant trait with incomplete penetrance. CONCLUSIONS: These findings extend the allelic heterogeneity in FRG and confirm previous observations of inheritance and genotype­phenotype correlation in patients with this disease.

[Sodium-glucose cotransporter type 2 inhibitors (SGLT2): from familial renal glucosuria to the treatment of type 2 diabetes mellitus]. / Inhibidores del cotransportador sodio-glucosa tipo 2 (SGLT2): de la glucosuria renal familiar al tratamiento de la diabetes mellitus tipo 2.

For centuries, the kidney has been considered primarily an organ of elimination and a regulator of salt and ion balance. Although once thought that the kidney was the structural cause of diabetes, which in recent years has been ignored as a regulator of glucose homeostasis, is now recognized as a major player in the field of metabolic regulation carbohydrate. During fasting, 55% of the glucose comes from gluconeogenesis. Only 2 organs have this capability: the liver and kidney. The latter is responsible for 20% of total glucose production and 40% of that produced by gluconeogenesis. Today we have a better understanding of the physiology of renal glucose transport via specific transporters, such as type 2 sodium-glucose cotransporter  (SGLT2). A natural compound, phlorizin, was isolated in early 1800 and for decades played an important role in diabetes and renal physiology research. Finally, at the nexus of these findings mentioned above, recognized the effect of phlorizin-like compounds in the renal glucose transporter, which has offered a new mechanism to treat hyperglycemia. This has led to the development of several potentially effective treatment modalities for the treatment of diabetes.

Heterogeneous stock rats: a new model to study the genetics of renal phenotypes.

Chronic kidney disease is a growing medical concern, with an estimated 25.6 million people in the United States exhibiting some degree of kidney injury and/or decline in kidney function. Animal models provide great insight into the study of the genetics of complex diseases. In particular, heterogeneous stock (HS) rats represent a unique genetic resource enabling rapid fine-mapping of complex traits. However, they have not been explored as a model to study renal phenotypes. To evaluate the usefulness of HS rats in the genetics of renal traits, a time course evaluation (weeks 8-40) was performed for several renal phenotypes. As expected, a large degree of variation was seen for most renal traits. By week 24, three (of 40) rats exhibited marked proteinuria that increased gradually until week 40 and ranged from 33.7 to 80.2 mg/24 h. Detailed histological analysis confirmed renal damage in these rats. In addition, several rats consistently exhibited significant hematuria (5/41). Interestingly, these rats were not the same rats that exhibited proteinuria, indicating that susceptibility to different types of kidney injury is likely segregating within the HS population. One HS rat exhibited unilateral renal agenesis (URA), which was accompanied by a significant degree of proteinuria and glomerular and tubulointerstitial injury. The parents of this HS rat were identified and bred further. Additional offspring of this pair were observed to exhibit URA at frequency between 40% and 60%. In summary, these novel data demonstrate that HS rats exhibit variation in proteinuria and other kidney-related traits, confirming that the model harbors susceptibility alleles for kidney injury and providing the basis for further genetic studies.

Familial renal glucosuria: SLC5A2 mutation analysis and evidence of salt-wasting.

Familial renal glucosuria (FRG) is an inherited renal tubular disorder characterized by persistent isolated glucosuria in the absence of hyperglycemia. Mutations in the sodium/glucose co-transporter SGLT2 coding gene, SLC5A2, were recently found to be responsible for the disorder. Here, we report the molecular and phenotype study of five unrelated FRG families. Five patients were identified and their family members screened for glucosuria. SLC5A2 coding region of index cases was polymerase chain reaction amplified and sequenced. Five different mutations are reported, including four novel alleles. The IVS12+1G>A and p.A102V alleles were identified in homozygosity in index patients of two unrelated families. A proband from another family was compound heterozygous for the p.R132H and p.A219T mutations, and the heterozygous p.Q167fsX186 frameshift allele was the only mutation detected in the affected individual from an additional pedigree. For the remaining family no mutations were detected. The patient homozygous for the p.A102V mutation had glucosuria of 65.6 g/1.73 m(2)/24 h, evidence of renal sodium wasting, mild volume depletion, and raised basal plasma renin and serum aldosterone levels. Our findings confirm previous observations that in FRG, transmitted as a codominant trait with incomplete penetrance, most mutations are private. In the only patient with massive glucosuria in our cohort there was evidence evocative of renin-angiotensin aldosterone system activation by extracellular volume depletion induced by natriuresis. Definite proof of renin-angiotensin aldosterone system activation in FGR should rely on evaluation of additional patients with massive glucosuria.

Renal glycosuria treated as diabetes mellitus: case report.

A case of renal glycosuria is reported. A 55 year old female was diagnosed and treated in an upcountry hospital for diabetes mellitus. She developed symptoms of hypoglycaemia while on an oral hypoglycaemic agent, leading to her admission in Mulago Hospital. Persistent glycosuria was noted despite treatment and normal serum glucose. Oral glucose tolerance test and timed urine glucose showed a normal curve but high urine sugar. A diagnosis of renal glycosuria was made, oral hypoglycaemic therapy was stopped, patient improved and was discharged. Though renal glycosuria is a benign condition, mistaken diagnosis for diabetes mellitus puts patients at risk of hypoglycaemia due to treatment. Diagnosis of the condition requires physicians' awareness of its existence in our community and the use of Marbles' criteria obviates confusion with diabetes mellitus though it does not absolutely exclude Fanconi syndrome.

Hypercalciuria in children with renal glycosuria: evidence of dual renal tubular reabsorptive defects.

During the past 5 years, we have identified idiopathic hypercalciuria in five of seven patients referred for evaluation of renal glycosuria between 1985 and 1991. The children, all boys, ranged in age from 6 to 12 years. Endocrine function was normal, and none of the patients had hyperparathyroidism, hypercalcemia, renal tubular acidosis, or other secondary causes of hypercalciuria. The calcium/creatinine ratio in a fasting urine specimen was elevated in all five children who had hypercalciuria, with a mean value (+/- SD) of 0.34 +/- 0.06 (normal, < 0.2). In one child who had renal colic with spontaneous passage of gravel-like material, the idiopathic hypercalciuria persisted after 1 week on a diet containing 2000 mg of sodium and 300 mg of calcium. On the basis of studies that examined the site along the nephron responsible for hypercalciuria in rats with streptozocin-induced diabetes, we speculate that in children with renal glycosuria, there is defective reabsorption of glucose and calcium in the straight portion of the proximal tubule or in the collecting duct. It is likely that a similar mechanism accounts for the idiopathic hypercalciuria in children with diabetes mellitus.

[ADH and cAMP receptor binding in the kidney medulla of mice insensitive to ADH]. / Retseptsiia ADG i tsAMF v medulliarnoi chasti pochki myshei, nechuvstvitel'nykh k ADG.

Specific binding of ADH by the membrane fraction of the kidney medulla was lower in the normal CBA mice than in mutant mice with nephrogenic diabetes. Gel filtration of the solubilized ADH receptors of mutants revealed the presence of an unidentified factor which caused cooperative binding of ADH. DEAE-chromatography revealed no difference between cytosolic cAMP receptors in normal and mutant animals. Assay of GTP-ase activity of the membrane fraction revealed that ADH increased this parameter in CBA mice but not in mutant animals. Cholera toxin significantly diminished membrane ATP-ase activity whereas membrane preparations from mutant mice developed a reactivity to ADH. GTP binding ability in these preparations was higher than inn intact ones. In CBA mice this ability increased dramatically. HPLC profiles of G-protein complexes with GNP were very different in CBA and mutant mice. Mutation seems to cause changes both in binding and in "cross-talk" link op-complex membrane receptor of ADH.

Complete absence of tubular glucose reabsorption: a new type of renal glucosuria (type 0).

Primary renal glucosuria is an inherited defect of tubular glucose reabsorption and usually classified in type A and type B. We now observed a new type in a 15-year-old boy who had a complete absence of tubular glucose reabsorption. His father had a daily glucosuria of 1.1 g/1.73 m2 and his mother of 2.7 g/1.73 m2. Two siblings excreted 0.4 g/1.73 m2 and 0.3 g/1.73 m2 glucose and one sister had no glucosuria. The proband excreted daily 136 to 160 g/1.73 m2 glucose accompanied by normal blood glucose levels between 75-105 mg/dl. The glomerular filtration rate (inulin clearance) was 148-153 ml/min/1.73 m2 and the endogenous glucose clearance was 112-160 ml/min/1.73 m2 when blood glucose levels were 72-82 mg/dl. Thus, glucose clearance was nearly identical to inulin-clearance. After intravenous glucose loading with a blood glucose concentration of 261-342 mg/dl, glucose clearance remained in the same range and tubular glucose reabsorption was virtually absent. There were no disturbances in tubular reabsorption of other substrates. This new type of primary renal glucosuria was not recognized thus far, and we propose to call it type O glucosuria. The family tree revealed consanguinity and most probably the proband is homozygous and both his parents are heterozygous for type O renal glucosuria.