Metachromatic leukodystrophy among the Navajo Indians: A study of the arylsulfatase A gene mutations present in this population

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder of myelin metabolism, resulting from the inability to properly degrade 3-sulfo-galactosylceramide (sulfatide). This metabolic block is often due to defective functioning of the lysosomal enzyme arylsulfatase A (ARSA). Unmetabolized sulfatide accumulates in the white matter of the central nervous system (CNS) and in the peripheral nerves, leading to progressive demyelination and death. Late infantile, juvenile and adult clinical variants of MLD have been described. A Navajo Indian child was diagnosed with late infantile MLD (LIMLD), and his ARSA gene was amplified by the polymerase chain reaction (PCR) and sequenced. A single mutation was found: a G $\to$ A transition at the first nucleotide of intron 4 (IVS4nt1) which abolishes the 5\sp\prime splice site consensus sequence. This mutation had never previously been reported. Negligible amounts of ARSA mRNA were observed in Northern blots. However, PCR amplification and sequencing of the complementary DNA (cDNA) showed that all ARSA transcripts from the patient have exon 4 deleted, resulting in a premature stop codon within exon 5. In addition, splicing intermediates and other aberrantly spliced mRNA species were detected. Both parents carry this mutation and the father also carries the common pseudodeficiency (PD) allele. Three additional unrelated Navajo LIMLD patients were found to be homozygous for the IVS4nt1 mutation by allele specific oligonucleotide (ASO) hybridization and/or cycle sequencing. Family members of affected Navajo children were found to be carriers of this mutation, and the pseudodeficient mother of an affected child was also a carrier of the common PD allele. The source of genomic DNA for PCR amplification only needed to be a drop of blood dried onto filter paper. The IVS4nt1 allele appeared to be unique to the Navajo until an Eskimo child with MLD was found to be homozygous for this mutation. Her parents and three unaffected siblings carried this mutation while a fourth asymptomatic sibling was also homozygous. The healthy mother had low ARSA activity but was not a carrier of the common PD allele. Rapid DNA-based diagnostic methods could be used for carrier and patient identification in these populations

Effect of aldosterone on BK channel expression in mammalian cortical collecting duct

Apical large-conductance Ca2+-activated K+ (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K+ secretion. Dietary K+ loading for 10–14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K+ secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922–F932, 2005). To test whether this adaptation was mediated by a K+-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na+ (LS) or high-Na+ (HS) diet for 7–10 days to alter circulating levels of aldosterone but not serum K+ concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, α-splice variants, β-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na+ (JNa) and K+ (JK) transport by microperfusion; kidneys were processed for immunolocalization of BK α-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na+ and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK α-, β2-, and β4-subunit mRNA and localization of immunodetectable α-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ∼1 to 5 nl·min−1·mm−1, the increase in JNa was greater in LS vs. HS rabbits, yet the flow-stimulated increase in JK was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K+ loading

Lack of Effects of Metformin and AICAR Chronic Infusion on the Development of Hypertension in Dahl Salt-Sensitive Rats

In the kidney, reabsorption via the epithelial sodium channel (ENaC) is involved in long-term blood pressure control. Previously we demonstrated that ENaC hyperactivity is associated with development of salt-sensitive (SS) hypertension in Dahl SS rats. AMP-activated kinase (AMPK), playing a role in cellular energy homeostasis, has been shown to decrease ENaC activity. Here, we tested whether metformin and AICAR, two drugs that activate AMPK, affect the development of salt-induced hypertension. High salt diet significantly increased mean arterial pressure (MAP) in Dahl SS rats. Blood pressure elevation was accompanied by a short-term decline of heart rate and increased circadian arterial pressure dipping. Metformin and AICAR were delivered intravenously at doses of 200 and 20 mg/kg/day, respectively. However, both control and drug-treated groups had similar development of high blood pressure within 3 weeks of 8% NaCl dietary salt intake. In the metformin-treated animals MAP reached 164.9 ± 9.1 mmHg, which was not significantly different from the control group (171.8 ± 5.6 mmHg). Patch clamp analysis revealed that the metformin-treated rats had no difference in the activity of ENaC. AICAR treatment also did not affect the development of hypertension and kidney injury. MAP reached 182.8 ± 4.8 and 178.0 ± 2.8 mmHg in AICAR and vehicle treated groups, respectively. Of note, we found that high-salt diet activated AMPK in the Dahl SS rats, and treatment with these AMPK activators had no significant further effect on AMPK activity. We conclude that AMPK activators, at least under these conditions, do not affect development of hypertension during high-salt diet in the Dahl SS rat model

Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCD(c14) cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK

Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors

Here, the authors model the collecting duct system in kidneys by taking ureteric bud (UB) progenitor cells from both mouse and human primary tissues, as well as from hESC and hiPSC to generate organoids, which can model congenital anomalies of the kidney and urinary tract