Renal proximal tubular reabsorption is reduced in adult spontaneously hypertensive rats: roles of superoxide and NHE3

Proximal tubule reabsorption is regulated by systemic and intrinsic mechanisms, including locally produced autacoids. Superoxide (O2−), produced by NADPH oxidase (NOX) enhances NaCl transport in the loop of Henle and the collecting duct, but its role in the PT is unclear. We measured PT fluid reabsorption (Jv) in WKY rats and compared that to Jv in SHR, a model of enhanced renal O2− generation. Rats were treated with the NOX inhibitor apocynin (Apo), or with small interfering RNA (siRNA) for p22phox, which is the critical subunit of NOX. Jv was lower in SHR compared to WKY (WKY: 2.4±0.3 vs SHR: 1.1±0.2 nl/min/mm, n=9–11, p<0.001). Apo and siRNA to p22phox normalized Jv in SHR yet had no effect in WKY. Jv was reduced in proximal tubules perfused with S-1611, a highly selective inhibitor of the Na+/H+ exchanger 3 (NHE3), the major Na+ uptake pathway in the proximal tubule, in WKY but not in SHR. Pretreatment with Apo restored an effect of S-1611 to reduce Jv in the SHR (SHR+Apo: 2.9±0.4 vs SHR+Apo+S-1611: 1.0±0.3 nl/min/mm, p<0.001). However, since expression of NHE3 was similar between SHR and WKY, this suggests that O2− affects NHE3 activity. Direct microperfusion of tempol or apocynin into the PT also restored Jv in SHR. In conclusion, O2− generated by NOX, inhibits proximal tubule fluid reabsorption in SHR. This finding implies that PT fluid reabsorption is regulated by redox balance, which may have profound effects on ion and fluid homeostasis in the hypertensive kidney. Keywords: Proximal reabsorption, superoxide, tempol, apocyni

Low salt intake increases adenosine type 1 receptor expression and function in the rat proximal tubule

Adenosine mediates Na+ reabsorption in the proximal tubule (PT) and other segments by activating adenosine type 1 receptors (A1-AR). We tested the hypothesis that A1-AR in the PT is regulated by salt intake and participates in the kidney adaptation to changes in salt intake. Absolute fluid reabsorption (Jv) was measured by direct in vivo microperfusion and recollection in rats maintained on low (LS; 0.03% Na, wt/wt)-, normal (NS; 0.3% Na)-, and high-salt (HS; 3.0% Na) diets for 1 wk. The effect of microperfusion of BG9719 a highly selective inhibitor of A1-ARs or adenosine deaminase (AD), which metabolizes adenosine, was measured in each group. Jv was higher in PT from LS rats (LA: 2.8 ± 0.2 vs. NS: 2.1 ± 0.2 nl·min−1·mm−1, P < 0.001). Jv in HS rats was not different from NS. BG9719 reduced Jv in LS rats by 66 ± 6% (LS: 2.8 ± 0.2 vs LS+CVT: 1.3 ± 0.3 nl·min−1·mm−1, P < 0.001), which was greater than its effect in NS (45 ± 4%) or HS (41 ± 4%) rats. AD reduced Jv similarly, suggesting that A1-ARs are activated by local production of adenosine. Expression of A1-AR mRNA and protein was higher (P < 0.01) in microdissected PTs in LS rats compared with NS and HS. We conclude that A1-ARs in the PT are increased by low salt intake and that A1-AR participates in the increased PT reabsorption of solute and fluid in response to low salt intake

Identification of DCAF1 by Clinical Exome Sequencing and Methylation Analysis as a Candidate Gene for Autism and Intellectual Disability: A Case Report

Autism spectrum disorder (ASD) comprises a heterogeneous group of neurodevelopmental disorders and occurs in all racial, ethnic, and socioeconomic groups. Cutting-edge technologies are contributing to understanding genetic underpinnings in ASD. The reported patient is a 32-year-old male and as an infant was noted to have microcephaly, hypospadias, pulmonary vascular anomaly, and small stature. He was diagnosed with Cornelia De Lange Syndrome (CDLS) at that time based on the clinical features. As a child, he had autistic features and intellectual disabilities and as diagnoses with autism and intellectual disability. He was referred as an adult to our neurodiversity clinic and a full exome trio sequencing with reflex to mitochondrial genes identified a de novo variant of uncertain significance in a candidate gene, DCAF1. The specific variant was c.137 C > T (p.Thr46Ile) in exon 4 in the DCAF1 gene. In silico analysis supports a deleterious effect on protein structure/function. DCAF1 participates with DDB1 and CUL4 as a part of the E3 ubiquitin ligase complex. The E3 ligase complex has been associated with a syndromic form of X-linked intellectual disability. The DDB1/CUL4 E3 ubiquitination complex plays a role in methylation-dependent ubiquitination. Next, a methylation study identified a signature similar to the methylation pattern found in X- linked intellectual disability type 93. This is associated with variants of the BRWD3 gene, which is linked with the functioning of the DDB1/CUL4 E3 ubiquitination complex. Taken together, this suggests that the de novo DCAF1 variant may be a newly identified molecular cause of autism and intellectual disability

Molecular Dysregulation in Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) comprises a heterogeneous group of neurodevelopmental disorders with a strong heritable genetic component. At present, ASD is diagnosed solely by behavioral criteria. Advances in genomic analysis have contributed to numerous candidate genes for the risk of ASD, where rare mutations and s common variants contribute to its susceptibility. Moreover, studies show rare de novo variants, copy number variation and single nucleotide polymorphisms (SNPs) also impact neurodevelopment signaling. Exploration of rare and common variants involved in common dysregulated pathways can provide new diagnostic and therapeutic strategies for ASD. Contributions of current innovative molecular strategies to understand etiology of ASD will be explored which are focused on whole exome sequencing (WES), whole genome sequencing (WGS), microRNA, long non-coding RNAs and CRISPR/Cas9 models. Some promising areas of pharmacogenomic and endophenotype directed therapies as novel personalized treatment and prevention will be discussed

MicroRNA Expression Profiles in Autism Spectrum Disorder: Role for miR-181 in Immunomodulation

Background: MicroRNAs (miRNAs) are important regulators of molecular pathways in psychiatric disease. Here, we examine differential miRNAs expression in lymphoblastoid cell lines (LCLs) derived from 10 individuals with autism spectrum disorder (ASD) and compare them to seven typically developing unrelated age- and gender-matched controls and 10 typically developing siblings. Small RNAseq analysis identified miRNAs, and selected miRNAs were validated using quantitative real-time polymerase reaction (qRT-PCR). KEGG analysis identified target pathways, and selected predicted mRNAs were validated using qRT-PCR. Results: Small RNAseq analysis identified that multiple miRNAs differentiated ASD from unrelated controls and ASD from typically developing siblings, with only one, hsa-miR-451a_R-1, being in common. Verification with qRT-PCR showed that miR-320a differentiated ASD from both sibling and unrelated controls and that several members of the miR-181 family differentiated ASD from unrelated controls. Differential expression of AKT2, AKT3, TNF α and CamKinase II predicted by KEGG analysis was verified by qRT-PCR. Expression of CamKinase II βwas found to be correlated with the severity of stereotyped behavior of the ASD participants. Conclusions: This study provides insight into the mechanisms regulating molecular pathways in individuals with ASD and identifies differentiated regulated genes involved in both the central nervous system and the immune system