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Impact of Angiotensin II-mediated stimulation of sodium transporters in the nephron assessed by computational modelling.

Angiotensin II (AngII) raises blood pressure (BP) partly by stimulating tubular Na+ reabsorption. The effects of AngII on tubular Na+ transporters (i.e., channels, pumps, cotransporters and exchangers) vary between short-term and long-term exposure. To better understand the physiological impact, we used a computational model of transport along the rat nephron to predict the effects of short- and long-term AngII-induced transporter activation on Na+ and K+ reabsorption/secretion, and to compare measured and calculated excretion rates. Three days AngII infusion at 200 ng/kg/min is non-pressor, yet stimulates transporter accumulation. The increase in abundance of Na+/H+ exchanger 3 (NHE3) or activated Na+-K+-2Cl- cotransporter 2 (NKCC2-P) predicted significant reductions in urinary Na+ excretion, yet there was no observed change in urine Na+. The lack of anti-natriuresis, despite Na+ transporter accumulation, was supported by Li+ and creatinine clearance measurements, leading to the conclusion that 3-day non-pressor AngII increases transporter abundance without proportional activation. Fourteen days AngII infusion at 400 ng/kg/min raises BP and increases Na+ transporter abundance along the distal nephron; proximal tubule and medullary loop transporters are decreased and urine Na+ and volume output are increased, evidence for pressure-natriuresis. Simulations indicate that decreases in NHE3 and NKCC2-P contribute significantly to reducing Na+ reabsorption along the nephron and to pressure-natriuresis. Our results also suggest that differential regulation of medullary (decrease) and cortical (increase) NKCC2-P is important to preserve K+ while minimizing Na+ retention during AngII infusion. Lastly, our model indicates that accumulation of active Na+-Cl- cotransporter (NCC-P) counteracts epithelial Na+ channel (ENaC)-induced urinary K+ loss.

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The single-cell transcriptomic landscape of early human diabetic nephropathy.

Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single-nucleus RNA sequencing (snRNA-seq) on cryopreserved human diabetic kidney samples to generate 23,980 single-nucleus transcriptomes from 3 control and 3 early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side-by-side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic thick ascending limb, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na+/K+-ATPase, WNK1, mineralocorticoid receptor, and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule, and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.

Copyright © 2019 the Author(s). Published by PNAS.

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Inhibition of endocytosis of clathrin-mediated angiotensin II receptor type 1 in podocytes augments glomerular injury.

Background: Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, β-arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis.

Method: We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII.

Results: We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR.

Conclusions: Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

Copyright © 2019 by the American Society of Nephrology.


endocytosis; podocyte; proteinuria

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Patient-customized oligonucleotide therapy for a rare genetic disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

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Search-and-replace genome editing without double-strand breaks or donor DNA.

Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutation without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct about 89% of known pathogenic human genetic variants.

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