Ouabain enhances renal cyst growth in a slowly progressive mouse model of autosomal dominant polycystic kidney disease.

Renal cyst progression in autosomal dominant polycystic kidney disease (ADPKD) is highly dependent on agents circulating in blood. We have previously shown, using different in vitro models, that one of these agents is the hormone ouabain. By binding to Na+-K+-ATPase (NKA), ouabain triggers a cascade of signal transduction events that enhance ADPKD cyst progression by stimulating cell proliferation, fluid secretion, and dedifferentiation of the renal tubular epithelial cells. Here, we determined the effects of ouabain in vivo. We show that daily administration of ouabain to Pkd1RC/RC ADPKD mice for 1-5 mo, at physiological levels, augmented kidney cyst area and number compared with saline-injected controls. Also, ouabain favored renal fibrosis; however, renal function was not significantly altered as determined by blood urea nitrogen levels. Ouabain did not have a sex preferential effect, with male and female mice being affected equally. By contrast, ouabain had no significant effect on wild-type mice. In addition, the actions of ouabain on Pkd1RC/RC mice were exacerbated when another mutation that increased the affinity of NKA for ouabain was introduced to the mice (Pkd1RC/RCNKAα1OS/OS mice). Altogether, this work highlights the role of ouabain as a procystogenic factor in the development of ADPKD in vivo, that the ouabain affinity site on NKA is critical for this effect, and that circulating ouabain is an epigenetic factor that worsens the ADPKD phenotype.NEW & NOTEWORTHY This work shows that the hormone ouabain enhances the progression of autosomal dominant polycystic kidney disease (ADPKD) in vivo. Ouabain augments the size and number of renal cysts, the kidney weight to body weight ratio, and kidney fibrosis in an ADPKD mouse model. The Na+-K+-ATPase affinity for ouabain plays a critical role in these effects. In addition, these outcomes are independent of the sex of the mice.

Generating single-stranded DNA circles with minimal resources.

Single-stranded circular oligonucleotides are heavily utilized in rolling circle amplification and rolling circle transcription technologies. Although various reported methodologies are available to synthesize circular, single-stranded DNA (ssDNA), the unduly complicated protocols and the associated cost minimize the utility of these methodologies to a non-expert or a beginner in the field. Our protocol provides the simplest yet robust synthesis of circular ssDNA templates to be utilized in various applications, using minimal resources.•In this manuscript, we describe the most basic approach to synthesize circular ssDNA.•Our method utilizes the minimal resources, yet it is robust.•The utility of the methodology is very high for a non-expert or a beginner in the field.