Wanda E. Filipiak

Podocyte Depletion Causes Glomerulosclerosis: Diphtheria Toxin–Induced Podocyte Depletion in Rats Expressing Human Diphtheria Toxin Receptor Transgene

Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.

Advances in transgenic rat production

Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models.

Targeted disruption of Adamts16 gene in a rat genetic model of hypertension

A disintegrin-like metalloproteinase with thrombospondin motifs–16 (Adamts16) is an important candidate gene for hypertension. The goal of the present study was to further assess the candidacy of Adamts16 by targeted disruption of this gene in a rat genetic model of hypertension. A rat model was generated by manipulating the genome of the Dahl Salt–sensitive (S) rat using zinc-finger nucleases, wherein the mutant rat had a 17 bp deletion in the first exon of Adamts16, introducing a stop codon in the transcript. Systolic blood pressure (BP) of the homozygous Adamts16mutant rats was lower by 36 mmHg compared with the BP of the S rats. The Adamts16mutant rats exhibited significantly lower aortic pulse wave velocity and vascular media thickness compared with S rats. Scanning electron and fluorescence microscopic studies indicated that the mechanosensory cilia of vascular endothelial cells from the Adamts16mutant rats were longer than that of the S rats. Furthermore, Adamts16mutant rats showed splitting and thickening of glomerular capillaries and had a longer survival rate, compared with the S rats. Taken together, these physiological observations functionally link Adamts16 to BP regulation and suggest the vasculature as the potential site of action of Adamts16 to lower BP.

Podocyte-specific overexpression of GLUT1 surprisingly reduces mesangial matrix expansion in diabetic nephropathy in mice.

Increased expression of the facilitative glucose transporter, GLUT1, leads to glomerulopathy that resembles diabetic nephropathy, whereas prevention of enhanced GLUT1 expression retards nephropathy. While many of the GLUT1-mediated effects are likely due to mesangial cell effects, we hypothesized that increased GLUT1 expression in podocytes also contributes to the progression of diabetic nephropathy. Therefore, we generated two podocyte-specific GLUT1 transgenic mouse lines (driven by a podocin promoter) on a db/m C57BLKS background. Progeny of the two founders were used to generate diabetic db/db and control db/m littermate mice. Immunoblots of glomerular lysates showed that transgenic mice had a 3.5-fold (line 1) and 2.1-fold (line 2) increase in GLUT1 content compared with wild-type mice. Both lines showed similar increases in fasting blood glucose and body weights at 24 wk of age compared with wild-type mice. Mesangial index (percent PAS-positive material in the mesangial tuft) increased 88% (line 1) and 75% (line 2) in the wild-type diabetic mice but only 48% (line 1) and 39% (line 2) in the diabetic transgenic mice (P < 0.05, transgenic vs. wild-type mice). This reduction in mesangial expansion was accompanied by a reduction in fibronectin accumulation, and vascular endothelial growth factor (VEGF) levels increased only half as much in the transgenic diabetic mice as in wild-type diabetic mice. Levels of nephrin, neph1, CD2AP, podocin, and GLUT4 were not significantly different in transgenic compared with wild-type mice. Taken together, increased podocyte GLUT1 expression in diabetic mice does not contribute to early diabetic nephropathy; surprisingly, it protects against mesangial expansion and fibronectin accumulation possibly by blunting podocyte VEGF increases.

Knock-in rats expressing Cre and Flp recombinases at the Parvalbumin locus.

Rats have the ability to learn and perform sophisticated behavioral tasks, making them very useful for investigating neural circuit functions. In contrast to the extensive mouse genetic toolkit, the paucity of recombinase-expressing rat models has limited the ability to monitor and manipulate molecularly-defined neural populations in this species. Here we report the generation and validation of two knock-in rat strains expressing either Cre or Flp recombinase under the control of Parvalbumin (Pvalb), a gene expressed in the critical “fast-spiking” subset of inhibitory interneurons (FSIs). These strains were generated with CRISPR-Cas9 gene editing and show highly specific and penetrant labeling of Pvalb-expressing neurons, as demonstrated by in situ hybridization and immunohistochemistry. We validated these models in both prefrontal cortex and striatum using both ex vivo and in vivo approaches, including whole-cell recording, optogenetics, extracellular physiology and photometry. Our results demonstrate the utility of these new transgenic models for a wide range of neuroscience experiments.

Knock-in rat lines with Cre recombinase at the dopamine D1 and adenosine 2a receptor loci.

Genetically-modified mice have become standard tools in neuroscience research. Our understanding of the basal ganglia in particular has been greatly assisted by BAC mutants with selective transgene expression in striatal neurons forming the direct or indirect pathways. However, for more sophisticated behavioral tasks and larger intracranial implants, rat models are preferred. Furthermore, BAC lines can show variable expression patterns depending upon genomic insertion site. We therefore used CRISPR/Cas9 to generate two novel knock-in rat lines specifically encoding Cre recombinase immediately after the dopamine D1 receptor (Drd1a) or adenosine 2a receptor (Adora2a) loci. Here we validate these lines using in situ hybridization and viral vector mediated transfection to demonstrate selective, functional Cre expression in the striatal direct and indirect pathways respectively. We used whole-genome sequencing to confirm the lack of off-target effects, and established that both rat lines have normal locomotor activity and learning in simple instrumental and Pavlovian tasks. We expect these new D1-Cre and A2a-Cre rat lines will be widely used to study both normal brain functions and neurological and psychiatric pathophysiology.

The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension

Measures of the adipokine chemerin are elevated in multiple cardiovascular diseases, including hypertension, but little mechanistic work has been done to implicate chemerin as being causative in such diseases. The chemerin knockout (KO) rat was created to test the hypothesis that removal of chemerin would reduce pressure in the normal and hypertensive state. Western analyses confirmed loss of chemerin in the plasma and tissues of the KO vs. wild‐type (WT) rats. Chemerin concentration in plasma and tissues was lower in WT females than in WT males, as determined by Western analysis. Conscious male and female KO rats had modest differences in baseline measures vs. the WT that included systolic, diastolic, mean arterial and pulse pressures, and heart rate, all measured telemetrically. The mineralocorticoid deoxycorticosterone acetate (DOCA) and salt water, combined with uninephrectomy as a hypertensive stimulus, elevated mean and systolic blood pressures of the male KO higher than the male WT. By contrast, all pressures in the female KO were lower than their WT throughout DOCA‐salt treatment. These results revealed an unexpected sex difference in chemerin expression and the ability of chemerin to modify blood pressure in response to a hypertensive challenge.—Watts, S. W., Darios, E. S., Mullick, A. E., Garver, H., Saunders, T. L., Hughes, E. D., Filipiak, W. E., Zeidler, M. G., McMullen, N., Sinal, C. J., Kumar, R. K., Ferland, D. J., Fink, G. D. The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension. FASEB J. 32, 6596–6614 (2018). www.fasebj.org