Biology subjects

Harris, P. C.

Publications and source records attributed to Harris, P. C..

3 recordsLinked to original sources

bioRxiv2025

Induced Proteinuria Enhances Adeno-Associated Virus Transduction of Renal Tubule Epithelial Cells After Intravenous Administration

A variety of genetic diseases of the kidney tubule are amenable to correction via gene therapy. However, gene delivery to renal tubule epithelial cells mediated by viral vectors via the blood is inefficient due to the permselectivity of the glomerular barrier. We hypothesized that effacement of podocyte foot processes would disrupt typical glomerular limitations on filtration and make renal tubule epithelial cells susceptible to transduction from viral vectors delivered intravenously. We determined that adeno-associated virus serotype 8 (AAV8) transduced significantly more epithelial cells in the kidney under the conditions of LPS-induced proteinuria. Use of AAV1 in tandem with LPS-induced proteinuria yielded an ideal two-pronged effect of both partially detargeting the liver and transducing the kidney with a higher bioluminescent signal than AAV8 did, and at half of the dose. Using adenovirus serotype 5 (Ad5) in conjunction with LPS-induced proteinuria showed that kidney transduction was enhanced, but only in glomerular cells. These studies mechanistically test the efficacy of different viral vectors and demonstrate their capacity to transduce kidney epithelial cells. This is a fundamental step in designing future treatments for kidney gene therapy. Significance StatementThe slit diaphragms in the glomerulus of the kidney are too narrow to allow most solute from the blood to enter the nephron. As a result, large particles such as viral vectors generally cannot access tubule epithelial cells necessary to correct genetic disorders. In this study, mice are induced into a state of proteinuria and subsequently administered viral vectors intravenously. This group of mice had higher levels of transduced kidney epithelial cells than the control (non-proteinuria) group. The induction of transient proteinuria before intravenous viral vector administration is a novel approach that could make tubulopathies treatable via gene therapy in both animal models and humans.

genetics↗

bioRxiv2024

Activation of PIEZO1 Inhibits Kidney Cystogenesis

The disruption of calcium signaling associated with polycystin deficiency has been proposed as the primary event underlying the increased abnormally patterned epithelial cell growth characteristic of Polycystic Kidney Disease. Calcium can be regulated through mechanotransduction, and the mechanosensitive cation channel Piezo1 has been implicated in sensing of intrarenal pressure and in urinary osmoregulation. However, a possible role for PIEZO1 in kidney cystogenesis remains undefined. We hypothesized that cystogenesis in ADPKD reflects altered mechanotransduction, suggesting activation of mechanosensitive cation channels as a therapeutic strategy for ADPKD. Here, we show that Yoda-1 activation of PIEZO1 increases intracellular Ca2+ and reduces forskolin-induced cAMP levels in mIMCD3 cells. Yoda-1 reduced forskolin-induced IMCD cyst surface area in vitro and in mouse metanephros ex vivo in a dose-dependent manner. Knockout of polycystin-2 dampened the efficacy of PIEZO1 activation in reducing both cAMP levels and cyst surface area in IMCD3 cells. However, collecting duct-specific Piezo1 knockout neither induced cystogenesis in wild-type mice nor affected cystogenesis in the Pkd1RC/RC model of ADPKD. Our study suggests that polycystin-2 and PIEZO1 play a role in mechanotransduction during cystogenesis in vitro, and ex vivo, but that in vivo cyst expansion may require inactivation or repression of additional suppressors of cystogenesis and/or growth. Our study provides a preliminary proof of concept for PIEZO1 activation as a possible component of combination chemotherapy to retard or halt cystogenesis and/or cyst growth.

cell biology↗

bioRxiv2019

mtor Haploinsufficiency Ameliorates Renal Cyst Formation in Adult Zebrafish tmem67 Mutants

Although zebrafish embryos have been utilized to study ciliogenesis and to model polycystic kidney disease (PKD), adult zebrafish remain unexplored. Here, we report the generation and characterization of a zebrafish mutant of tmem67, a homologue of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Although a small population of homozygous embryos exhibited pronephric cysts, all mutants were able to survive to adulthood and developed progressive mesonephric cysts with full penetrance. The cysts in the adult zebrafish kidneys manifested features of mammalian PKD, including switching of cyst origin from the proximal tubules to the collecting ducts, increased proliferation of cyst-lining epithelial cells, and hyperactive mTOR signaling. Consistent ciliary abnormalities were observed in both the embryonic and adult zebrafish mutants compared with the wild-type fish, including shorter and fewer single cilia in the distal pronephros and all segments of the mesonephros and greater numbers of multiciliated cells (MCCs). Lack of single cilium preceded cystogenesis, suggestive of a primary defect. Expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of cystogenesis in young adult zebrafish, suggesting an adaptive action. Interestingly, mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants. In summary, we have established a tmem67 mutant as the first adult zebrafish PKD model, revealed a novel aspect of cilium regulation, and identified sustained mTOR inhibition as a candidate therapeutic strategy for tmem67-based PKD. Significance StatementWhile zebrafish embryos are well recognized for their value in studying ciliogenesis and polycystic kidney disease (PKD), adult zebrafish have not commonly been used. Here, we report the establishment of the first adult zebrafish model for PKD, which exhibits characteristics of mammalian PKD and shows kidney ciliary abnormalities consistent with those observed in an embryonic model. We also provide evidence for mTOR inhibition as a therapeutic strategy for this particular type of cystogenesis. Compared to the embryonic model, the adult fish model exhibits a spectrum of progressive pathogeneses and enables ciliary abnormalities to be discerned as either primary or secondary to cystogenesis. We believe that this novel adult fish model will facilitate mechanistic studies and therapeutic development for PKD.

genetics↗