Actin cytoskeleton and associated myosin motors within the renal epithelium.

This review highlights the complex membrane architectures and organelles observed along the renal tubular segments through careful review of ultrastructural and physiological studies published over the past several decades. We also showcase the vital role(s) played by the actin cytoskeleton and actin associated myosin motor proteins in regulating cell type-specific physiological functions within cells of the renal epithelium. The purpose of this review is to provide a fresh conceptual framework to explain the structure-function relationships that exist between the actin cytoskeleton, organelle structure, and cargo transport within the mammalian kidney. We believe that with recent advances in technologies to visualize the actin cytoskeleton and associated proteins within intact kidneys, it is imperative to reimagine the functional role(s) for these proteins in situ, which will provide a rationale for their unique, cell type specific function(s), necessary to build and maintain complex physiological processes.

Genetic dissection of the Mom5 modifier locus and evaluation of Mom5 candidate genes.

Germline mutations in the adenomatous polyposis coli (APC) gene cause familial adenomatous polyposis (FAP), a hereditary colon cancer syndrome in which affected individuals may develop 100-1000s of colonic adenomas. In families affected by FAP, adenoma number can vary markedly between individuals, despite the fact that these individuals carry the same APC mutation. In at least some FAP pedigrees, evidence suggests that these phenotypic differences are caused by segregating modifier alleles that impact adenoma number. However, identifying these modifiers in the human population is difficult, therefore mouse models are essential. Using the Apc (Min/+) mouse colon cancer model, we previously mapped one such modifier, Mom5, to a 25 Mbp region of chromosome 5 that contains hundreds of genes. The purpose of the present study was to refine the Mom5 interval and evaluate candidate genes for the Mom5 modifier of intestinal neoplasia. Recombinant mice were used to narrow the Mom5 interval to 8.1 Mbp containing 70 genes. In silico and gene expression analyses were utilized to identify and evaluate potential candidate genes that reside within this interval. These analyses identified seven genes within the Mom5 interval that contain variants between the B6 and 129P2 strains. These genes represent the most likely candidates for the Mom5 modifier.

Conditional Myh9 and Myh10 inactivation in adult mouse renal epithelium results in progressive kidney disease.

Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions, including endocytosis and organelle transport pathways. Cell type-specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the glycosylphosphatidylinositol-anchored protein uromodulin (UMOD) and gradual loss of Na+ K+ 2Cl- cotransporter from the apical membrane of the thick ascending limb epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules and activation of ER stress and unfolded protein response pathways in Myh9&10-cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress-mediated progressive renal tubulointerstitial disease. These observations establish cell type-specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin.

Evaluation of Rint1 as a modifier of intestinal tumorigenesis and cancer risk.

The Rad50 Interacting Protein 1 (Rint1) influences cellular homeostasis through maintenance of endoplasmic reticulum, Golgi and centrosome integrity and regulation of vesicle transport, autophagy and the G2/M checkpoint. Rint1 has been postulated to function as a tumor suppressor as well as an oncogene, with its role depending perhaps upon the precise cellular and/or experimental context. In humans, heterozygosity for germline missense variants in RINT1 have, in some studies, been associated with increased risk of both breast and Lynch syndrome type cancers. However, it is not known if these germline variants represent loss of function alleles or gain of function alleles. Based upon these findings, as well as our initial consideration of Rint1 as a potential candidate for Mom5, a genetic modifier of intestinal tumorigenesis in ApcMin/+ mice, we sought to explicitly examine the impact of Rint1 on tumorigenesis in ApcMin/+ mice. However, heterozygosity for a knockout of Rint1 had no impact on tumorigenesis in Rint1+/-; ApcMin/+ mice. Likewise, we found no evidence to suggest that the remaining Rint1 allele was lost somatically in intestinal tumors in ApcMin/+ mice. Interestingly, in contrast to what has been observed in Rint1+/- mice on a mixed genetic background, Rint1+/- mice on a pure C57BL/6J background did not show spontaneous tumor development. We also evaluated colorectal cancer data available in the COSMIC and ONCOMINE databases and found that RINT1 overexpression, as well as the presence of somatic missense mutations in RINT1 were associated with colorectal cancer development. In vitro evaluation of two missense variants in RINT1 suggested that such variants do have the potential to impact RINT1 function.

Nonmuscle myosin 2 proteins encoded by Myh9, Myh10, and Myh14 are uniquely distributed in the tubular segments of murine kidney.

The diverse epithelial cell types of the kidneys are segregated into nephron segments and the collecting ducts in order to endow each tubular segment with unique functions. The rich diversity of the epithelial cell types is highlighted by the unique membrane channels and receptors expressed within each nephron segment. Our previous work identified a critical role for Myh9 and Myh10 in mammalian endocytosis. Here, we examined the expression patterns of Nonmuscle myosin 2 (NM2) heavy chains encoded by Myh9, Myh10, and Myh14 in mouse kidneys as these genes may confer unique nephron segment-specific membrane transport properties. Interestingly, we found that each segment of the renal tubules predominately expressed only two of the three NM2 isoforms, with isoform-specific subcellular localization, and different levels of expression within a nephron segment. Additionally, we identify Myh14 to be restricted to the intercalated cells and Myh10 to be restricted to the principal cells within the collecting ducts and connecting segments. We speculate that the distinct expression pattern of the NM2 proteins likely reflects the diversity of the intracellular trafficking machinery present within the different renal tubular epithelial segments.