Purpose: Incidence and mortality rates for renal cell carcinoma (RCC) have been rising for decades. Unfortunately, the molecular events that support RCC carcinogenesis remain poorly understood. In an effort to gain a better understanding of signaling events in clear cell RCC (cRCC), we investigated the antitumor activity of secreted frizzled-related protein 1 (sFRP1), a negative regulator of Wnt signaling. Experimental Design: Genomic profiling of cRCC tumors and patient-matched normal tissues was done and confirmed using quantitative PCR and immunohistochemistry. Methylation-specific PCR was done on patient samples to evaluate the mechanism responsible for sFRP1loss. sFRP1 expression was restored in cRCC cells and the effects on tumor phenotype were characterized. Results: Genomic profiling, quantitative PCR, and immunohistochemistry indicated that loss of sFRP1occurred in cRCC and papillary RCC patient tissues. Twelve Wnt-regulated genes were up-regulated in cRCC tissues, including c-myc and cyclin D1, potentiators of cell proliferation and survival. Methylation of the sFRP1 gene was one mechanism identified for attenuation of sFRP1 mRNA. Stable reexpression of sFRP1in cRCC cells resulted in decreased expression of Wnt target genes, decreased growth in cell culture, inhibition of anchorage-independent growth, and decreased tumor growth in athymic nude mice. Conclusions: To our knowledge, this is the first report to show that stable restoration of sFRP1 expression in cRCC cells attenuates the cRCC tumor phenotype. Our data support a role for sFRP1as a tumor suppressor in cRCC and that perhaps loss of sFRP1is an early, aberrant molecular event in renal cell carcinogenesis.

No abstract

The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the α subunit of the epithelial sodium channel (αENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1. Here, we investigated the role of Period 1 in mediating the downstream effects of aldosterone in renal cells. Aldosterone treatment stimulated expression of Period 1 (Per1) mRNA in renal collecting duct cell lines and in the rodent kidney. RNA silencing of Period 1 dramatically decreased expression of mRNA encoding αENaC in the presence or absence of aldosterone. Furthermore, expression of αENaC-encoding mRNA was attenuated in the renal medulla of mice with disruption of the Per1 gene, and these mice exhibited increased urinary sodium excretion. Renal αENaC-encoding mRNA was expressed in an apparent circadian pattern, and this pattern was dramatically altered in mice lacking functional Period genes. These results suggest a role for Period 1 in the regulation of the renal epithelial sodium channel and more broadly implicate the circadian clock in control of sodium balance.

Most bodily functions vary over the course of a 24h day. Circadian rhythms in body temperature, sleep-wake cycles, metabolism, and blood pressure (BP) are just a few examples. These circadian rhythms are controlled by the central clock in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral clocks located throughout the body. Light and food cues entrain these clocks to the time of day and this synchronicity contributes to the regulation of a variety of physiological processes with effects on overall health. The kidney, brain, nervous system, vasculature, and heart have been identified through the use of mouse models and clinical trials as peripheral clock regulators of BP. The dysregulation of this circadian pattern of BP, with or without hypertension, is associated with increased risk for cardiovascular disease. The mechanism of this dysregulation is unknown and is a growing area of research. In this review, we highlight research of human and mouse circadian models that has provided insight into the roles of these molecular clocks and their effects on physiological functions. Additional tissue-specific studies of the molecular clock mechanism are needed, as well as clinical studies including more diverse populations (different races, female patients, etc.), which will be critical to fully understand the mechanism of circadian regulation of BP. Understanding how these molecular clocks regulate the circadian rhythm of BP is critical in the treatment of circadian BP dysregulation and hypertension.

The circadian clock protein Period 1 (Per1) contributes to the regulation of expression of the α subunit of the renal epithelial sodium channel (αENaC) at the basal level and in response to the mineralocorticoid hormone aldosterone. The goals of the present study were to define the role of Per1 in the regulation of additional renal sodium handling genes in cortical collecting duct cells and to evaluate BP in mice lacking functional Per1. To determine if Per1 regulates additional genes important in renal sodium handling, a candidate gene approach was employed. Immortalized collecting duct cells were transfected with a non-target siRNA or a Per1 specific siRNA. Expression of the genes for αENaC and Fxyd5, a positive regulator of Na, K-ATPase activity, decreased in response to Per1 knockdown. Conversely, mRNA expression of caveolin-1, Ube2e3 and ET-1, all negative effectors of ENaC, was induced following Per1 knockdown. These results led us to evaluate BP in Per1 KO mice. Mice lacking Per1 exhibit significantly reduced BP and elevated renal ET-1 levels compared to wild type animals. Given the established role of renal ET-1 in ENaC inhibition and blood pressure control, elevated renal ET-1 is one possible explanation for the lower blood pressure observed in Per1 KO mice. These data support a role for the circadian clock protein Per1 in the coordinate regulation of genes involved in renal sodium reabsorption. Importantly, the lower BP observed in Per1 KO mice compared to wild type suggests a role for Per1 in BP control as well.

In the past decade, it has become increasingly evident that the circadian clock system plays an important role in many physiological processes. The circadian clock can be divided into 2 parts: the central clock, residing in the suprachiasmatic nucleus of the hypothalamus, which receives light cues, and the peripheral clocks that reside in various tissues throughout the body. The peripheral clocks play an integral and unique role in each of their respective tissues, driving the circadian expression of specific genes involved in a variety of physiological functions. The goal of this review is to provide an introduction to and overview of the peripheral clocks, including potential mechanisms, targets, and implications for disease states. The peripheral clocks include the cardiovascular, metabolic, endocrine, immune, and reproductive systems.

Circadian variations in renal function were first described in the 19th century, and GFR, renal blood flow, urine production, and electrolyte excretion exhibit daily oscillations. These clinical observations are well established, but the underlying mechanisms that govern circadian fluctuations in kidney are not fully understood. Here we provide a brief overview of the machinery governing the circadian clock and examine the clinical and molecular evidence supporting a critical role for circadian rhythm in the kidney. There is a connection between BP oscillation and renal disease that supports the use of chronotherapy in the treatment of hypertension or correction of nondipping BP. Such studies support a developing model of clock controlled sodium and water transport in renal epithelial cells. Recent advances in identifying novel clock-controlled genes using rodent and cellular models also shed light on the molecular mechanisms by which the circadian clock controls renal function; however, the field is new and much more work remains.

The mineralocorticoid aldosterone is a major regulator of Na+ and acid-base balance and control of blood pressure. Although the long-term effects of aldosterone have been extensively studied, the early aldosterone-responsive genes remain largely unknown. Using DNA array technology, we have characterized changes in gene expression after 1 h of exposure to aldosterone in a mouse inner medullary collecting duct cell line, mIMCD-3. Results from three independent microarray experiments revealed that the expression of many transcripts was affected by aldosterone treatment. Northern blot analysis confirmed the upregulation of four distinct transcripts identified by the microarray analysis, namely, the serum and glucose-regulated kinase sgk, connective tissue growth factor, period homolog, and preproendothelin. Immunoblot analysis for preproendothelin demonstrated increased protein expression. Following the levels of the four transcripts over time showed that each had a unique pattern of expression, suggesting that the cellular response to aldosterone is complex. The results presented here represent a novel list of early aldosterone-responsive transcripts and provide new avenues for elucidating the mechanism of acute aldosterone action in the kidney.