Ivana Y. Kuo

Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2.

Distinguishing Right from Left In most vertebrates during embryonic development, rotational movement of the cilia within a structure in the embryo, known as the node, generates unidirectional flow required for future left-right asymmetry of the internal organs. The flow may transport a determinant molecule or provide mechanical force. However, it is not clear how the flow is sensed. Yoshiba et al. (p. 226, published online 13 September; see the Perspective by Norris and Grimes) show that nodal flow in mouse embryos is sensed by the cilia of perinodal cells located at the edge of the node, in a manner dependent on Pkd2, a Ca2+-permeable cation channel that has been implicated in polycystic kidney disease in humans. A Ca2+ channel implicated in polycystic kidney disease helps to establish the left-right body axis of the mammalian embryo. Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca2+ channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.

Polycystin-2 mutations lead to impaired calcium cycling in the heart and predispose to dilated cardiomyopathy

Mutations in PKD1 and PKD2, the genes encoding the proteins polycystin-1 (PC1) and polycystin-2 (PC2), cause autosomal dominant polycystic kidney disease (ADPKD). Although the leading cause of mortality in ADPKD is cardiovascular disease, the relationship between these conditions remains poorly understood. PC2 is an intracellular calcium channel expressed in renal epithelial cells and in cardiomyocytes, and is thus hypothesized to modulate intracellular calcium signaling and affect cardiac function. Our first aim was to study cardiac function in a zebrafish model lacking PC2 (pkd2 mutants). Next, we aimed to explore the relevance of this zebrafish model to human ADPKD by examining the Mayo Clinics ADPKD database for an association between ADPKD and idiopathic dilated cardiomyopathy (IDCM). Pkd2 mutant zebrafish showed low cardiac output and atrioventricular block. Isolated pkd2 mutant hearts displayed impaired intracellular calcium cycling and calcium alternans. These results indicate heart failure in the pkd2 mutants. In human ADPKD patients, we found IDCM to coexist frequently with ADPKD. This association was strongest in patients with PKD2 mutations. Our results demonstrate that PC2 modulates intracellular calcium cycling, contributing to the development of heart failure. In human subjects we found an association between ADPKD and IDCM and suggest that PKD mutations contribute to the development of heart failure.

Decreased polycystin 2 expression alters calcium-contraction coupling and changes β-adrenergic signaling pathways

Significance The main cause of death in autosomal-dominant polycystic kidney disease (ADPKD) patients is cardiac-related. However, the reasons why remain unclear. We show that mice lacking one copy of polycystin 2, a protein mutated in ADPKD, have altered calcium signaling and desensitized calcium-contraction coupling in cardiomyocytes. We also show that decreased polycystin 2 levels affect cardiac function by altering responses to adrenergic stimulus. We propose that altering polycystin levels in the heart directly contributes to remodeling of the heart in patients with ADPKD in the absence of renal failure or high blood pressure. Cardiac disorders are the main cause of mortality in autosomal-dominant polycystic kidney disease (ADPKD). However, how mutated polycystins predispose patients with ADPKD to cardiac pathologies before development of renal dysfunction is unknown. We investigate the effect of decreased levels of polycystin 2 (PC2), a calcium channel that interacts with the ryanodine receptor, on myocardial function. We hypothesize that heterozygous PC2 mice (Pkd2+/−) undergo cardiac remodeling as a result of changes in calcium handling, separate from renal complications. We found that Pkd2+/− cardiomyocytes have altered calcium handling, independent of desensitized calcium-contraction coupling. Paradoxically, in Pkd2+/− mice, protein kinase A (PKA) phosphorylation of phospholamban (PLB) was decreased, whereas PKA phosphorylation of troponin I was increased, explaining the decoupling between calcium signaling and contractility. In silico modeling supported this relationship. Echocardiography measurements showed that Pkd2+/− mice have increased left ventricular ejection fraction after stimulation with isoproterenol (ISO), a β-adrenergic receptor (βAR) agonist. Blockers of βAR-1 and βAR-2 inhibited the ISO response in Pkd2+/− mice, suggesting that the dephosphorylated state of PLB is primarily by βAR-2 signaling. Importantly, the Pkd2+/− mice were normotensive and had no evidence of renal cysts. Our results showed that decreased PC2 levels shifted the βAR pathway balance and changed expression of calcium handling proteins, which resulted in altered cardiac contractility. We propose that PC2 levels in the heart may directly contribute to cardiac remodeling in patients with ADPKD in the absence of renal dysfunction.

Signaling in muscle contraction.

Signaling pathways regulate contraction of striated (skeletal and cardiac) and smooth muscle. Although these are similar, there are striking differences in the pathways that can be attributed to the distinct functional roles of the different muscle types. Muscles contract in response to depolarization, activation of G-protein-coupled receptors and other stimuli. The actomyosin fibers responsible for contraction require an increase in the cytosolic levels of calcium, which signaling pathways induce by promoting influx from extracellular sources or release from intracellular stores. Rises in cytosolic calcium stimulate numerous downstream calcium-dependent signaling pathways, which can also regulate contraction. Alterations to the signaling pathways that initiate and sustain contraction and relaxation occur as a consequence of exercise and pathophysiological conditions.

Cyst formation following disruption of intracellular calcium signaling.

Significance Autosomal dominant polycystic kidney disease is the most common cause of fluid-filled cysts within the kidney. However, how cyst formation occurs is not well understood. It is thought that proteins disrupted by this disease, such as polycystin 2, change calcium signaling, leading to the formation of cysts. In this study, we grow LLC-PK1 cells in a protein gel environment to enable the study of cysts in culture, which cannot be observed in traditional cell culture techniques. We demonstrate that loss of intracellular calcium release channels result in cyst growth and are correlated with a loss of a functional cellular component known as the primary cilia. These results demonstrate that calcium signaling is an important component in cyst development. Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca2+) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca2+-activated Ca2+ channel of the endoplasmic reticulum. We hypothesize that Ca2+ signaling through PC2, or other intracellular Ca2+ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca2+ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca2+ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca2+ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca2+ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca2+ signaling lead to ADPKD cysts.

The number and location of EF hand motifs dictates the calcium dependence of polycystin-2 function

Polycystin 2 (PC2) is a calcium‐dependent calcium channel, and mutations to human PC2 (hPC2) are associated with polycystic kidney disease. The C‐terminal tail of hPC2 contains 2 EF hand motifs, but only the second binds calcium. Here, we investigate whether these EF hand motifs serve as a calcium sensor responsible for the calcium dependence of PC2 function. Using NMR and bioinformatics, we show that the overall fold is highly conserved, but in evolutionarily earlier species, both EF hands bind calcium. To test whether the EF hand motif is truly a calcium sensor controlling PC2 channel function, we altered the number of calcium binding sites in hPC2. NMR studies confirmed that modified hPC2 binds an additional calcium ion. Single‐channel recordings demonstrated a leftward shift in the calcium dependence, and imaging studies in cells showed that calcium transients were enhanced compared with wild‐type hPC2. However, biophysics and functional studies showed that the first EF hand can only bind calcium and be functionally active if the second (native) calcium‐binding EF hand is intact. These results suggest that the number and location of calcium‐binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function.—Kuo, I. Y., Keeler, C., Corbin, R., Ćelić, A., Petri, E. T., Hodsdon, M. E., Ehrlich, B. E. The number and location of EF hand motifs dictates the calcium dependence of polycystin‐2 function. FASEB J. 28, 2332–2346 (2014). www.fasebj.org

The polycystins are modulated by cellular oxygen-sensing pathways and regulate mitochondrial function

The polycystin proteins are encoded by the genes mutated in autosomal dominant polycystic kidney disease. A new role for these proteins in oxygen sensing and cell metabolism is proposed. Oxygen regulates the trafficking and channel activity of the polycystin complex, which modulates mitochondrial function by altering mitochondrial calcium uptake.

An Explicit Formulation Approach for the Analysis of Calcium Binding to EF-Hand Proteins Using Isothermal Titration Calorimetry

We present an improved and extended version of a recently proposed mathematical approach for modeling isotherms of ligand-to-macromolecule binding from isothermal titration calorimetry. Our approach uses ordinary differential equations, solved implicitly and numerically as initial value problems, to provide a quantitative description of the fraction bound of each competing member of a complex mixture of macromolecules from the basis of general binding polynomials. This approach greatly simplifies the formulation of complex binding models. In addition to our generalized, model-free approach, we have introduced a mathematical treatment for the case where ligand is present before the onset of the titration, essential for data analysis when complete removal of the binding partner may disrupt the structural and functional characteristics of the macromolecule. Demonstration programs playable on a freely available software platform are provided. Our method is experimentally validated with classic calcium (Ca(2+)) ion-selective potentiometry and isotherms of Ca(2+) binding to a mixture of chelators with and without residual ligand present in the reaction vessel. Finally, we simulate and compare experimental data fits for the binding isotherms of Ca(2+) binding to its canonical binding site (EF-hand domain) of polycystin 2, a Ca(2+)-dependent channel with relevance to polycystic kidney disease.

Ion Channels in Renal Disease

The cells of the kidney contain many specialized ion channels and transporters, which act in concert to regulate volume and ionic concentration by absorption or secretion of ions into the urine. Each region of the kidney involved in filtration and concentration of ions expresses a particular subset of ion channels. Together, these ion channels ensure appropriate electrolyte homeostasis. However, a number of hereditary and genetic mutations render these channels mis-or non-functional. Mutations to one or more of these ion channels are associated with a variety of symptoms including proteinuria, progressive loss of renal function, and renal hypertension. The progressive loss of renal function, culminating in end-stage renal disease is typically treated by dialysis or transplantation. End-stage renal disease is an increasing health problem, both in terms of prevalence and economic burden. The scope of this review is to first provide a general overview of the kidney and function, and then specifically address the ion channels that, when mutated, lead to kidney disease. 1.1. Physiology of renal ion handling The basic unit of the kidney is the nephron, and its function is to balance the ionic composition of the blood by filtering the blood, retrieving the necessary ions, secreting excess ions, and conserving water to concentrate the urine. Renal disease can be a manifestation of genetic mutations to renal channels (the focus of this review) or transporters (not discussed here, but there are many excellent reviews1). The correlation between distribution of a particular ion channel and its function for the kidney is a critical factor in the localization of disease. Most of these ion channels are tightly regulated and linked to a particular region of the nephron. Malfunctions in these channels can lead to impaired absorption of ions, and ultimately alter the osmotic balance in the kidney, with consequences on the ionic balance of the blood and tissues of the body. Specifically, mutations to a particular ion channel can have large effects beyond the kidney, as the ionic balance regulates a plethora of cotransporters required for transport of additional ions and other nutrients as well. The nephron can be divided into the renal corpuscle, responsible for initial filtration, and the renal tubule, responsible for secretion and reabsorption of ions. The outline below describes the path of fluid filtration and concentration through the kidney, and identifies the ion channels that will be the subject of further discussion in Section 2. The order in which the ion channels are discussed in Section 2 reflects the fluid path through the kidney (Table 1). Figure 1 provides a schematic of the kidney filtration and concentration apparatus, and localizes the ion channels that will be addressed in this review. Figure 1 Overview of the kidney nephron and the distribution of ion channels discussed in this review. Fluid enters the glomerulus, then down the convoluted proximal tubule. After passing through the loop of Henle, the fluid is further concentrated in the distal ... Table 1 Summary of the renal ion channels discussed in this review. The renal corpuscle is comprised of the glomerulus, which filters the blood, and the Bowman’s capsule. The Bowman’s capsule is composed of an inner layer of podocytes and an outer single layer of epithelial cells. Podocytes are specialized glomerular epithelial cells that surround the glomerular capillaries. Fluids from blood in the glomerulus are filtered through gaps between the podocytes, and the resulting fluid passed to the renal tubule. The concentration of the major ions, sodium (Na+), potassium (K+), chloride (Cl−), carbonate (HCO3−), calcium (Ca2+), through the Bowman’s space is the same as in whole blood2. Mutations to the transient receptor potential (TRP) canonical channel TRPC6 (See Section2.1) found in this region result in Focal Segmental Glomerulosclerosis. After exiting the Bowman’s capsule, fluid enters the proximal convoluted tubule. Through this region, up to 67% of filtered Na+ and K+ is reabsorbed. The loop of Henle is comprised of the descending and ascending limbs. The ascending limb of the loop of Henle consists of the thin ascending limb, and a distal portion known as the thick ascending limb of the loop of Henle. Defects in the normal function of the ion channels within the thick ascending loop of Henle are relevant to Na+, K+, and Cl− imbalances, and impair the absorption of these ions. In the thick ascending limb of the loop of Henle, NaCl enters the cell via the bumetanide- sensitive Na+-K+-2Cl−-cotransporter (NKCC2 or BSC1, a transporter which will not be discussed further here), whereas K+ is recycled into the lumen via an adenosine triphosphate (ATP)-sensitive K channel (ROMK, see Section 2.3); this channel, ROMK, provides the K+ necessary for NKCC2 activity. Cl− leaves the cell by the basolateral membrane through either the chloride channel (ClC-Kb, see Section 2.2.3.1) or is cotransported with K+ using NKCC2 or other transporters. Na+, on the other hand, exits the cell through the Na+-K+-ATPase (an ATP driven pump which will not be dealt with in this review). Recirculation of K+ to the lumen together with the exit of Cl− across the basolateral membrane provides the lumen-positive transepithelial voltage gradient that drives Na+, K+, Ca2+, and Mg2+ reabsorption (see Section 2.1). In the thick ascending limb of the loop of Henle, 20% of filtered Na+ and K+ is reabsorbed. Together, the upstream proximal tubule and thick ascending limb of loop of Henle reabsorb 90% of filtered Ca2+. After leaving the proximal tubule, the fluid enters the distal tubule. In the early distal convoluted tubule, NaCl reabsorption is mediated by the luminal NaCl cotransporter (another transporter which will not be dealt with in this review) and leaves the cell through ClC-Kb associated with barttin (mutations of which are discussed in Section 2.2), and through the Na+-K+-ATPase as well. Finally, the modified filtrate enters the collecting system before it passes to the urinary bladder. This part of the nephron is comprised of connecting tubules, cortical collecting ducts, and medullary collecting ducts. Na+ in this region is reabsorbed via the epithelial Na+ channel (ENaC, see Section 2.4) on the luminal side and again exits the cell by the Na+-K+-ATPase. The inwardly rectifying potassium channel Kir4.1 (see Section 2.3.3), found on the basolateral membrane is believed to provide the K+ that drives the Na+-K+-ATPase pump. At the same time as Na+ absorption, K+ is extruded by the Ca2+-activated big conductance K+ channel (BK, not further discussed here, but see the following reviews3) and ROMK (see Section 2.3). The activity of the channels can be enhanced with aldosterone, which stimulates the mineralocorticoid receptor, and increases ENaC, the Na+-K+-ATPase, and ROMK channel expression and activity. Furthermore, mineralocorticoids increase the serum-and glucocorticoid-inducible kinase transcription, which also activates ENaC, the Na+-K+-ATPase, and ROMK. In addition to the plasma membrane ion channels, one important intracellular ion channel, polycystin 2 will also be discussed (see Section 2.5). As a member of the TRP family, this channel primarily resides in the endoplasmic reticulum, where it can act as a Ca2+ release channel. Due to the widespread distribution of polycystin 2, mutations can result in the development cysts in any of the nephron segments (Figures 1 and ​and22). Figure 2 Distribution of the transient receptor potential (TRP) channels in the kidney. Note that the distribution of TRP channels is different depending on the region. For example, polycystin 2 is most prominently expressed in the distal convoluted tubule. 1.2. Treatment Strategies As alluded to above, most patients with end-stage renal disease are given kidney dialysis treatment, or, if available, a transplant. Other treatment strategies include angiotensin-converting enzyme inhibitors that act to prevent proteinuria, and slow or halt the progression of proteinuric nephropathies. Frontline treatment of renal hypertension is through treatment with Ca2+ channel blockers primarily targeting the voltage-gated Ca2+ channels4 found on vascular smooth muscle cells in the peripheral resistance vessels. Additionally, the drugs alter the degree of constriction of the renal afferent arterioles. The voltage-gated Ca2+ channels are not included here, as this review is mainly addressing the genetic diseases associated with ion channels of the kidney, specifically with renal channelopathies. For more detailed information about these vascular voltage-gated Ca2+ channels, the reader is directed to reviews addressing the pharmacology of Ca2+ channel blockers, primarily the dihydropyridine blockers4. Although these treatment strategies are currently used today, it is anticipated that the identification of the ion channel genes involved in specific kidney disease and the consequential modifications to channel and kidney function will result in novel and more kidney specific therapeutic approaches to delay or even prevent dialysis or kidney transplantation.

Decreased Polycystin 2 Levels Result in Non-Renal Cardiac Dysfunction with Aging

Mutations in the gene for polycystin 2 (Pkd2) lead to polycystic kidney disease, however the main cause of mortality in humans is cardiac related. We previously showed that 5 month old Pkd2+/- mice have altered calcium-contractile activity in cardiomyocytes, but have preserved cardiac function. Here, we examined 1 and 9 month old Pkd2+/- mice to determine if decreased amounts of functional polycystin 2 leads to impaired cardiac function with aging. We observed changes in calcium handling proteins in 1 month old Pkd2+/- mice, and these changes were exacerbated in 9 month old Pkd2+/- mice. Anatomically, the 9 month old Pkd2+/- mice had thinner left ventricular walls, consistent with dilated cardiomyopathy, and the left ventricular ejection fraction was decreased. Intriguingly, in response to acute isoproterenol stimulation to examine β-adrenergic responses, the 9 month old Pkd2+/- mice exhibited a stronger contractile response, which also coincided with preserved localization of the β2 adrenergic receptor. Importantly, the Pkd2+/- mice did not have any renal impairment. We conclude that the cardiac-related impact of decreased polycystin 2 progresses over time towards cardiac dysfunction and altered adrenergic signaling. These results provide further evidence that polycystin 2 provides a critical function in the heart, independent of renal involvement.