*Citation: Potthoff SA, Stegbauer J, Becker J, Wagenhaeuser PJ, Duvnjak B, Rump LC and Vonend O (2013) P2Y*2 *receptor deficiency aggravates chronic kidney disease progression. Front. Physiol. 4:234. doi: 10.3389/fphys.2013.00234*

*This article was submitted to Renal and Epithelial Physiology, a section of the journal Frontiers in Physiology.*

*Copyright © 2013 Potthoff, Stegbauer, Becker, Wagenhaeuser, Duvnjak, Rump and Vonend. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Role of extracellular ATP and P2 receptor signaling in regulating renal cyst growth and interstitial inflammation in polycystic kidney disease

## *Gopi Rangan\**

*Michael Stern Translational Laboratory for Polycystic Kidney Disease, Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney, Sydney, NSW, Australia*

#### *Edited by:*

*Robert J. Unwin, University College London, UK*

#### *Reviewed by:*

*Lisa M. Harrison-Bernardm, Louisiana State University, USA Rajeev Rohatgi, Mount Sinai School of Medicine, USA*

#### *\*Correspondence:*

*Gopi Rangan, Centre for Transplant and Renal Research, Clinical Sciences Block, Westmead Hospital, Sydney, NSW 2145, Australia e-mail<EMAIL_ADDRESS>
Polycystic kidney diseases (PKD) are a group of inherited ciliopathies in which the formation and growth of multiple cysts derived from the distal nephron and collecting duct leads to the disruption of normal kidney architecture, chronic interstitial inflammation/fibrosis and hypertension. Kidney failure is the most life-threatening complication of PKD, and is the consequence of cyst expansion, renal interstitial disease and loss of normal kidney tissue. Over the last decade, accumulating evidence suggests that the autocrine and paracrine effects of ATP (through its receptor family P2X and P2Y), could be detrimental for the progression of PKD. (2009). *In vitro,* ATP-P2 signaling promotes cystic epithelial cell proliferation, chloride-driven fluid secretion and apoptosis. Furthermore, dysfunction of the polycystin signal transduction pathways promotes the secretagogue activity of extracellular ATP by activating a calcium-activated chloride channel *via* purinergic receptors. Finally, ATP is a danger signal and could potentially contribute to interstitial inflammation associated with PKD. These data suggest that ATP-P2 signaling worsens the progression of cyst enlargement and interstitial inflammation in PKD.

**Keywords: polycystic kidney disease, cyst, interstitial inflammation, purinergic, ATP**

#### **INTRODUCTION**

Polycystic kidney diseases (PKD) are a group of inherited ciliopathies in which the formation and growth of multiple fluid-filled renal cysts leads to disruption of normal kidney architecture, chronic renal interstitial inflammation and fibrosis, and hypertension (Harris and Torres, 2009). Kidney failure is the most serious and life-threatening complication of PKD (Rangan et al., 2005), and is the consequence of cyst expansion, renal interstitial disease and loss of normal kidney tissue (Grantham et al., 2011). Cyst growth in PKD is due to a combination of proliferation of cyst-lining epithelial cells of the distal nephron and collecting ducts, chloride-driven fluid secretion and extracellular matrix defects (Grantham et al., 1989). Although there is no treatment to currently prevent kidney failure due to PKD, rapid progress is being made (Harris and Torres, 2009). For example, in 2012, a 3-year Phase 3 clinical trial using a small molecule inhibitor (tolvaptan, a vasopressin type 2 receptor antagonist) successfully attenuated the rate of kidney enlargement in humans with PKD (Kelsey, 2013). The premise for the use of vasopressin receptor antagonism was based on preclinical data showing that vasopressin altered in intracellular purinergic (cAMP) signaling in PKD (Wang et al., 2005).

It has been hypothesized that a multi-drug approach, targeting cyst growth and interstitial inflammation, will be needed to effectively prevent kidney failure in PKD (Aguiari et al., 2013), and therefore further therapies are needed (Chang and Ong, 2013). For over a decade, it has been postulated that the autocrine and paracrine effects of extracellular nucleotides and their metabolites could be detrimental for the progression of PKD, and that strategies to attenuate this activation using inhibitors of nucleotide release, nucleotide scavengers or nucleotide antagonists could be useful for the therapy of PKD (Wilson et al., 1999; Schwiebert, 2001; Leipziger, 2003; Hillman et al., 2005; Hovater et al., 2008; Xu et al., 2009). Currently, knowledge regarding the role of extracellular nucleotides in the pathogenesis of kidney failure due to PKD remains in the early phase of clinical translation. This is highlighted by the paucity of published articles, as a PubMed search performed on the 29th April 2013, using the terms "ATP and polycystic kidney disease," revealed 42 publications, only 15 of which were original scientific articles and 6 were review articles that were directly relevant to this field of research. Nevertheless, significant insights have been gained from this limited information to support that extracellular nucleotide signaling is worthy of further pursuit in PKD. In fact, it has been hypothesized that this approach might act synergistically with vasopressin receptor antagonism (Buchholz et al., 2011; Luft, 2011). Extracellular nucleotides and metabolites can be classified according to the base from which they are derived and the number of associated number of phosphate groups (adenosine-derived; ATP, ADP; AMP; guanosine-derived: GTP, GDP, GMP; cytidine-derived: CTP, CDP, CMP; 5-methyluridine- and uridine-derived). The majority of the available data regarding extracellular nucleotides in PKD is based on ATP-P2 receptor signaling. Therefore, this mini-review will focus primarily on the role of ATP and its P2 receptors, in the pathogenesis and progression of cyst enlargement and interstitial inflammation in PKD.

## **OVERVIEW OF PKD**

PKD consists of several variants which differ according to their mode of inheritance, underlying genetic mutation and pattern of cyst formation. These variants include automosal dominant PKD (ADPKD), autosomal recessive PKD (ARPKD) and other autosomal recessive cystic renal diseases, including nephronophthisis (NPHP). ADPKD is the most common type of cystic renal disease, with an incidence of approximately one in every 1000 live births, and estimated to affect 6.5 million people world-wide (Bisceglia et al., 2006; Harris and Torres, 2009). Structurally, in ADPKD, the cysts are spherical in shape and occur in both kidneys. Cyst growth is not synchronized. Therefore the end-stage kidney in ADPKD contains thousands of cysts that vary in size from 100µm to several centimeters in diameter, resulting in a large (weighing up to 3–5 kg, compared to 125–170 g in normal males) and irregularly shaped organ. In ADPKD, the cysts start to form in early life (possibly *in utero* or during the early postnatal period) due to the clonal proliferation of focal epithelial cells lining the distal nephron and collecting duct, which leads to diverticular-like protrusions extending into interstitium (Baert, 1978). With continued growth, the latter detach from the parent nephron when their diameter exceeds 100 µm, and form "encapsulated cysts" (Hovater et al., 2008). Once in the interstitium the cysts continue to slowly expand over many decades (Grantham et al., 2011). Kidney failure therefore occurs after a long latent period, usually by the 5th decade of life (Baert, 1978), when a sufficient number of cysts (possibly *>* 1000) (Luft, 2011) have collectively grown to disrupt normal kidney architecture and function. In contrast, ARPKD is a less frequent, childhood disease (1:20 000 live births) (Sweeney and Avner, 2006). It is characterized by the synchronized microcystic dilation of collecting ducts. Detachment of the dilated cystic collecting ducts from the nephron does not occur (Baxter, 1961). The kidneys are large but maintain their reniform shape, and kidney failure typically occurs during the neonatal period (Sweeney and Avner, 2006). NPHP has an autosomal recessive mode of inheritance, and is characterized by tubulointerstitial nephropathy and corticomedullary duct ectasia, but kidney enlargement does not occur (Wolf and Hildebrandt, 2011). NPHP is one of the most frequent genetic disorders causing kidney failure in children and adolescents (Wolf and Hildebrandt, 2011).

ADPKD, ARPKD and NPHP are caused by mutations in the *Pkd1/Pkd2*, *Pkhd1* and *Nphp* (Grantham et al., 1989, 2011; Wilson et al., 1999; Schwiebert, 2001; Rangan et al., 2005; Wang et al., 2005; Harris and Torres, 2009; Xu et al., 2009; Aguiari et al., 2013; Chang and Ong, 2013; Kelsey, 2013) genes respectively, which encode the proteins, polycystin (PC)-1/PC-2, fibrocystin and nephrocystin (Sweeney and Avner, 2006; Harris and Torres, 2009). These so-called "cystproteins" (Hildebrandt and Otto, 2005) have all been found to co-localize to the primary renal cilia (an antenna-like sensory organelle involved in mechanosensation), and interact with each other at a molecular and functional level (Kaimori and Germino, 2008; Fedeles et al., 2011). In physiological states, the intact cystoprotein complex maintains the normal function of the cilium, negatively regulates the cell-cycle (Bhunia et al., 2002) and promotes intracellular calcium transport (Cowley, 2008) and cellular differentiation as well as normal renal tubular morphogenesis (Boletta and Germino, 2003). The intracellular level of PC-1 plays a central role in both ADPKD and ARPKD, as it is the rate-limiting component that ultimately determines cyst formation (Fedeles et al., 2011). Interestingly, ADPKD is a focal disease, as only 1–2% of nephrons in a kidney develop cysts (Martinez and Grantham, 1995). It has therefore been postulated that a heterozygote germ-line mutation in *Pkd1* or *Pkd2,* combined with postnatal disruption of the second normal allele is required for cyst formation (the two-hit mechanism of the Knudson theory) (Nauli et al., 2006). An additional molecular explanation for the postnatal onset of ADPKD is an age-related decline in the dosage of functional PC-1 protein (Rossetti et al., 2009). In contrast to ADPKD, postnatal somatic mutations do not have a role in cyst formation in ARPKD (Sweeney and Avner, 2006). Despite their importance in PKD, therapeutic approaches to modulate the genetic expression of PCs has been problematic because: (1) there is significant heterogeneity in the genetic mutations of PC-1 (Harris and Torres, 2009); (2) over- as well as under-expression of PC-1 can cause PKD (Harris and Torres, 2009); (3) there are difficulties with the delivery of viral vectors for gene therapy due to the poor endocytosis capability of the epithelial cells lining cysts (Witzgall et al., 2002). Therefore, treatments to prevent kidney failure due to PKD have focused on the abnormalities in cellular function and signal transduction arising from the dysfunction of cystproteins (Torres et al., 2007). These abnormalities are largely a consequence of reduced intracellular calcium which leads to increased intracellular levels of cAMP. As discussed earlier, cAMP levels are also increased by circulating vasopressin.

Halting the growth of cysts is one of the main therapeutic goals in preventing the decline of kidney function in PKD. The mechanisms of cyst growth involve three factors (Torres et al., 2007; Terryn et al., 2011): (i) increased proliferation of CECs: (ii) the gradual and incremental accumulation of fluid inside cysts; and (iii) abnormalities in extracellular matrix formation and degradation which are permissive for cyst expansion. The accumulation of fluid in cysts is an important mechanism of disease progression in PKD, and as discussed below, may be induced by purinergic signaling (Terryn et al., 2011). It is due to the net transepithelial secretion of chloride across apical membranes of CECs *via* chloride channels [cystic fibrosis transmembrane conductance regulator in response to the induction of cAMP-mediated protein kinase A (CFTR-PKA); and calcium-activated chloride channels]. This chloride efflux then induces Na+ (as a result electric coupling) and water (as a result of osmotic coupling via aquaporin channels) efflux, resulting in the progressive accumulation of fluid within the cysts (Terryn et al., 2011).

#### **POTENTIAL MECHANISMS OF EXTRACELLULAR ATP RELEASE AND P2 RECEPTOR SIGNALING IN PKD**

The intracellular concentration of ATP ranges between 1 to 10 mM (Beis and Newsholme, 1975), and ∼0.1% of this reservoir (Schwiebert, 2001) may theoretically be released into the extracellular space (up to 10 µmol/L) of the renal microenvironment in PKD, which includes the interior of encapsulated cysts (as in ADPKD) (Schwiebert, 2001), the renal interstitial space and/or the nephron lumen. The mechanisms of the extracellular release could hypothetically involve (Jacobson and Boeynaems, 2010): (1) apoptosis and necrosis of cystic epithelial cells (CECs) as well as destruction of normal renal epithelial cells with disease progression (Goilav, 2011); (2) non-lytic mechanisms (Bowler et al., 2001) involving CECs, that requires the exocytosis of secretary granules, vesicular transport and membrane channels and include ATP-binding cassette transporters, pannexins and connexins (Lohman et al., 2012), possibly in response to mechanical stretch [as shown in bladder epithelial cells (Ferguson et al., 1997)], hypoxia (Bergfeld and Forrester, 1992), dysfunction of cystoproteins (Schwiebert et al., 2002), increased cellular metabolism (Wilson, 1997; Sullivan et al., 1998) and other ATP release mechanisms that may be abnormal in PKD (Schwiebert et al., 2002); and (3) release of ATP from other local sources such as infiltrating inflammatory cells and renal nerves (Bailey et al., 2000).

Once in the extracellular fluid, ATP is capable of activating purinergic receptors, which are one of the most abundant receptor families in mammalian tissues (Abbracchio et al., 2009). There are two groups of ATP-responsive (P2) receptors: (i) P2Y receptors are G-protein coupled which act through a second messenger, and respond to a wide variety of nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) They consist of eight members (subtypes 1, 2, 4, 6, 11, 12, 13, and 14, which vary depending on the type of G protein involved and specificity of the ligand) (Jacobson and Boeynaems, 2010) and modulate intracellular calcium and cAMP; and (ii) P2X receptors are rapidly acting non-selective cation channels that are calcium permeable, and which open primarily after binding to ATP (Schwiebert, 2001). They consist of eight subtypes, and in response to ATP-ligand binding, cause plasma membrane depolarization which allows calcium influx from external stores (Schwiebert, 2001).

Extracellular ATP is rapidly degraded in the plasma (Gorman et al., 2007) and in order for it to have a physiological or pathological function, it requires accumulation in the local microenvironment. The net build-up of ATP in PKD is likely to be dependent on a number of factors (Di Virgilio, 2012): (i) the pattern of P2 receptor expression in cystic and non-cystic renal tissue as well as in infiltrating inflammatory cells; (ii) the level of expression of nucleotide hydrolyzing enzymes (CD39 and CD73), which breakdown ATP and generate adenosine, which has anti-inflammatory and anti-tumor effects (Di Virgilio, 2012).

#### **EVIDENCE FOR ATP RELEASE INTO THE LOCAL MICROENVIRONMENT AND P2 RECEPTOR ALTERATION IN PKD**

Fluid extracted from micro-dissected cysts from ADPKD patients was found to contain pharmacologically relevant levels of ATP (up to 10 µM) (Wilson et al., 1999), which is a thousand times higher than that found in peripheral blood (Lazarowski et al., 2001). Primary cultures of renal epithelial cells from ADPKD and ARPKD patients release between two to five times more ATP, both across the apical and basolateral membrane, under resting conditions and with hypotonic challenge, compared to non-ADPKD cells (Wilson et al., 1999; Schwiebert et al., 2002). This might, in part, be due to the slower degradation of ATP in ADPKD cells due to a reduced expression of CD39 (Xu et al., 2009) or other mechanisms. ATP, in concentrations of 4 nM, was also detectable

in cyst fluid from *cpk* mice (a murine model that phenotypically resembles ARPKD, and is due to a mutation in *cystin*) (Hillman et al., 2004). However, the presence of ATP in the interstitial space, nephron lumen or urine of patients and animals with PKD has not been reported and is not known. In the microenvironment of tumors (which are thought to share some analogy to the pathogenesis of PKD) (Ghiringhelli et al., 2009), ATP has been shown to accumulate, and under these circumstances, thought to be primarily due to increased cancer cell metabolism and tumorassociated inflammation (Ghiringhelli et al., 2009; Di Virgilio, 2012). This could potentially also be the case in PKD.

Several studies have examined the expression of P2 receptors *in vitro* and *in vivo* in the cyst lining epithelium in PKD. In primary cultures of ADPKD and ARPKD cells from humans, mRNA for both P2X and P2Y receptors was found to be expressed (Xu et al., 2009). In *cpk* mice, immunohistology showed that collecting duct cysts were positive for P2X7 protein (Hillman et al., 2002). Similarly, in human ARPKD, P2X7 was also expressed in the epithelial cells of dilated collecting ducts and cysts but not present in normal human fetal kidneys (Hillman et al., 2004). In the Han:Sprd rat model of PKD, CECs from heterozygous rats were positive for P2Y (Y2 and Y6), P2X5 and P2X7 (Turner et al., 2004). Taken together, these data suggest that ATP accumulates in the PKD microenvironment, and that CECs possess the appropriate receptors to be able to respond to the presence of the ligand.

## **CILIARY DYSFUNCTION IN PKD ALTERS THE AUTOCRINE ATP-P2 AXIS, INTRACELLULAR CALCIUM AND CHLORIDE SECRETION IN CYSTIC EPITHELIAL CELLS**

Growing evidence suggests that extracellular ATP has a physiological role in maintaining the health and function of renal epithelial cells, and that this is dependent on an intact and functioning cilium. In immortalized normal human kidney tubular epithelial cells (HK-2 cell line), P2X7 receptors were localized to the primary cilia, supporting the possibility that ATP-P2 signaling is involved in mechanosensation and cyst formation (Chang and Ong, 2013). In addition, in *C. Elegans*, ATP synthase (which produces ATP from ADP) physically associates with the polycystins, LOV-1 and PKD2, suggesting that ATP synthase activity could be dysfunctional in PKD and might be a factor contributing to extracellular accumulation of ATP (Hu and Barr, 2005). The importance of a functioning cilia for ATP-purinergic signaling was further revealed from *in vitro* studies of collecting duct principal cells from Oak Ridge Polycystic kidney disease (orpktg737) mice [a model of ARPKD in which cells lack a cilium due to a mutation in *polaris* (Hovater et al., 2008)]. These studies showed that the complete absence of an apical cilia impairs ATP secretion in response to mechanical, chemical and osmotic stimuli, in collecting duct principal cells (Hovater et al., 2008).

In normal renal epithelial cells extracellular ATP promotes chloride secretion and regulates intracellular calcium (Wildman et al., 2003). In PKD mutant cells, accumulating evidence suggests that chloride secretion in response to ATP is exacerbated. For example, over-expression of the cytoplasmic COOH-terminus of PC-1 in mouse cortical collecting duct cells prolonged the duration of chloride conductance in response to ATP, supporting the hypothesis that PKD mutant cells are more sensitive to some of the physiological effects of ATP (Hooper et al., 2003; Wildman et al., 2003). Therefore, it has been hypothesized that the dysfunction of the PC signal transduction pathways promotes the "secretagogue activity" of extracellular ATP by stimulating a calcium-activated chloride channel *via* purinergic receptors (Hooper et al., 2003; Wildman et al., 2003). In addition to chloride, ATP also acts in an autocrine fashion to stimulate intracellular calcium *via* purinergic receptor signaling. However, the intracellular calcium response to ATP is impaired in both murine and human ADPKD cells (Hovater et al., 2008; Xu et al., 2009). The latter was also associated with a reduced expression P2X7 and CD39 (Xu et al., 2009).

## **FUNCTIONAL EVIDENCE SUGGESTING THAT EXTRACELLULAR ATP-P2 SIGNALING DIRECTLY PROMOTES CYST EXPANSION AND INTERSTITIAL INFLAMMATION IN PKD**

#### **CYST EXPANSION (CHLORIDE-DRIVEN FLUID SECRETION AND PROLIFERATION OF CECs)**

In normal inner medullary collecting duct cells extracellular purinergic agonists can be mitogenic or co-stimulatory with other growth factors (Ishikawa et al., 1997). Therefore, in PKD, ATP has been postulated to have a role in the expansion of encapsulated renal cysts (as in ADPKD) or cystic tubular expansion (as in the ARPKD). It has been suggested that because cysts in the former are "encapsulated," ATP accumulation may have a more important pathogenic role because of sequestration within the cyst interior, but this has not been proven (Schwiebert, 2001).

To date the functional roles of ATP-P2 signaling on the mechanisms of cyst expansion has been examined *in vitro* in cultured cells and in a zebrafish model. Collectively, these studies have indicated that ATP-P2 signaling may either accelerate or attenuate cyst growth. Similar to normal tubular epithelial cells (Kishore et al., 1995, 2000), in primary cultures of human PKD cells, ATP agonists (ATP, Bz-ATP and UTP) increased intracellular calcium levels and secretory anion transport by activating chloridedependent secretion (that was independent of cAMP/PKA/CFTR) (Schwiebert et al., 2002). In contrast, Hillman and colleagues examined the role of ATP-P2 signaling in the initial steps of cyst formation, by growing CEC aggregates *ex vivo* from *cpk* mice (Hillman et al., 2004). In this study, the exposure of these cells to 2 - and 3 -*O*-(4-benzoylbenozoyl)-adensosine 5 -triphosphate (BzATP, a P2X7 agonist) reduced the number of cysts that formed by approximately one-third (Hillman et al., 2004). Cyst size and proliferation was not altered but there was a non-significant increase in caspase-3 activity (Lazarowski et al., 2001). Exogenous ATP and UTP also reduced cyst number in this model but to a much lesser extent (nearly 10%) (Lazarowski et al., 2001). In comparison to *cpk* mice CECs, in MDCK cell-derived cysts, treatment with non-specific P2 receptor antagonists (Reactive Blue 2, suramin) or removal of ATP from culture medium with apyrase (by 50%) attenuated the cAMP-ERK-dependant growth by ∼50% (Turner et al., 2007). In this model, cyst growth was not affected by treatment with a non-selective P2X inhibitor, Coomassie Brilliant Blue G), suggesting a role for P2Y receptors (Turner et al., 2007). Similarly, in a separate report, in MDCK

cysts derived from principal cells (Clone C7), ATP-dependent cyst growth was driven by fluid secretion rather than cell proliferation and was synergistic with cAMP (Buchholz et al., 2011). The latter was largely dependent on extracellular ATP and attenuated by the P2 receptor antagonist, suramin (Buchholz et al., 2011), and the effects were not evident in MDCK cells derived from intercalated cells (Buchholz et al., 2011). Consistent with the data in MDCK cells, in a zebrafish model of ADPKD (morpholino induced knockdown of *Pkd2*), a P2X7 antagonist (oxidized ATP) markedly reduced the cystic dilatation and peritubular oedema in pronephric ducts compared to the control (no treatment) or to a P2X7 agonist (Bz-ATP) (Chang et al., 2011). This was associated with a reduction in cell proliferation and p-ERK activity (Chang et al., 2011).

The contrasting results mentioned above are most likely due to differences in the experimental design (particularly the stage of cyst growth), the cellular model and the type of PKD examined. Perhaps, ATP-P2 signaling reduces cyst *formation* (as in *cpk* CEC aggregates) but promotes expansion once the cysts have actually formed. Further *in vivo* data using genetically orthologous models of PKD are awaiting to provide clarification regarding the role of ATP-P2 signaling in initiation and growth of cysts in PKD.

#### **RENAL INTERSTITIAL INFLAMMATION**

Renal interstitial inflammation is recognized as an important factor in the progression in PKD (Ta et al., 2013). Macrophage accumulation may promote cyst growth and interstitial inflammation, and be detrimental to the progression of PKD (Swenson-Fields et al., 2013; Ta et al., 2013). The release of excess extracellular ATP is a "danger signal" that is likely to lead to interstitial inflammation *via* inflammatory signaling pathways (Idzko et al., 2007; Ivison et al., 2011). To date, the role of ATP-P2 signaling in mediating interstitial inflammation associated with PKD has not been examined. Based on data from other experimental models, one might hypothesize that extracellular ATP (released into the renal interstitium by inflammatory cells and CECs) could have a proinflammatory effect (Deplano et al., 2013). For example, in antibody-mediated autoimmune glomerulonephritis, renal injury, glomerular macrophage accumulation and the urinary excretion of the monocyte chemoattractant protein-1 was attenuated by P2X7 deficiency (in mice) or by administration of a P2X7 antagonist (A-438079) (in rats) (Taylor et al., 2009) Similarly, in a murine model of renal interstitial fibrosis (unilateral ureteral obstruction), interstitial macrophage and myofibroblast accumulation, interstitial fibrosis and tubular cell apoptosis was also attenuated by P2X7 deficiency (Goncalves et al., 2006). Lastly, preliminary data shows that treatment of rats with unilateral ureteral obstruction with a P2X7 antagonist (Brilliant Blue G) also attenuated interstitial inflammation and fibrosis but increased tubule cell proliferation (Leite et al., 2012). The latter raises the possibility that the ATP-P2 system could have complex and divergent effects *in vivo* in PKD.

## **FUTURE DIRECTIONS AND POTENTIAL FOR ATP-P2 SIGNALING IN THE THERAPY OF HUMAN PKD**

Clinical trials (Phase 1 and 2) to determine the safety and efficacy of P2X7 receptor antagonists in chronic inflammatory diseases (rheumatoid arthritis, inflammatory bowel disease) are presently in progress [reviewed in Arulkumaran et al. (2011)]. In PKD, further preclinical evidence is required before clinical trials of P2X7 receptor antagonists can be considered. Experiments comparing disease progression in compound/double knockout mice using P2X−*/*− 7 mice and genetically orthologous/nonorthologous murine models of PKD are required (Hillman et al., 2005). In addition, in experimental models of cancer, the overexpression of either CD39 or CD73 promotes tumor progression due to loss of tumor-associated inflammation (Synnestvedt et al., 2002; Eltzschig et al., 2009). On the other hand, in PKD, the loss of interstitial inflammation due to the transgenic expression of CD39 or CD73, might be renoprotective, as shown in experimental renal ischaemia (Crikis et al., 2010). Studies to determine the effects small molecule inhibitors (such as Brilliant Blue G, suramin, A-438079 or preferably those used in clinical trials) during the early as well as the established phases of PKD in small animal models are also needed. In particular, the stageand disease-specific effects (relevant to human disease) of ATPpurinergic signaling in preclinical models needs to be understood (Schwiebert et al., 2002). The effects of natural and existing small molecule modulators of ATP-P2 signaling in the pathogenesis of PKD should not be forgotten, as this may accelerate translation to the clinic. For example, the methylxanthine, caffeine, induced ATP release in smooth muscle cells (Katsuragi et al., 2008), and it is possible that effects on P2 receptors underlie its ability to promote chloride driven fluid secretion in human ADPKD cells (Belibi et al., 2002). Similarly, the effects of existing pharmacological inhibitors, such as clopidogrel and ticlopidine which affect P2Y12 receptors (Savi and Herbert, 2005), on cyst growth in *in vitro* models, could be screened.

### **CONCLUSION**

Evidence accumulated to date supports that ATP-P2 signaling is potentially important in the pathogenesis of PKD. However, the study of ATP-P2 signaling in PKD is presently in an early phase of investigation (**Figure 1**) and further work, particularly preclinical

#### **REFERENCES**

a microdissection study of two cases at an early stage of the disease. *Kidney Int.* 13, 519–525. doi: 10.1038/ki.1978.75

**FIGURE 1 | Hypothetical diagram showing the potential role of ATP-P2 signaling in the pathogenesis of cyst growth and interstitial inflammation in PKD.** Current data suggests that extracellular ATP is increased in the microenvironment of the kidney affected by PKD, due to apical/basolateral release by cystic epithelial cells. Reduced degradation or increased cellular sensitivity to ATP may also contribute to net accumulation of ATP in the local environment. The presence of extracellular ATP has paracrine and autocrine effects on numerous cells, including cystic epithelial cells and interstitial inflammatory cells. In cystic epithelial cells, *in vitro* data suggests that P2 receptor activation promotes chloride-driven fluid secretion (independent of PKA-cAMP-CFTR mechanism) and proliferation (via p-ERK), both of which lead to cyst growth. In addition, preclinical data from non-PKD animal models suggests that P2 signaling promotes the chemotaxis of macrophages and lymphocytes, promoting interstitial inflammation. However, the hierarchy by which ATP-P2 signaling mediates proliferation, chloride secretion and interstitial inflammation in PKD is not known. In addition, the localization of P2 receptors (basolateral vs. apical distribution) or whether specific receptors have unique functions is not known.

*in vivo* studies, are needed before randomized controlled trials using small molecular inhibitors of P2 receptor antagonists in humans with PKD can be considered.

review. *Adv. Anat. Pathol.* 13, 26–56. doi: 10.1097/01.pap.0000201831. 77472.d3

*J. Mol. Med. (Berl.)* 89, 251–261. doi: 10.1007/s00109-010-0715-1

adaptive immunity against tumors. *Nat. Med.* 15, 1170–1178. doi: 10.1038/nm.2028

3:4 trial results. *Nat. Rev. Nephrol.* 9, 1. doi: 10.1038/nrneph.2012.236

penetrant PKD1 alleles suggest a role for gene dosage in cyst initiation in polycystic kidney disease. *Kidney Int.* 75, 848–855. doi: 10.1038/ki.2008.686

*Int.* 83, 855–864. doi: 10.1038/ki. 2012.446

J. (2004). Altered ATP-sensitive P2 receptor subtype expression in the Han:SPRD cy/+ rat, a model of autosomal dominant polycystic kidney disease. *Cells Tissues Organs* 178, 168–179. doi: 10.1159/000082247

26, 181–194. doi: 10.1007/s00467- 010-1585-z

Xu, C., Shmukler, B. E., Nishimura, K., Kaczmarek, E., Rossetti, S., Harris, P. C., et al. (2009). Attenuated, flowinduced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells. *Am. J. Physiol.* 296, F1464–F1476.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 13 May 2013; accepted: 28 July 2013; published online: 16 August 2013. Citation: Rangan G (2013) Role of extracellular ATP and P2 receptor signaling in regulating renal cyst growth and interstitial inflammation in polycystic kidney disease. Front. Physiol. 4:218. doi: 10.3389/fphys.2013.00218*

*This article was submitted to Renal and Epithelial Physiology, a section of the journal Frontiers in Physiology.*

*Copyright © 2013 Rangan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Emerging key roles for P2X receptors in the kidney

#### *R. E. Birch1, E. M. Schwiebert 2, C. M. Peppiatt-Wildman1† and S. S. Wildman1 \*†*

*1 Medway School of Pharmacy, The Universities of Kent and Greenwich, Kent, UK 2 Discovery BioMed Inc., Birmingham, AL, USA*

*Edited by: Volker Vallon, University of California San Diegio and VA San Diego Healthcare System, USA*

#### *Reviewed by:*

*Roger Evans, Monash University, Australia*

*Janos Peti-Peterdi, University of Southern California, USA*

#### *\*Correspondence:*

*S. S. Wildman , Urinary System Physiology Unit, Medway School of Pharmacy, The Universities of Kent and Greenwich at Medway, Anson Building, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK e-mail<EMAIL_ADDRESS>
*†Joint senior authors.*

P2X ionotropic non-selective cation channels are expressed throughout the kidney and are activated in a paracrine or autocrine manner following the binding of extracellular ATP and related extracellular nucleotides. Whilst there is a wealth of literature describing a regulatory role of P2 receptors (P2R) in the kidney, there are significantly less data on the regulatory role of P2X receptors (P2XR) compared with that described for metabotropic P2Y. Much of the historical literature describing a role for P2XR in the kidney has focused heavily on the role of P2X1R in the autoregulation of renal blood flow. More recently, however, there has been a plethora of manuscripts providing compelling evidence for additional roles for P2XR in both kidney health and disease. This review summarizes the current evidence for the involvement of P2XR in the regulation of renal tubular and vascular function, and highlights the novel data describing their putative roles in regulating physiological and pathophysiological processes in the kidney.

**Keywords: P2X receptor, P2 receptors, kidney, renal circulation, renal tubular transport, inborn errors, P2X7 receptor, pathology**

## **INTRODUCTION**

Extracellular ATP and related nucleotides have been shown to contribute to complex autocrine and/or paracrine signaling networks throughout the body following their activation of P2 receptors (P2R; formerly termed purinoceptor; Burnstock and Knight, 2004). The P2 family of receptors is divided into metabotropic G protein-coupled P2Y receptors (P2YR) and ionotropic ligandgated P2X receptors (P2XR), which act as non-selective cation channels (Fredholm et al., 1994). There are eight pharmacologically distinct P2YR (P2Y1*,*2*,*4*,*6*,*11−14) and seven unique P2XR subunits (P2X1-7), which can form seven homomeric assemblies, seven established heteromeric assemblies (P2X1/2, 1/4, 1/5, 2/3, 2/6, 4/6, 4/7), as well as several predicted heteromeric assemblies. Heterologous expression systems have been used to demonstrate that P2XR can readily form functional trimeric or hexameric assemblies (North, 2002). The actual number of subunits present in native cell multimeric assemblies is still debated, however, the trimer is favored (North, 2002). It is established that there are an abundance of P2R in a variety of cell types throughout the body, linked to numerous physiological processes such as regulation of other membrane-located ion channels, cell-to-cell communication, secretion of cytokines, metabolic processes such as glugoneogenesis, cell proliferation, and cell death (Burnstock and Knight, 2004).

It is perhaps not surprising that P2R are ubiquitously expressed throughout the kidney, in both cortical and medullary vascular and tubular compartments. They contribute to a diverse range of physiological and pathophysiological processes and yet the localization of specific P2R subtypes can be highly regional, overlapping, and often debated. Much of the renal literature published to date focuses on the role of metabotropic P2YR, and in particular in their regulation of sodium (Na) and water transport in the collecting duct (CD; Kishore et al., 2009; Wildman et al., 2009; Vallon and Rieg, 2011), with P2XR being somewhat overlooked (with the exception of their role in regulating afferent arteriole diameter; see summary **Figure 1**). Arguably a dogma exists that P2XR play little or no role in kidney function. However, a recent glut of manuscripts provides compelling evidence for P2XR playing key roles in the kidney. This review will discuss the most recent advances in the field of "renal P2XR" [i.e., the key literature of the last 3-years (2010–2013)], focusing on the tubular and vascular localization of P2XR and their function(s) in both the physiological and pathological setting (see summary **Figure 2**).

#### **NEW ROLES FOR P2XR IN RENAL TUBULAR TRANSPORT**

Expression of P2XR varies throughout the nephron. P2X4R and P2X6R are expressed in the proximal tubule, distal tubule, loop of Henle and CD, making these receptor subtypes the most widely distributed (Unwin et al., 2003). P2X1R and P2X7R are localized predominantly in Bowman's capsule and the CD and are more widely distributed in vascular networks (as discussed in the next section; Inscho et al., 2003; Vitzthum et al., 2004; Osmond and Inscho, 2010; Crawford et al., 2011). Immunohistochemical studies have also demonstrated low levels of expression for P2X2R and P2X5R in the cortical and medullary CDs (Turner et al., 2003; Wildman et al., 2008). The putative roles previously described for P2XR in the nephron have included inhibition of fluid reabsorption in the proximal tubule, inhibition of magnesium absorption in the distal tubule, and inhibition of AQP2-mediated water absorption and modulation (inhibiting and potentiating) of ENaC-mediated Na+ absorption/reabsorption in the CD (Bailey et al., 2012; see summary **Figure 1**).

Novel data obtained from pharmacological experiments that utilized P2XR selective agonists, complimented by studies in knockout mice (−*/*−), now provide compelling evidence for a functional role for P2XR in the medullary thick ascending limb (mTAL) (Marques et al., 2012). It is well-established that NaCl enters cells of the TAL via the apical Na/H exchanger and Na-K-2Cl co-transporter and leaves the cell via basolateral Na-K-ATPase, and nitric oxide (NO) inhibits both the Na/H exchanger and Na-K-ATPase to regulate ion transport. Experiments to determine the effect of basolateral ATP on NaCl absorption in isolated perfused mouse mTALs, using the electrical measurement of equivalent short-circuit current, demonstrated that basolateral ATP attenuated the absorptive short-circuit current (Marques et al., 2012). Authors used P2XR selective agonists and antagonists to demonstrate the effect was mediated, not via P2YR as might have been expected, but via P2XR. Experiments reproduced in both P2X7R−*/*− and P2X4R−*/*− concluded that the ATP-inhibition of transport was reduced in the P2X4R−*/*− animals thus indicating a key role for P2X4R. This finding was further corroborated by PCR experiments, which confirmed the presence of P2X4R mRNA, together with P2X1R and P2X5R mRNA in isolated mTAL (Marques et al., 2012). In addition, complimentary studies seeking to determine the factors responsible for flow-mediated NO production in the mTAL identified ATP as a candidate. Researchers used P2YR and P2XR selective antagonists to demonstrate a role for basolateral P2XR (and apical P2YR) in ATP-mediated, flow-induced production of NO in the mTAL (Cabral et al., 2012). Collectively, data from these studies suggest that ATP, released by increased tubular flow rate, acts on basolaterally-expressed P2X4R (potentially a heteromeric assembly, with either P2X1R or P2X5R), to increase NO production, which ultimately inhibits NaCl reabsorption in the mTAL. However, the mechanism by which luminal ATP activates basolaterally-expressed P2X4 receptors is yet to be elucidated.

Studies performed in our laboratory and with collaborators have similarly identified roles for P2X4R, and potentially P2X7R, in the regulation of Na reabsorption. However, our studies have focused on the CD rather than the TAL. Initially using M1 cells, an immortalized mouse cortical CD cell line, in combination with electrophysiology techniques we identified ionotropic P2XRmediated channel activity (Birch et al., 2013a). Application of ATP to outside-out patches activated single-channel openings, from multiple receptor subtypes, with single-channel properties similar to those of P2XR previously identified in other cell types (Evans, 1996; Birch et al., 2013a). Characterization of the exact

P2XR subtypes mediating the single-channel activity is a current focus of the group. Initial observations in rat CD principal cells *in vitro* demonstrated that apical P2X4R modulates ENaC (epithelial Na channel) activity: high concentrations of luminal ATP evoked P2X4R-mediated inhibition of ENaC activity, whereas low concentrations conversely potentiated ENaC activity (Wildman et al., 2008). In addition to our *in vitro* functional data we have used P2X4R−*/*− mice to demonstrate an *in vivo* role for P2X4R in Na reabsorption and salt sensitivity (Craigie et al., 2012). Data from our most recent *in vivo* renal clearance studies have revealed a raised mean arterial blood pressure (MABP) in P2X4R−*/*− when compared with wild-type (WT) littermates. When dietary Na was altered to what is termed a low Na diet (0.03% compared to 0.3% in a standard diet), the reduction in dietary Na significantly reduced MABP in the P2X4R−*/*− but not in WT. Interestingly, when comparing the ENaC-mediated reabsorption for WT and P2X4R−*/*− on a standard diet there was no significant difference. However, when on a low Na diet the P2X4R−*/*− mice exhibited a significantly reduced fractional Na excretion compared with WT, indicating this group of animals were unable to increase ENaC-mediated Na reabsorption in the absence of P2X4R. These data collectively suggest that P2X4R−*/*− mice have salt-sensitive blood pressure and are unable to increase their ENaC-mediated sodium reabsorption when dietary sodium is low. The latter finding is compatible with our previous *in vitro* data that describes a P2X4-mediated potentiation of ENaC activity when extracellular sodium is low. Thus, P2X4 may act as an intrinsic regulator of ENaC activity in the aldosterone-sensitive distal nephron to moderate Na reabsorption in response to changes is Na load. Perhaps of note, *in vivo* pharmacological studies performed in rats, whereby P2XR agonists were intravenously infused showed a marked, progressivelyincreasing diuresis, together with an increase in the Na excretion rate. Authors concluded that activation of P2XR increased renal Na and water excretion in rats via a Na-K-ATPase-dependent mechanism (Jankowski et al., 2011). Evidently, P2X4R in the CD may feature in the complexity of essential hypertension.

Whilst the precise mechanism(s) responsible for P2XRmediated increases in Na excretion require clarification, the evidence in favor of significant functional roles for P2XR in the nephron is increasing. This is further confirmed by pathophysiological findings that provide evidence for the involvement of P2XR in kidney dysfunction. P2X4R have been highlighted as key receptors in inflammasome activation and have been linked to diabetic nephropathy (DN) (Chen et al., 2013). It is wellestablished that tubulointerstitial inflammation is involved in the development of DN, and is mediated by key inflammatory cytokines. Chen and colleagues demonstrated that extracellular ATP causes P2X4R to activate the NOD-like receptor 3 (NLRP3) inflammasome, causing cytokine maturation and release (Chen et al., 2013). In light of the potential link between P2X4R, inflammasome activation, and DN, P2X4R signaling in NLRP3 inflammasomes was investigated using renal biopsies from patients with type-2 diabetes. Authors reported increased expression of P2X4R in renal tubular epithelial cells of patients with type-2 diabetes compared with non-diabetic patients, and that P2X4R expression correlated with expression of IL-1 and IL-18 (Chen et al., 2013). This finding is perhaps not surprising since ATP is one of the best established damage-associated molecular patterns (DAMPs), and as such plays an important role in inflammation and immunity by functioning as a signal of cell damage, stress or death during injury and disease (Chen and Nunez, 2010). A cautionary note: whilst these studies substantiate the hypothesis that P2X4R blockade would be a successful therapeutic strategy in attenuating DN, there is evidence to suggest this may lead to hypertension by disrupting sodium transport in the distal nephron (as previously discussed). This highlights the fact that although P2XR have emerged as attractive therapeutic targets for many pathophysiological conditions, their widespread expression and diverse roles means the manipulation of these receptors is extremely complex.

Activation of P2X7R (albeit via a more established mechanism), like P2X4R, induces downstream inflammatory events, including the NALP3 inflammasome/caspase-1-dependent maturation of IL-1β and IL-18, and their subsequent release from various myeloid cell types (Idzko et al., 2007). There is now also evidence for Madin-Darby canine kidney (MDCK) epithelial cells expressing functional P2X7R, as well as TLR4 and molecules associated with the NALP3 inflammasome (Jalilian et al., 2012), perhaps highlighting their importance in *renal* inflammatory injury and *renal* disease. P2X7R are perhaps the P2XR subtype most linked to pathophysiology of the kidney. Historically, P2X7R (which can form pores under certain circumstances) are known to be pro-inflammatory, and whilst they are extensively expressed in cells of the immune system they are not *highly* expressed in healthy kidney tissue (Hillman et al., 2005). Recent additional lines of evidence that describe alternative roles for P2X7R in kidney dysfunction have revealed a role for P2X7R in: the disrupted calcium homeostasis in peripheral blood mononuclear cells of chronic kidney disease patients (Lajdova et al., 2012), P2XRmediated cytogenesis in polycystic kidney disease (Chang et al., 2011), interstitial fibroblast cell death following ATP release from necrotic renal epithelial cells (Ponnusamy et al., 2011), and a crucial role in driving hypertension and renal injury (Ji et al., 2012b). It seems that both P2X4R and P2X7R are evermore commonly associated with kidney dysfunction. With this in mind we have utilized P2X4R−*/*− and P2X7R−*/*− mouse models and noted that in the P2X4R−*/*−, mRNA for P2X7R was reduced and vice versa, which collectively indicates an interaction of P2X4R with P2X7R at the expressional level to (patho)physiologically regulate renal function (Birch et al., 2013b; Craigie et al., 2013). The specific role of P2XR in renal pathophysiology will be discussed below in greater detail.

#### **NEW ROLES FOR P2XR AND RENAL VASCULATURE**

In addition to tubular epithelial cell expression, P2R are expressed in renal vascular and glomerular cells of the medulla and cortex (Bailey et al., 2012). The role of P2R in renal vasculature is complex and dependent on many factors including species, vascular tone, and variable P2R expression patterns. Despite this, numerous functional studies have previously established that P2R have a regulatory role in the renal vasculature (Inscho et al., 1992; Weihprecht et al., 1992; Eltze and Ullrich, 1996; Inscho, 2001; Rost et al., 2002; Eppel et al., 2006a). Much of the historical evidence surrounding a role for P2XR in the regulation of renal blood flow focuses on the P2X1R, most likely a reflection of this being the key receptor expressed by vascular smooth muscle cells (VSMCs). P2X1R are expressed in afferent arterioles but not in efferent arterioles (Osmond and Inscho, 2010) and functional data suggest that ATP-mediated activation of P2X1R represents an important mechanism for regulating renal blood flow and glomerular capillary pressure by regulating afferent arteriolar resistance and in pressure-dependent autoregulatory adjustments in afferent arteriolar diameter (Inscho et al., 2003). *In vivo* studies in which rabbits were infused intrarenally with α, β-methylene ATP described a reduction in both cortical and medullary blood flow that implicated P2XR in the regulation of regional renal blood flow (Eppel et al., 2006b). The interpretation of these former studies have, however, been challenged by recent mouse micropuncture experiments, that have raised questions as to the role of P2XR specifically in tubuloglomerular feedback (TGF), which is the mechanism responsible for matching glomerular filtration to perfusion in the loop of Henle (Schnermann, 2011). The author of this study reported that both intravenous infusion and luminal perfusion of inhibitors of P2R (at a dose that significantly reduced the blood pressure response to α, β-methylene ATP), failed to affect the TGF response to saturating increases in loop perfusion rate. Moreover, macula densa release of nucleotides appeared not to be involved in the vasoconstriction that accompanies increased loop flow *in vivo* (Schnermann, 2011). These findings are in contrast to an *in vivo* study in which anaesthetized rats were treated with Ip5I, a potent P2X1R antagonist, and showed a significant loss of autoregulatory ability, whereas in anaesthetized rats treated with DPCPX, a potent A1R antagonist, autoregulatory control of renal blood flow remained intact (Osmond and Inscho, 2010). The disparity noted above may reflect the acknowledged differences in the relative expression of P2XR between species. Collectively these data suggest that P2X1R are implicated in renal autoregulatory (and maybe not TGF) responses, although the relative contribution of P2XR to these complex physiological processes requires further clarification and may be species dependent. Furthermore, it is possible that ATP and adenosine signaling pathways integrate to mediate TGF. ATP released in response to increased perfusion pressure and/or increases in tubular concentrations could mediate vasoconstriction of the afferent arteriole via P2XR, but might also be dephosphorylated to form adenosine, which may compound the afferent arteriole vasoconstriction.

In a recent study utilizing the live kidney slice model to investigate regulation of medullary blood flow (MBF) we reported a role for P2XR (and P2YR) in the regulation of vasa recta diameter via contractile pericytes (Crawford et al., 2011). Data described an ATP-mediated constriction of *in situ* vasa recta via contractile pericytes and also demonstrated attenuation of the ATP-mediated constriction in the presence of the P2XR antagonist PPADS. Additionally pericytes constricted vasa recta diameter when live slices were superfused with 2meSATP and BzATP, which act at P2XR. PCR experiments performed on isolated vasa recta to investigate P2R mRNA expression combined with the pharmacological data acquired in functional experiments indicated a putative role for both P2X1R and P2X7R in the regulation of MBF (Crawford et al., 2011). Since pericytes are considered to be relatively undifferentiated having the ability to differentiate further in to VSMCs, it is interesting to note that calcium entry via P2XR has also recently been implicated in the contractile mechanism of renal VSMCs isolated from arcuate arteries and interlobular arteries (Povstyan et al., 2011), thus providing additional evidence for P2XR having a functional role in mediating VSMC contraction in other vascular cells of the kidney. Regarding the functional role of P2XR in pericytes *per se*, P2XR (particularly P2X7R) have also been implicated in the regulation of the retinal pericytes (Sugiyama et al., 2004, 2005) and in the regulation of pericytes of spiral ligament capillaries in the cochlea (Wu et al., 2011).

Given the established role for P2XR in the regulation of vascular cells throughout the body it is not surprising that there are emergent studies in the renal field also highlighting their significance in renal blood flow regulation. What is striking, however, is the accumulating evidence that points toward P2XR playing a critical role in renal pathophysiology, whether the etiology is vascular or tubular in origin.

#### **P2XR AND RENAL PATHOPHYSIOLOGY**

With current funding bodies placing ever increasing importance on translational "bench to bedside" research it is particularly exciting when a molecule or group of molecules emerge as having prominent roles in organ pathology. As eluded to already in this article, there is a compelling mass of evidence that indicates P2XR play crucial and divergent roles in renal pathophysiology. As such, P2XR now represent novel putative targets for renal disease intervention and treatment strategies (North and Jarvis, 2013), and are currently the subject of avid investigation by multiple major international pharmaceutical companies.

Much of the current focus on the potential role of P2XR in renal disease is focused on the homomeric P2X7R, this ligandgated cation channel being unique in terms of both its structure and function. Unlike its other family members it is thought to exist only as a homomer and has an extended C-terminus with 200 extra amino acid residues, which is thought to be pivotal in regulating its function, being involved in: determining cellular localization, stimulation of various signaling cascades, proteinprotein interactions, and post-translational modification of the receptor itself (Costa-Junior et al., 2011). Sustained stimulation of P2X7R with high agonist concentrations (mM) is classically associated with the formation of large transmembrane pores that disrupt the ion gradients within cells resulting in subsequent cell death by either apoptosis or necrosis (Surprenant et al., 1996). The P2X7R is typically expressed by immune cells and mediates release of pro-inflammatory cytokines, prominently interleukin-1 beta (IL-1β), via activation of caspase 1 (Ferrari et al., 1997; Costa-Junior et al., 2011). In contrast to the healthy kidney in which P2X7R is virtually undetected, P2X7R expression is readily detected in inflammatory renal diseases, such as glomerulonephritis (Vonend et al., 2004). The role of P2X7R in renal pathophysiology is not however limited to inflammatory disease. Indeed current topical studies demonstrate that P2X7R plays a detrimental role in a broad range of renal pathologies that will be discussed below.

A recent study has suggested that the pro-inflammatory nature of P2X7R could contribute to the development of hypertension and consequential renal injury. The functional role of P2X7R in hypertension was investigated using WT and P2X7−*/*− mice rendered hypertensive by a high salt diet and deoxycorticosterone acetate (DOCA) treatment (Ji et al., 2012b). After DOCA-salt treatment, P2X7R mRNA and protein expression was increased in WT mice, accompanied by a greater increase in systolic blood pressure (SBP), significantly greater than that of P2X7−*/*− mice. Inflammatory cell infiltration was reduced in P2X7−*/*− mice, and IL-1β release from macrophages was inhibited. Furthermore, the downstream effectors of IL-1β signaling, COX-2 and ROS were also affected; COX-2 expression being reduced in P2X7−*/*− mice and anti-oxidant levels being increased in their serum. Authors of the study suggested that blockade of the P2X7Rmediated inflammatory responses protect renal function against P2X7R-associated renal injury, this being reflected by reduced albuminuria and renal interstitial fibrosis, and increased creatinine clearance (Ji et al., 2012b). With regard to hypertension, both WT and P2X7−*/*− exhibited initial increases in SBP following DOCA-salt treatment, and only at day-4 of treatment were differences in SBP observed between the two animal groups; P2X7−*/*− mice having significantly lower SBP than WT mice. This study built on previous observations demonstrating that immune cell infiltration was pivotal to the development of salt-sensitive hypertension (Franco et al., 2007), authors of this preceding study reported P2X7R expression by immune cells exacerbated renal injury by instigating a vicious cycle of inflammation. Additional *in vivo* evidence to support this has been attained from pharmacological studies performed in Dahl salt-sensitive (DS) rats (Ji et al., 2012a). Hypertensive rats were infused with two P2X7R selective antagonists (brilliant blue G and A-438079); the saltsensitive hypertension, urinary protein or albumin excretion, renal interstitial fibrosis, and macrophage and T-cell infiltration in the DS rats was markedly reduced, whilst creatinine clearance significantly improved (Ji et al., 2012a). Collectively these novel studies provide persuasive evidence that the P2X7R is critical in hypertension and renal injury progression, thus highlighting the therapeutic potential for this receptor in a number of disease states underlying salt-sensitive hypertension, including but not limited to, hyperaldosteronism.

As previously mentioned, blockade of P2X7R has been shown to inhibit IL-1β release from renal macrophages (Ji et al., 2012b), and this may have significant consequences for the underlying mechanisms of certain forms of renal disease. Renal fibroblasts are activated by IL-1β amongst other cytokines and due to the P2X7R/IL-1β axis it seems pertinent to assume P2X7R-mediated attenuation of fibroblast activation could occur. Whilst this may have beneficial effects in terms of reducing renal interstitial fibrosis, activation of renal fibroblasts post acute kidney injury (AKI) is vital for normal renal repair. AKI is typically characterized by renal tubular cell death and associated release of high concentrations of ATP into the interstitium, combined with the associated damage to the tubular basement membrane, to which interstitial fibroblasts are directly connected; this milieu provides an ideal setting for P2X7R to play a prominent role in either AKI associated fibrosis or renal repair. To investigate this paradigm, a recent study investigated the effects of supernatant from necrotic renal proximal tubular cells (RPTC) on the viability of renal fibroblasts (Ponnusamy et al., 2011). The supernatant from necrotic RPTC was shown to induce the expression of P2X7R in cultured rat renal interstitial fibroblast cells (NRK-49F), and the ATP released from necrotic RPTC induced NRK-49F cell death by a P2X7R-mediated mechanism, therefore demonstrating a deleterious direct renal epithelial-fibroblast cross-talk pathway (Ponnusamy et al., 2011). It was concluded that damaged renal epithelial cells could directly induce the death of renal interstitial fibroblasts by ATP activation of P2X7R. This evidence was not, however, in accordance with a previous study which showed that the number of Epo-TAg (a genetic marker for cortical interstitial fibroblasts) expressing fibroblasts is reduced in the injured kidney, but upon intense stimulation such as severe anemia or hypoxia, there was a greater increase in Epo-TAg expressing fibroblasts in the injured kidney which consequently narrowed the disproportion between the injured and uninjured kidney. Authors conclude that rather than fibroblasts being destroyed in the event of AKI, the threshold for stimulated gene expression is increased (Maxwell et al., 1997), which could possibly be mediated by P2X7R. So it is unclear if AKI results in a reduced number of renal interstitial fibroblasts or an alteration in cell properties.

Renal interstitial fibroblasts have both beneficial and deleterious effects on the kidney by playing a key role in renal repair and renal fibrosis, respectively. Consequently, interstitial fibroblast cell proliferation, activation, and death are tightly regulated by numerous factors. It is certainly plausible that ATP is one such factor and P2X7R is an important mediator. It has been shown *in vitro* that P2X7R activation directly influences interstitial fibroblast cell death (Ponnusamy et al., 2011). However, the possibility exists that P2X7R also indirectly influences fibroblast activation by inducing IL-1β release from infiltrating immune cells (Ji et al., 2012b). Both studies propose P2X7R as a therapeutic target. Although pharmacological blockade of P2X7R was highly effective at attenuating renal fibrosis, the implications this may have on the repair response post-AKI need to be considered.

Lastly, in addition to the pro-inflammatory and pro-death functions of P2X7R discussed in preceding paragraphs there seems also to be a role for P2X7R in autosomal dominant polycystic kidney disease (ADPKD). This common, yet complex, genetic renal disease occurs in response to mutations in either the PKD1 or PKD2 gene (Mochizuki et al., 2013), and is characterized by progressive cyst enlargement due to excess fluid secretion and proliferation of renal tubular epithelial cells (Torres et al., 2009). Whilst this condition is a major cause of end stage renal disease, there still lacks a clinically approved therapy, primarily due to the slow progression of the disease and complexity of its development. The current focus is to assess the potential of combination therapies targeting either cyst development through inhibition of cell proliferation, and/or cyst growth through attenuation of fluid secretion. Recent data yielded from the zebrafish model of ADPKD where PKD2 is knocked-down, described increased P2X7R mRNA expression at an early stage, which coincided with cyst formation in the glomerulus and tubular regions in 75% of PKD2 morphants. Pharmacological blockade with a P2X7R antagonist OxATP reduced the frequency of cyst formation to 35%, suggesting that P2X7R contributes in some way to cyst formation in ADPKD (Chang et al., 2011). PKD2 morphants demonstrate increased ERK activation in primary cilia, which is linked to enhanced cell proliferation and fluid secretion and hence cyst enlargement in PKD. Previous studies have demonstrated that P2X7R acts as a flow sensor for ERK activation in osteoblasts (Okumura et al., 2008). Given that authors describe inhibition of P2X7R as suppressing the elevated ERK activation in PKD2 morphants by over 50%, coupled with the fact cilium bending has been shown to trigger ATP release, suggests a similar P2X7Rcoupled mechanism may be present in renal epithelial cells. This hypothesis is further substantiated by findings that demonstrated inhibition of P2X7R did not suppress cyst progression through the traditional anti-inflammatory mechanism (Chang et al., 2011; Praetorius and Leipziger, 2013). Whilst extremely novel, these data are, however, at odds with previously published data collected from studies using the MDCK model of cyst formation. Here authors agreed that ERK activation is an important factor in cyst growth. However, they found no evidence for the involvement of a P2XR, and concluded that a P2YR is responsible for cAMP-dependent activation of the ERK pathway and cyst growth (Turner et al., 2007).

## **SUMMARY**

P2XR represent a receptor group important in the regulation of tubular and vascular function in both kidney health and disease (see **Figures 1**, **2** for a summary). Much of the recent data providing compelling functional evidence that substantiate this phenomenon has been generated from knockout mouse studies, which further attests to the importance of these models in improving our knowledge of how kidney function is regulated. As a result of these studies there appear to be three key P2XR subunits/subtypes, P2X1, 4, and 7, which have emerged as having numerous roles in the regulation of kidney function. As such, these receptor subtypes not only represent favorable novel therapeutic targets in multiple pathological settings, but also represent a novel focus for future research on the role of nucleotides in the kidney.

## **ACKNOWLEDGMENTS**

We gratefully acknowledge the financial support of the BBSRC, the MRC, and The Wellcome Trust.

## **REFERENCES**

Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells. *Nat. Med.* 13, 913–919. doi: 10.1038/nm1617

*Blood Press. Res.* 35, 48–57. doi: 10.1159/000330349

*Semin. Cell Dev. Biol.* 24, 3–10. doi: 10.1016/j.semcdb.2012.10.003

(P2X7). *Science* 272, 735–738. doi: 10.1126/science.272.5262.735

receptor in capillary endothelial cells of the cochlear spiral ligament and its role in regulating the capillary diameter. *Am. J. Physiol. Heart Circ. Physiol.* 301, H69–H78. doi: 10.1152/ajpheart. 01035.2010

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 05 July 2013; accepted: 05 September 2013; published online: 27 September 2013.*

*Citation: Birch RE, Schwiebert EM, Peppiatt-Wildman CM and Wildman SS (2013) Emerging key roles for P2X receptors in the kidney. Front. Physiol. 4:262. doi: 10.3389/fphys.2013.00262*

*This article was submitted to Renal and Epithelial Physiology, a section of the journal Frontiers in Physiology.*

*Copyright © 2013 Birch, Schwiebert, Peppiatt-Wildman and Wildman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


**Conflict of interest**

The authors whose names are listed in this paper certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.


Section 3 Applications

measures for social networks under active attack. Information Sciences.

*Security and Privacy From a Legal, Ethical, and Technical Perspective*

[19] Johnson DS. Approximation algorithms for combinatorial problems. Journal of Computer and System Sciences. 1974;**9**(3):256-278

[20] Zachary WW. An information flow model for conflict and fission in small groups. Journal of Anthropological Research. 1977;**33**(4):452-473

[21] Loomis CP, Morales JO, Clifford RA, Leonard OE. Turrialba: Social Systems and the Introduction of Change. Glencoe, IL: Free Press; 1953

[22] Gleiser PM, Danon L. Community structure in jazz. Advances in Complex

[23] Guimera R, Danon L, Diaz-Guilera A, Giralt F, Arenas A. Self-similar community structure in a network of human interactions. Physical Review E.

[24] Enron email network. Available from: UC Berkeley Enron Email Analysis website http://bailando.sims. berkeley.edu/enron\_email.html

[25] Paranjape A, Benson AR, Leskovec J.

[26] Panzarasa P, Opsahl T, Carley KM. Patterns and dynamics of users' behavior and interaction: Network analysis of an online community. Journal of the American Society for Information Science and Technology.

[27] Hamsterster friendships network dataset–KONECT, April 2017. Available from: http://konect.uni-koblenz.de/ networks/petster-friendships-hamster)

Motifs in temporal networks. In: Proceedings of the 10th ACM International Conference on Web Search and Data Mining; ACM. 2017.

Systems. 2003;**6**(04):565-573

2003;**68**(6):065103

pp. 601-610

2009;**60**(5):911-932

**138**

2019;**473**:87-100

**141**

**Chapter 9**

**Abstract**

Networks

in both academia and industry.

comes with significant limitation (e.g., [1]).

**1. Introduction**

following:

that mechanism.

Beyond Differential Privacy:

with Deep Generative Neural

*Ofer Mendelevitch and Michael D. Lesh*

Synthetic Micro-Data Generation

Recent advances in generative modeling, based on large scale deep neural networks, provide a novel approach for sharing individual-level datasets (micro-data) without privacy concerns. Unlike differential privacy, which enforces a specific query mechanism on data to ensure privacy, generative models can accurately learn the statistical patterns of such micro-data and then be used to generate "synthetic data" that accurately reflects these statistical patterns, yet contain none of the original data itself, and thus can be safely shared for analysis and modeling without compromising privacy. The successful application of these techniques to various industries including healthcare, finance, and autonomous vehicles is promising and results in continued investment in research and development of generative models

**Keywords:** generative models, synthetic data, deep neural networks, micro-data

Differential privacy, created more than a decade ago, continues to play an important role in protecting privacy of micro-data while enabling statistical analysis. Initially applied by statistics agencies such as the US census bureau, it is now well recognized that, although useful for some applications, differential privacy

To understand some of the limitations of differential privacy, consider the

• Differential privacy is defined around the concept of a *mechanism*; as such, it is not intended to create "sharable datasets," but instead allows a user (analyst) to submit various types of queries (via the defined *query mechanism*), requesting some kind of aggregate statistics, like summary statistics of the original data. This limits the usability of differential privacy to queries that are supported by

• An appropriate *privacy budget* needs to be decided upon, and in practice it's often difficult to agree on what that budget needs to be. In fact, practical


*Article* **Changes in Dietary Habits and Exercise Pattern of Korean Adolescents from Prior to during the COVID-19 Pandemic**

**So Young Kim 1 , Dae Myoung Yoo 2 , Chanyang Min 2,3 and Hyo Geun Choi 2,4,\***

**Abstract:** This study aimed to investigate changes in the exercise pattern and dietary habits in adolescents during the COVID-19 pandemic. The 12–18-year-old population in the Korea Youth Risk Behavior Web-Based Survey data of 2019 and 2020 was enrolled. The exercise pattern and dietary habits of 105,600 participants (53,461 in the 2019 group and 52,139 in the 2020 group) were compared. The odds ratios (ORs) for the dietary habits and exercise pattern of the 2020 group compared to the 2019 group were analyzed using multiple logistic regression analysis with complex sampling. The odds of eating fruit, drinking soda, drinking sweet drinks, and consuming fast food were lower in the 2020 group than in the 2019 group (all *p* < 0.001). The odds of eating breakfast were higher in the 2020 group than in the 2019 group (all *p* < 0.001). The 2020 group showed lower odds of frequent vigorous and moderate aerobic exercise and higher odds of frequent anaerobic exercise than the 2019 group (all *p* < 0.001). During the COVID-19 pandemic, adolescents consumed less fruit, soda, and sweet drinks, while they had more breakfast. The frequency of aerobic exercise was lower, while the frequency of anaerobic exercise were higher during the COVID-19 pandemic period.

**Keywords:** COVID-19; dietary habits; exercise pattern; adolescents; cohort study

#### **1. Introduction**

The outbreak of COVID-19 had various impacts on our daily lives [1]. Due to the high infectivity of SARS-CoV-2, the prevention of contagious infection and the isolation of COVID-19 patients have been among the most important strategies in response to the COVID-19 pandemic. The quarantine maneuvers included social distancing, restriction of social activities, and lockdown. These limitations on social circumferences increased the time spent indoors and eating home-cooked meals, while they decreased outdoor activities and eating out [2]. The lockdown of schools and workplaces changed the schedules of daily life for many people.

A number of researchers have been concerned about the changes in dietary habits and exercise pattern during the COVID-19 pandemic [3–7]. A cross-sectional survey in Poland found that approximately 43% and 52% of adults consumed more food and snacks, respectively, during the COVID-19 lockdown [3]. Approximately 30% of participants gained weight because of the frequent consumption of fast food (3.0 ± 1.6 kg) during the COVID-19 lockdown [3]. Another survey in Italy reported that approximately 33.5% of participants changed their eating habits, and approximately 81% of participants increased frozen food consumption during the COVID-19 lockdown [5]. This survey estimated that home confinement during the COVID-19 crisis was negatively associated with physical activity intensity and positively associated with sedentary time during the COVID-19 lockdown [5].

**Citation:** Kim, S.Y.; Yoo, D.M.; Min, C.; Choi, H.G. Changes in Dietary Habits and Exercise Pattern of Korean Adolescents from Prior to during the COVID-19 Pandemic. *Nutrients* **2021**, *13*, 3314. https://doi.org/10.3390/ nu13103314

Academic Editor: Carlos Vasconcelos

Received: 18 August 2021 Accepted: 19 September 2021 Published: 23 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Importantly, adolescents are in periods of development and are susceptible to acquiring poor lifestyle patterns, including habits related to diet and exercise pattern [5,8,9]. Indeed, an international online survey reported that adolescents consumed more fast food and sweet food during home confinement due to the COVID-19 lockdown (44.6% vs. 64% for fast food; 14% vs. 20.7% for sweet food) [5]. In addition, 70.5% of participants reduced their exercise pattern during the COVID-19 lockdown [5]. An international online survey demonstrated reduced physical activity levels (vigorous, moderate, walking, and overall) during home confinement due to the COVID-19 crisis (5.04 ± 2.51 day/week vs. 3.83 ± 2.82 day/week of physical activities, *p* < 0.001) [6]. However, the information on the respondents was very limited, and the analysis did not consider possible confounders, such as economic status, sleep time, and obesity. As multiple lifestyle factors influence dietary habits and exercise pattern, these factors should be concurrently considered in analyses of the impact of the COVID-19 pandemic on diet and exercise pattern.

As changes in dietary habits and exercise pattern during the COVID-19 pandemic were suggested to increase the risk of metabolic disorders in a previous study [10], we hypothesized that there may have also been changes in the dietary habits and physical activity during the COVID-19 pandemic in Korean adolescents. To test this hypothesis, the dietary habits and exercise pattern were analyzed in adolescents in before and during the COVID-19 pandemic periods. The first patient with COVID-19 was diagnosed on 19 February 2020. Thus, this study compared the 2019 group as a prepandemic population (surveyed from 3 June 2019 to 12 July 2019) with the 2020 group as a COVID-19 pandemic population (surveyed from 3 August 2020 to 13 November 2020 during the social distancing strategies that had been maintained in Korea without complete lockdowns).

#### **2. Materials and Methods**

#### *2.1. Study Population and Data Collection*

This cross-sectional study used data from the Korea Youth Risk Behavior Web-based Survey (KYRBWS) and covered the nation using statistical methods based on the designed sampling and adjusted weighted values. The KYRBWS obtained data from South Korean adolescents using stratified, two-stage (schools and classes) clustered sampling based on data from the Education Ministry. Sampling was weighted by statisticians, who performed poststratification analyses and considered the nonresponse rates and extreme values. Data from the 2019 and 2020 KYRBWS were analyzed. The details of the sampling methods are described on the KYRBWS website [11]. The KCDC collected the data, and Korean adolescents from 7th through 12th grade voluntarily and anonymously completed the self-administered questionnaire. The validity and reliability of the KYRBWS have been documented by other studies [12,13].

Of the 112,251 total participants (57,303 in 2019; 54,948 in 2020), the following were excluded from this study: participants without information on age (*n* = 373), height or weight (*n* = 2596), and sedentary time (*n* = 3682). Finally, 105,600 participants (53,461 in 2019; 52,139 in 2020), who were 12 through 18 years old, were included in this study (Figure 1).

#### *2.2. Survey*

#### 2.2.1. Exposure

In each of the 2019 and 2020 surveys, adolescent participants were selected as stated above to represent the entire adolescent population in Korea. The participants from 2019 were not followed up with. The participants of 2020 were newly selected from the entire Korean adolescent population.

**Figure 1.** *F*lowchart of the major steps involved in the conduction of this study. **Figure 1.** Flowchart of the major steps involved in the conduction of this study.

#### 2.2.2. Outcomes

*2.2. Survey* 2.2.1. Exposure In each of the 2019 and 2020 surveys, adolescent participants were selected as stated above to represent the entire adolescent population in Korea. The participants from 2019 were not followed up with. The participants of 2020 were newly selected from the entire Korean adolescent population. Dietary habits were surveyed by asking about the frequency of particular dietary habits in the last week. The frequencies of breakfast, fresh fruit, soda beverage (except for pure soda drinks and caffeinated drinks), sweet drinks, and fast food intake were assessed using questionnares. "How many days did you eat breakfast, fresh fruit, soda beverage (except for pure soda drinks and caffeinated drinks), sweet drink, and fast food in the recent 7 days, respectively?".

2.2.2. Outcomes Dietary habits were surveyed by asking about the frequency of particular dietary habits in the last week. The frequencies of breakfast, fresh fruit, soda beverage (except for pure soda drinks and caffeinated drinks), sweet drinks, and fast food intake were assessed using questionnares. "How many days did you eat breakfast, fresh fruit, soda beverage (except for pure soda drinks and caffeinated drinks), sweet drink, and fast food in the recent 7 days, respectively?". Regarding exercise pattern, aerobic exercise involving vigorous physical activity was assessed with the following question: "How many days did you exercise with high intense enough to sweat more than 20 min in the recent 7 days?". Aerobic exercise involving moderate physical activity was assessed by asking: "How many days did you exercise until heart rate increase or be short of breath more than 60 min in the recent 7 days?". Anaerobic strength exercises were assessed by asking: "How many days did you exercise to increase muscle power such as sit-up, lift weight, or chin-up bar in the recent 7 days?".

> Regarding exercise pattern, aerobic exercise involving vigorous physical activity was assessed with the following question: "How many days did you exercise with high intense

#### 2.2.3. Covariates

enough to sweat more than 20 min in the recent 7 days?". Aerobic exercise involving moderate physical activity was assessed by asking: "How many days did you exercise until Body mass index (BMI, kg/m2 ) was calculated using height and weight. Mean sedentary time (h/day) and leisure time were calculated as 5/7 of the time on weekdays plus 2/7 of the time on weekends [14,15]. Sleep times were calculated as 5/7 of the time on weekdays plus 2/7 of the time on weekends [14,15]. The self-reported economic level was categorized into 3 levels: high, middle, and low. Subjective self-reported health status was categorized into 4 levels that ranged from very healthy to unhealthy. Subjective body shape image was categorized into 3 levels, including thin, normal, and obese. Smoking

in the last 30 days was assessed and categorized into 3 levels: 0, 1–19, and ≥20 days [16]. Drinking alcohol in the last 30 days was categorized into 3 levels: 0, 1–2, and ≥3 days [16].

#### *2.3. Statistical Analysis*

The general characteristics of the data from the 2019 and 2020 surveys were compared using *t*-tests and chi-square tests.

The odds ratios (ORs) for particular dietary habits (breakfast, fresh fruit consumption, soda beverage, sweet drinks, and fast food intake) and exercise pattern (vigorous physical activity, moderate physical activity, and strength exercise) from 2020 compared to 2019 were calculated using multiple logistic regression analysis with complex sampling.

Crude and partially adjusted (age, BMI, sedentary time for study or leisure, sex, economic level, sleep time, subjective health status, subjective body shape image, and smoking and alcohol consumption) and fully adjusted (partial model plus dietary habits and exercise pattern) models were designed. Subgroups determined by sex and school level (middle school vs. high school) were analyzed.

Two-tailed analyses were conducted, and *p*-values lower than 0.05 were considered to indicate significance; 95% confidence intervals (Cis) were also calculated. The weights recommended by the KYRBWS were applied, and thus complex sampling was applied. The data were analyzed using SPSS ver. 25.0 (IBM, Armonk, NY, USA).

#### *2.4. Ethics Approval*

The ethics committee of Hallym University approved the use of these data. The study was exempted from the need for written informed consent by the Institutional Review Board (2019-09-005). All Korea Youth Risk Behavior Web-Based Survey (KYRBWS) data analyses were conducted in accordance with the guidelines and regulations provided by the Institutional Review Board of the Centers for Disease Control and Prevention of Korea (KCDC). The understanding, reliability, and validity of each question were investigated by the KCDC to verify the applicability of the surveys [11].

#### **3. Results**

The average sedentary time reported in the study was 6.6 (±3.7) hours/day in the 2019 group and 5.9 (±3.4) hours/day in the 2020 group (*p* < 0.001; Table 1). The average sedentary time for leisure was 3.3 (±2.3) hours/day in the 2019 group and 4.3 (±2.8) hours/day in the 2020 group (*p* < 0.001). Reporting subjective body shape image as obese was lower in the 2019 group than in the 2020 group (37.5% vs. 38.5%, *p* < 0.001). The frequencies of smoking and alcohol consumption were higher in the 2019 group than in the 2020 group (*p* < 0.001). The distributions of BMI, sleep time, and subjective health status were different between the 2019 group and the 2020 group (all *p* < 0.001).

In the fully adjusted model, the 2020 group demonstrated lower odds of eating fruit, fast food, drinking soda, and drinking sweet drinks than the 2019 group (all *p* < 0.001; Table 2). On the other hand, the frequency of breakfast was lower in the 2019 group than in the 2020 group (*p* < 0.001). In middle school students, the frequencies of eating fruit, drinking soda, drinking sweet drinks, and eating fast food were lower in the 2020 group than in the 2019 group (all *p* < 0.001; Table S1). In high school students, the frequencies of eating fruit, drinking soda, and drinking sweet drinks were lower in the 2020 group than in the 2019 group (all *p* < 0.001, Table S2). When analyzed by sex, men showed lower frequencies of drinking soda and sweet drinks in the 2020 group than in the 2019 group (all *p* < 0.001; Table S3). In women, the frequencies of eating fruit, drinking sweet drinks, and eating fast food were lower in the 2020 group than in the 2019 group (all *p* < 0.05, Table S4).

**Table 1.** General Characteristics of Participants.

\* Independent *t*-test, Significance at *p* < 0.05. † Chi-square test, Significance at *p* < 0.05.

Regarding exercise pattern, the frequencies of vigorous and moderate aerobic exercise were lower in the 2020 group than in the 2019 group (all *p* < 0.001; Table 3). The 2020 group showed lower odds regarding the frequency of vigorous and moderate aerobic exercise than the 2019 group (all *p* < 0.001). In contrast, the frequency of anaerobic exercise was higher in the 2020 group than in the 2019 group (*p* < 0.001). The 2020 group showed higher odds regarding the frequency of anerobic exercise than the 2019 group (*p* < 0.001). Middle school students showed a lower frequency of vigorous aerobic exercise and a higher frequency of anaerobic exercise in the 2020 group than in the 2019 group (all *p* < 0.001; Table S1). High school students showed lower frequencies of vigorous and moderate aerobic exercise and a higher frequency of anaerobic exercise in the 2020 group than in the 2019 group (all *p* < 0.001; Table S2). When analyzed by sex, both men and women showed a lower frequency of vigorous aerobic exercise and a higher frequency of anaerobic exercise in the 2020 group than in the 2019 group (all *p* < 0.001; Tables S3 and S4).

**Table 2.** Odd ratios of dietary habits in 2020 compared to 2019 in total participants.

\* Multiple logistic regression analysis with complex sampling, Significance at *p* < 0.05; † Adjusted for age, BMI, sedentary time for study or leisure, sex, economic level, sleep time, subjective health status, subjective body shape image, smoking, and drinking alcohol histories. ‡ Adjusted for partial model plus dietary habit and exercise pattern.

**Table 3.** Odd ratios of exercise pattern in 2020 compared to 2019 in total participants.

\* Multiple logistic regression analysis with complex sampling, Significance at *p* < 0.05. † Adjusted for age, BMI, sedentary time for study or leisure, sex, economic level, sleep time, subjective health status, subjective body shape image, smoking, and drinking alcohol histories. ‡ Adjusted for partial model plus dietary habit and physical activities.

#### **4. Discussion**

The consumption of fast food, fruit, and drinks such as soda was lower, while the consumption of breakfast was higher during the COVID-19 crisis than in the prepandemic era. The pattern of exercise showed bidirectional changes, with lower aerobic exercise and higher anaerobic exercise during the COVID-19 pandemic when compared with the prepandemic period in this cohort of Korean adolescents. This study improved previous findings on the impact of the COVID-19 pandemic on dietary habits and exercise pattern by concurrently analyzing both the dietary habits and exercise pattern in an adolescent population. In addition, age and sex were subgrouped, and the impact of the COVID-19 pandemic on diet and exercise pattern was analyzed in each subgroup.

The COVID-19 pandemic was associated with decreased intakes of fast food, fruit, soda, and sweet drinks in this cohort of Koreran adolescents. On the other hand, the frequency of breakfast was increased during the COVID-19 pandemic. The decreased consumption of soda, sweet drinks, and fast food could be explained by less frequent eating out due to home confinement. The consumption of fast food and soda intake have mostly been associated with eating out in Korean adolescents [17]. In addition, healthseeking behavior could have decreased the consumption of unhealthy food during the COVID-19 pandemic in this study. The COVID-19 pandemic could have encouraged adolescents to address health-related issues because they were repeatedly exposed to

health campaigns and education regarding quarantine maneuvers from school and the mass media. Moreover, the increased time with family and frequency of home cooking could have provided an opportunity for adolescents to discuss healthy behavior with their parents and to alter their unhealthy dietary habits [18,19]. The traditional Korean diet at home includes cooked rice and a number of banchan, such as kimchi. Thus, Koreans could consume sufficient nutrients from vegetables, legumes, and fish, with a low intake of red meat [20]. Frequently cooking dinner was related to a higher Healthy Eating Index-2015 in the US (≥7 times/week: +3.57 points, *p* < 0.001) [19]. During the COVID-19 crisis, a cross-sectional survey in Italy demonstrated that approximately 3.3% of respondents quit smoking and 38.3% of respondents increased physical activities, such as bodyweight training [4]. The lockdown and other instances of quarantine could have resulted in difficulties acquiring food, especially fresh food, such as fruit, in some adolescents without caregivers. Indeed, it was reported that over 18% of adults lost weight during the COVID-19 lockdown (−2.9 ± 1.5 kg) [3]. In our cohort, although the BMI was slightly higher in the COVID-19 pandemic group than in the prepandemic group, it was not high enough to have a clinical impact. On the other hand, the increasd time to eat due to the lockdown of schools could have resulted in increasing breakfast consumption, which was observed in this study. Because the restriction of outdoor activities can increase the time available to eat, the increase in breakfast consumption could have been relative to the irregular meal time during school hours.

Anaerobic exercise was higher in the COVID-19 pandemic group than in the prepandemic group in this study. In contrast, vigorous aerobic exercise was lower in the COVID-19 pandemic group than in the prepandemic group. A previous survey estimated 1.3-fold and 1.2-fold higher rates of moderate-to-vigorous physical activity in male and middle-aged populations during the COVID-19 pandemic [21]. The increase in anaerobic exercise could have been a compensation for the decrease in aerobic exercise during the COVID-19 pandemic that was found in the study. A cross-sectional study reported that children spent less time in extracurricular sports (23.5%), and 94.5% of children watched screens for 1.5 (0.5–3.0) hours/day [22]. Quarantine and social distancing maneuvers might have limited outdoor activities [23]. The restrictions on physical activities during the COVID-19 pandemic may have been caused by both individual and community aspects [24,25]. Physical restrictions on outdoor activities may have reduced individuals' aerobic exercise. In addition, the social distancing policy included restrictions on group sports activities, such as football leagues, swimming, and baseball games, all of which are forms of aerobic exercise. On the other hand, the increased time spent at home may have encouraged people to spend time at home training. Moreover, awareness of health-related issues via health promotion campaigns encouraged people to engage in home-based exercises [23,26]. Indeed, a surveillance in Thailand reported a 1.5-fold higher rate of moderate-to-vigorous physical activity in adults who were exposed to fit-from-home campaigns [21]. Furthermore, physical activity was reported to be greatly influenced by personal factors in children, while sedentary behavior was influenced by environmental factors [27]. Thus, physical activity was less influenced by the COVID-19 pandemic, but the geographic area of activity decreased.

The present cohort was composed of a large, nationwide, representative population of Korean adolescents. The validation and regulation of the data were managed by KCDC. Many surveyed items of sedentary time for study, sedentary time for leisure, economic level, sleep time, subjective health status, subjective body shape image, smoking, alcohol consumption, and anthropometric index of BMI were included for analyses. We included these variables as they might have been able to affect diet and exercise pattern. However, because this study was based on the survey, recall bias may have existed. In addition, the amount of food consumption could not be evaluated. The detailed types of physical exercise could not be identified. Furthermore, the study participants could not be longitudinally followed up with; instead, new participants were enrolled in each year. Although numerous factors were surveyed and adjusted for, influential variables may have been unmeasured, such as comorbidities that restrict dietary consumption or exercise pattern. Lastly, because

this study included a Korean population, ethnic or regional differences could be present regarding dietary habits or exercise pattern of other ethnic groups [28].

#### **5. Conclusions**

There were changes in both the dietary habits and pattern of exercise between the pre- and during COVID-19 pandemic periods in this cohort of Korean adolescents. The consumption of fruit, fast food, soda, and sweet drinks was lower, while the consumption of breakfast was higher during the COVID-19 pandemic than in the prepandemic period. The frequencies of vigorous and moderate aerobic exercise were lower, while the frequency of anerobic exercise was higher during the COVID-19 pandemic than in the prepandemic period.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/nu13103314/s1, Table S1. Odd ratios of dietary habit or exercise pattern in 2020 compared to 2019 in middle school student. Table S2. Odd ratios of dietary habit or exercise pattern in 2020 compared to 2019 in high school student. Table S3. Odd ratios of dietary habit or exercise pattern in 2020 compared to 2019 in men. Table S4. Odd ratios of dietary habit or physical activities in 2020 compared to 2019 in women.

**Author Contributions:** H.G.C. designed the study; D.M.Y., C.M. and H.G.C. analyzed the data; S.Y.K. and H.G.C. drafted and revised the paper; and H.G.C. drew the figures. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by research grants (NRF-2018-R1D1A1A02085328 and 2021-R1C1C100498611) from the National Research Foundation (NRF) of Korea. The APC was funded by NRF-2018-R1D1A1A02085328.

**Institutional Review Board Statement:** The ethics committee of Hallym University (2020-07-022) permitted this study following the guidelines and regulations.

**Informed Consent Statement:** Written informed consent was waived by the Institutional Review Board.

**Data Availability Statement:** Releasing of the data by the researcher is not legally permitted. All data are available from the database of the Korea Center for Disease Control and Prevention. The Korea Center for Disease Control and Prevention allows data access, at a particular cost, for any researcher who promises to follow the research ethics. The data of this article can be downloaded from the website after agreeing to follow the research ethics.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**5. Conclusions**

An IA suitable for bioimpedance-based IoT applications and based on the ICF technique has been presented. The SSF structure has been incorporated in the design of the IA in order to reduce input referred noise and silicon area. In addition, a FD implementation has been selected to enhance the overall performance of the circuit. The proposed ICF FD IA been designed and fabricated in 180 nm CMOS technology to operate with a supply voltage of 1.8 V and provide a voltage gain of 4 V/V. Measurements on 10 different samples of the silicon prototype showed wide bandwidth, high CMRR and linear signal processing, thus confirming the suitability of the proposed solution for the intended application.

**Author Contributions:** Conceptualization, I.C., J.M.C. and J.L.A.; methodology, I.C., J.M.C. and J.L.A.; software, I.C. and J.M.C.; formal analysis, J.M.C. and R.P.-A.; investigation, I.C., J.M.C. and J.L.A.; resources, I.C., J.M.C. and M.Á.D.; data curation, I.C., J.M.C., M.Á.D. and R.P.A.; writing original draft preparation, I.C., J.M.C. and J.L.A.; writing—review and editing, I.C., J.M.C., J.L.A., M.Á.D., R.P.-A. and J.F.D.-C.; visualization, I.C. and J.M.C.; supervision, J.M.C. and J.L.A.; project administration, J.F.D.-C.; funding acquisition, J.L.A. and J.F.D.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** Work funded by projects RTI2018-095994-B-I00, from MCIN/AEI/10.13039/501100011033, and IB18079, from *Junta de Extremadura* R&D Plan, and by Fondo Europeo de Desarrollo Regional (FEDER) Una manera de hacer Europa. Silicon samples granted by EUROPRACTICE MPW and design tool support.

**Conflicts of Interest:** The authors declare no conflict of interests.