Hyperpolarization‐activated cation and T‐type calcium ion channel expression in porcine and human renal pacemaker tissues

Abstract

Renal pacemaker activity triggers peristaltic upper urinary tract contractions that propel waste from the kidney to the bladder, a process prone to congenital defects that are the leading cause of pediatric kidney failure. Recently, studies have discovered that hyperpolarization‐activated cation (HCN) and T‐type calcium (TTC) channel conductances underlie murine renal pacemaker activity, setting the origin and frequency and coordinating upper urinary tract peristalsis. Here, we determined whether this ion channel expression is conserved in the porcine and human urinary tracts, which share a distinct multicalyceal anatomy with multiple pacemaker sites. Double chromagenic immunohistochemistry revealed that HCN isoform 3 is highly expressed at the porcine minor calyces, the renal pacemaker tissues, whereas the kidney and urinary tract smooth muscle lacked this HCN expression. Immunofluorescent staining demonstrated that HCN ⁺ cells are integrated within the porcine calyx smooth muscle, and that they co‐express TTC channel isoform Cav3.2. In humans, the anatomic structure of the minor calyx pacemaker was assayed via hematoxylin and eosin analyses, and enabled the visualization of the calyx smooth muscle surrounding adjacent papillae. Strikingly, immunofluorescence revealed that HCN3⁺/Cav3.2⁺ cells are also localized to the human minor calyx smooth muscle. Collectively, these data have elucidated a conserved molecular signature of HCN and TTC channel expression in porcine and human calyx pacemaker tissues. These findings provide evidence for the mechanisms that can drive renal pacemaker activity in the multi‐calyceal urinary tract, and potential causes of obstructive uropathies.

Introduction

Pacemaker tissues coordinate autonomic muscle activity by setting the origin and frequency of contractions (Weiss et al. 2006; Huizinga & Lammers, 2009; Monfredi et al. 2010). The spontaneous, rhythmic depolarizations elicited by pacemakers propagate through the muscular syncytia, triggering contractile waves emanating from the pacemaker. In the urinary tract, renal pacemakers trigger proximal‐to‐distal smooth muscle contractions that propel waste from the kidney to the bladder (Bohnenpoll & Kispert, 2014; Feeney & Rosenblum, 2014). The mechanisms underlying this process are clinically significant, as defects in urinary tract peristalsis occur at a high incidence and are the leading cause of pediatric kidney failure (Rosen et al. 2008; Alberti, 2012).

In mammals, the complexity in the anatomical structure of the urinary tract, including the anatomic site of renal pacemaker activity (Lang et al. 1998; Weiss et al. 2006; Feeney & Rosenblum, 2014), varies depending on the species (Sperber, 1944; Oliver, 1968; Gosling & Dixon, 1974; Kriz, 1981). In small mammals such as the mouse (Fig. 1A, schematic; Fig. 1B, whole mount; Fig. 1C, high magnification), the proximal urinary tract has a flared funnel shape and encases a single elongated renal papilla. Contractions initiate where the pelvis joins the kidney, termed the pelvis–kidney junction (PKJ), transporting waste from the collecting ducts of the papilla to the bladder as contractile waves propagate distally down the urinary tract (Schmidt‐Nielsen, 1987; Dwyer & Schmidt‐Nielsen, 2003). Physiological studies have established that renal pacemaker activity is localized to the PKJ, and that morphologically distinct atypical smooth muscle cells at this site elicit pacemaker depolarizations that precede peristalsis (Bozler, 1942; Gosling, 1970; Klemm et al. 1999; Lang et al. 2007, 2010). Recent studies by our group assaying the ion channels required for coordinated peristalsis have discovered that this PKJ pacemaker population is a subset of modified smooth muscle cells co‐expressing hyperpolarization‐activated cation (HCN) and T‐type calcium (TTC) channels (Hurtado et al. 2010, 2014). Analyses of intact upper urinary tract explants and micro‐dissected PKJ tissues revealed that renal pacemaker depolarizations are abolished upon HCN channel block, and that HCN channel conductance underlies the pacemaker excitation that sets the origin of contraction to the PKJ and coordinates proximal‐to‐distal urinary tract peristalsis. Moreover, TTC channel activity was shown to play a role in setting contraction frequency. These findings were consistent with the known role of HCN channels in underlying neuronal and cardiac pacemaker activities (Santoro et al. 1997; Ludwig et al. 1998; Ishii et al. 1999; Biel et al. 2009), bioengineering studies demonstrating transfection of HCN channels is sufficient to induce de novo pacemaker activity in quiescent cell types (Qu et al. 2003; Plotnikov et al. 2004; Potapova et al. 2004; Cho et al. 2007), and studies demonstrating HCN⁺ cardiac pacemakers co‐express TTC channels that modulate contraction frequency (Hagiwara et al. 1988; Masumiya et al. 1998; Bohn et al. 2000; Madle et al. 2001; Niwa et al. 2004; Chandler et al. 2009; Mesirca et al. 2014). Moreover, these findings have provided insight into the numerous studies denoting electrophysiological similarities in pacemaker activity of the heart and urinary tract, revealing for the first time that these similarities may be attributed to analogous ion channel expression and function (Orbelli & Von Brucke, 1910; Bozler, 1942; Irisawa & Kobayashi, 1962; Weiss et al. 1967; Lang et al. 2007).

Figure 1.

The anatomy of the upper urinary tract varies amongst mammalian species. (A‐C) The murine upper urinary tract (A, schematic; B, whole mount image; C, high magnification) has an anatomical structure that is common to small mammalian species. This continuous tubular muscle has a funnel‐shaped morphology with a flared kidney‐proximal end, termed the pelvis, and a narrower distal segment, termed the ureter. The pelvis joins the connective tissue core of the kidney at the pelvis‐kidney junction (PKJ). A single elongated papilla extends into and is surrounded by the pelvis. The papilla (pa) contains the renal collecting ducts that carry out the final modification of wastes filtered by the kidney. Renal pacemaker activity in the unicalyceal urinary tract is localized to the PKJ, (D‐F) The porcine upper urinary tract (D, schematic; E, whole mount image; F, high magnification) exhibits the stereotypic anatomy and physiology of larger mammalian species, including humans. The funnel‐shaped morphology of the muscle is conserved in the porcine urinary tract, which also contains a flared kidney‐proximal pelvis and narrowed distal ureter. However, in contrast to the urinary tract of smaller mammals, the proximal end of the porcine pelvis is segmented into branches of muscle (arrowheads) that extend toward and join the connective tissue core of the kidney. The kidney has numerous renal papillae (pa) that are individually encased by these branched ends of the pelvis, forming an anatomical structure termed the renal calyx. (B) Scale bars:  1 mm (B), 10 mm (E).

In large mammals the anatomical structure of the urinary tract can be more complex than those of small mammals (Sperber, 1944; Oliver, 1968; Kriz, 1981). In the porcine and human urinary tracts, the renal pelvis is segmented into branches of muscle that extend toward and join the connective tissue core of the kidney (Dixon & Gosling, 1973, 1982). As can be seen in Fig. 1D‐F (D, schematic; E, whole mount; F, high magnification) the kidneys of these mammals have multiple renal papillae, and the branched ends of the urinary tract form extended processes that encase individual papillae. These anatomical structures, consisting of urinary tract muscle encasing papillae, are termed minor calyces. Peristalsis initiates at these calyces and propels waste from the papillary collecting ducts into the pelvis, and down to the bladder as contractile waves propagate distally down the urinary tract (Dixon & Gosling, 1973, 1982). Studies analyzing peristalsis in the multi‐calyceal urinary tract, including those in humans, have established that renal pacemaker activity localizes to the minor calyces. Specifically, anatomical analyses have demonstrated that atypical smooth muscle cells localize to the minor calyx muscle, and physiological studies have shown that spontaneous rhythmic pacemaker depolarizations which precede contractions are elicited at these sites (Gosling, 1970; Longrigg, 1975, 1982; Hannappel et al. 1982; Morita et al. 1986; Yamaguchi & Constantinou, 1989).

Notably, the discovery of atypical smooth muscle cells in renal pacemaker tissues across mammalian species is consistent with striking similarities in mammalian renal pacemaker excitation. Specifically, physiological studies in a wide range of mammalian species have revealed that spontaneous renal pacemaker depolarizations occur via a gradual depolarization after repolarization, which is also observed in HCN⁺ cardiac pacemakers (Orbelli & Von Brucke, 1910; Bozler, 1942; Irisawa & Kobayashi, 1962; Weiss et al. 1967; Lang et al. 2007). These similarities in cell types and electrical activity detected in renal pacemaker tissues of different mammals have led to prevailing hypotheses that indicate there is a common renal pacemaker population (Weiss et al. 2006; Bohnenpoll & Kispert, 2014; Feeney & Rosenblum, 2014). Thus, it is possible that the HCN and TTC channels that drive murine renal pacemaker activity are conserved in higher‐order mammalian species, although to date, an analogous ion channel expression has not been revealed in mammalian renal pacemaker tissues.

In this study, we discovered that HCN and TTC channels are expressed in renal pacemaker tissues of the porcine and human multi‐calyceal urinary tracts, which share a close anatomical structure and physiology (Morita et al. 1986; Soin et al. 2001; Bischoff et al. 2009). Whole mount, double chromagenic immunohistochemistry of proximal porcine urinary tract sections revealed that HCN3 is highly expressed and localized to the minor calyx pacemaker tissue between adjoining papillae. In contrast, the porcine kidney and urinary tract smooth muscle lacked this HCN expression. Immunofluorescent staining done to resolve the spatial distribution of the cell types at the porcine minor calyx found that HCN3⁺ cells are integrated within the calyx smooth muscle, and that these HCN3⁺ cells co‐express TTC channel isoform Cav3.2. For our studies of human tissue, the anatomy of the minor calyx was first assayed via hematoxylin and eosin (H&E) histology, which enabled the visualization of the calyx smooth muscle and adjacent papillae. Strikingly, we found that the human minor calyx exhibits an ion channel expression pattern analogous to that of the porcine urinary tract. Immunofluorescent staining demonstrated that HCN3⁺/Cav3.2⁺ cells also localize to the smooth muscle of the human minor calyx. Thus, we have discovered a molecular signature of HCN and TTC channel expression conserved in porcine and human renal pacemaker tissues. These studies provide much needed insight into the mechanisms that can drive pacemaker excitation in the multi‐calyceal urinary tract, and have implications for understanding normal and pathological urinary tract motility.

Methods

Biological materials

Mice utilized for gross anatomical analyses of the murine urinary tract were purchased from Taconic Farms (Germantown, NY, USA) and housed in the Weill Medical College of Cornell University Animal Facility according to the Research Animal Resource Center Guidelines. Whole pig kidneys with intact ureters were purchased from Pel Freez Biologics (Rodgers, AR, USA). Gross anatomical analyses of porcine and murine urinary tracts were done by bisecting kidneys along the frontal plane to expose the interior of the organ, and imaged with a digital camera (Coolpix 995; Nikon, Melville, NY, USA). Normal adult human proximal urinary tract tissues commercially available (Origene, Rockville, MD, USA) were assayed for smooth muscle and ion channel protein expression.

Histochemistry

For thick section chromagenic immunohistochemistry, porcine urinary tract tissues were embedded in 7% low melting point agarose, vibratome sectioned at 100 μm thickness, and then permeabilized and blocked with 0.5% TritonX‐100, 1% normal donkey serum in Tris‐buffered saline (TBS) overnight at room temperature, then at 37 °C for 3 h. For these studies we utilized the TLL6C5 anti‐HCN3 antibody (rat monoclonal), which was originally described by Muller et al. (2003) and was raised against the HCN3‐specific immunogen peptide (640) L L A R S A R R S A G S P A S P L V P V R A G P L L A R G P W A S T S (675) of rat that is highly conserved across mammalian species. Western blot analyses of human cell lines and rat tissue lysates have validated the specificity of this antibody, demonstrating that it identifies a band of ~ 90 kDa representing the known molecular weight of HCN3 (Muller et al. 2003), which we have confirmed in porcine cerebellum and human whole brain tissue lysates (data not shown). These findings demonstrating the specificity of TLL6C5, which is now available commercially from different companies, have been further substantiated in several studies utilizing western blot and immunohistochemistry analyses, including those of taste receptors (Stevens et al. 2001), the upper urinary tract (Cain et al. 2011), and thalamocortical neurons (Cain et al. 2015). TLL6C5 Anti‐HCN3 (Millipore, Billerica, MA, USA) and anti‐SMA (monoclonal anti‐actin, α‐smooth muscle, clone 1A4, Sigma Aldrich, St. Louis, MO, USA) were used at a concentration of 1/700 in blocking solution for 6 h at room temperature. After washing overnight in 0.5% TritonX‐100 in phosphate‐buffered saline (PBS), sections were incubated with alkaline phosphatase conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) at a concentration of 1/1200 in blocking solution for 3 h at room temperature. After washing with 0.5% TritonX‐100, chromagenic detection was done by NBT‐BCIP and FR‐BCIP staining, as previously described in detail (Hurtado & Mikawa, 2006). H&E staining was performed according to standard protocols.

Immunofluorescence

Immunofluorescent analyses were performed as previously described (Hurtado et al. 2010, 2014). In short, porcine urinary tract tissues were fixed in 4% paraformaldehyde (PFA), washed in PBS, cryoprotected by 30% sucrose in PBS and embedded in optimal cutting temperature (OCT) compound. Frozen tissue blocks of porcine and human urinary tract tissues were cut at 8 μm thickness. Sections were then rehydrated with PBS, permeabilized with 0.2% TritonX‐100 in PBS for 20 min, and blocked with 1% normal donkey serum in PBS for 1 h. TLL6C5 anti‐HCN3 antibody was used at a concentration of 1/700 on porcine tissues, and the specificity of this immunostaining was confirmed via immunizing peptide competition assay using 10 μg of blocking peptide per 1 μg of antibody (antibody and blocking peptide incubated overnight at 4 °C). Mouse anti‐smooth muscle actin conjugated to Cy3 (monoclonal anti‐actin, α‐smooth muscle, clone 1A4‐Cy3 conjugated, Sigma) was used at 1/2200, and anti‐uroplakin (H‐180; Santa Cruz Biotechnology, Dallas, TX, USA) at 1/700. In these studies we used the ACC‐025 anti‐Cav3.2 antibody (rabbit polyclonal; Alomone Labs, Jerusalem, Israel) raised against the Cav3.2 specific immunogen peptide (581) C H V E G P Q E R A R V A H S (595) of rat that is highly conserved in mammalian species. The specificity of this antibody has been validated by western blot analyses in different mammalian species including mouse (Markandeya et al. 2011), rat (Becker et al. 2008; Makarenko et al. 2015) and human (Harraz et al. 2015), which demonstrate that this antibody detects a band of ~ 250 kDa representing the known molecular weight of Cav3.2. Moreover, the specificity of ACC‐025 anti‐Cav3.2 has been established further in wide ranging immunohistochemical analyses of different species including dopaminergic neurons of rat (Dufour et al. 2014), carotid bodies of rat and mouse (Makarenko et al. 2015), and adrenal tissues of human (Scholl et al. 2015). We used this antibody at a concentration of 1/700, and confirmed the specificity of its immunostaining in our studies by immunizing peptide competition assay using 10 μg of blocking peptide per 1 μg of antibody (antibody and blocking peptide incubated overnight at 4 °C). Anti‐HCN3 utilized in the Human Atlas Project (rabbit polyclonal, HPA026584, Sigma) was used on human tissues at 1/500. All antibodies were diluted in 1% normal donkey serum in PBS, at 37 °C for 3 h. When co‐staining for HCN3 and Cav3.2 on human tissues, Anti‐Cav3.2 was first pre‐conjugated to Alexa‐594 (Antibody labeling kit, Molecular Probes; Life Technologies, Grand Island, NY, USA) and added last. After five washes with PBS, sections were incubated with Alexa Fluor‐conjugated secondary antibodies (Jackson ImmunoResearch), as noted, at a concentration of 1/1000 diluted in 1% normal donkey serum in PBS, at 37 °C for 1.5 h.

Results

HCN channels are highly expressed and localized to the porcine calyx pacemaker tissue

The porcine upper urinary tract was assayed to determine the spatial distribution of HCN channels (Fig. 2). Porcine kidneys were bisected along the frontal plane to expose the interior of the organ, and then further dissected to isolate the minor calyces, urinary tract smooth muscle, adjoining papillae and kidney. Isolated tissues were processed as whole mount vibratome sections (0.1 mm thick) and then analyzed by chromagenic immunohistochemistry, which provides a high signal‐to‐noise ratio, as background is readily quenched (Hauptmann, 2001; Hurtado & Mikawa, 2006). In these studies (Fig. 2A‐E) we assayed for HCN3, which is abundantly expressed in murine renal pacemaker tissues (Hurtado et al. 2010, 2014). Single chromagenic immunohistochemistry revealed that HCN3 (purple precipitate) is highly expressed and localized to the minor calyx tissue directly encasing the renal papillae (Fig. 2A, IgG control; Fig. 2B and C, low and high magnification HCN3 staining, respectively), at the junction where urinary tract joins the kidney, termed the calyx–kidney junction (CKJ) (Fig. 2D). In contrast, HCN3 expression was not detectable in control tissues, the renal papillae, adjacent kidney tissues or distal urinary tract segments.

Figure 2.

HCN3 is highly expressed in the minor calyx pacemaker tissues of the porcine upper urinary tract. (A‐D) Chromagenic immunohistochemistry was performed on the proximal region of the porcine upper urinary tract. Tissues assayed included the renal parenchyma and papillae (pa), as well as the calyx muscle surrounding the papillae. HCN isoform 3 (purple precipitate) was found to be highly expressed and localized to the calyx muscle in between adjacent papillae (A, IgG control; B, HCN3 immunohistochemistry), at the site where the calyx joins the kidney or at the calyx‐kidney junction (CKJ) (C,D, high magnification images). In contrast, HCN3 was not detectable in renal parenchyma and papillae tissues adjacent the CKJ, or in urinary tract segments distal to the CKJ. (E) Double chromagenic immunohistochemistry demonstrated HCN3 expression (purple precipitate) localizes within the calyx smooth muscle layer, adjacent to the definitive smooth muscle (turquoise precipitate) that lacked HCN expression. Scale bar:  1 mm.

To define the spatial relationship between HCN‐expressing tissue and the smooth muscle of the minor calyx, double chromogenic immunohistochemistry for HCN3 and smooth muscle actin (SMA) was performed (Fig. 2E). This confirmed that HCN3 (purple precipitate) is highly expressed at the CKJ (Fig. 2D), and demonstrated that HCN3 localizes within the smooth muscle coat (SMA, turquoise precipitate), adjacent to the definitive smooth muscle that lacks HCN expression (Fig. 2E). Taken together, these protein expression data reveal that HCN channels are abundantly expressed within the calyx pacemaker tissue of the porcine urinary tract, in a location adjacent to the definitive smooth muscle.

HCN‐expressing cells of the porcine calyx pacemaker are integrated within the smooth muscle coat and co‐express TTC channels

The spatial organization of different cell types of the porcine minor calyx was assayed next via immunofluorescent analyses (Fig. 3). To determine the feasibility of these experiments, we first assayed the autofluorescence of porcine minor calyx tissues (Fig. 3A,B). Results of these experiments revealed readily detectable green (Fig. 3A) and red (Fig. 3B) autofluorescence in the urothelium and tubular compartments of the papillae, whereas the adjacent calyx smooth muscle coat lacked a corresponding background autofluorescence. These data are consistent with studies that have identified background fluorescence in tubular structures of the kidney (Dunn et al. 2002; Duffield & Bonventre, 2005; Russo et al. 2007) but low background fluorescence in the upper urinary tract smooth muscle (Mahoney et al. 2006; Hurtado et al. 2010, 2014; Cain et al. 2011), and demonstrate that fluorescent immunohistochemical analyses of the CKJ smooth muscle layer are not precluded by high background noise.

Figure 3.

HCN ⁺ cells are integrated within the smooth muscle coat of the porcine calyx pacemaker. (A,B) Autofluorescence of the porcine proximal urinary tract was examined. Green (A) and red (B) autofluorescence was evident in the tubular structures of the renal papillae (pa). In contrast, the calyx‐kidney junction (CKJ) smooth muscle, which surrounds the luminal urothelium (u) and is adjacent to the papillary tissue, had minimal background autofluorescence. (C‐G) Low magnification immunofluorescence was performed for HCN3 (green), SMA (red) and uroplakin (blue) that marks the urothelium. HCN3‐expressing tissue (C, IgG control; E, HCN3/uroplakin) was found to be integrated within the smooth muscle coat of the CKJ (D, IgG control; F, SMA/uroplakin), adjacent to the definitive smooth muscle that lacked HCN expression (G, HCN3/uroplakin/SMA merged). (H‐M) High magnification immunofluorescence for HCN3 (green), SMA (red) and DAPI (blue) was performed to further define the HCN‐expressing cells of the CKJ. These data revealed that HCN3⁺ cells (H, HCN3/DAPI) are in direct contact with adjacent smooth muscle cells (I, SMA/DAPI) that lack HCN expression (J, HCN3/SMA/DAPI merged), which was confirmed at the single‐cell level (K, HCN3/DAPI; L, SMA/DAPI; M, HCN3/SMA/DAPI merged).

Next, we assayed the distribution of HCN‐expressing tissue relative to the smooth muscle of the CKJ via low magnification immunofluorescence (Fig. 3C‐G). Calyx tissues isolated as described above were cryoprotected and cryosectioned along the frontal plane into 8‐μm thin sections, and then assayed for HCN3 (green), SMA (red), and uroplakin (blue). Uroplakins are protein components of urothelial membrane plaques, thus marking the luminal surface of the calyx tissue encasing the renal papillae. These immunohistochemical analyses efficiently identified the specialized tissues of the upper urinary tract, denoting the smooth muscle layer surrounding the luminal urothelium (Fig. 3D, IgG control; F, SMA and uroplakin). Moreover, results of these experiments corroborated the data obtained via chromagenic immunohistochemistry (Fig. 2), demonstrating that HCN3⁺ tissue is integrated within the calyx smooth muscle coat, adjacent to the smooth muscle which lacked HCN channel expression (Fig. 3C, IgG control; E, HCN3 and uroplakin; G, HCN3, SMA and uroplakin merged). Critically, HCN immunostaining of calyx pacemaker tissues is abolished in the presence of blocking peptide, corroborating the specificity of these immunohistochemical analyses (Supporting Information Fig. S1).

HCN channel expression of the porcine minor calyx was further characterized at the cellular level via high magnification immunofluorescence for HCN3 (green), SMA (red) and DAPI (blue) (Fig. 3H‐M). These immunohistochemical analyses enabled the detection of the calyx smooth muscle coat at the CKJ, with SMA staining the stress fibers of elongated smooth muscle cells (Fig. 3I, SMA and DAPI). Moreover, HCN3 staining (Fig. 3H, HCN3 and DAPI) marked the cell membrane of a subset of cells adjacent to CKJ smooth muscle cells which did not express HCN channels (Fig. 3J, HCN3, SMA and DAPI). Thus, HCN3⁺ cells are integrated within the smooth muscle layer of the porcine minor calyx at the CKJ, which was evident at the single‐cell level (Fig. 3K‐M).

We next performed experiments to determine whether HCN⁺ cells of the porcine minor calyx exhibit an ion channel expression analogous to HCN⁺ murine renal pacemakers (Fig. 4A‐H). Specifically, our previous studies have revealed that murine renal pacemakers, like mammalian cardiac pacemakers, express TTC channels. To test this hypothesis, calyx pacemaker tissues were assayed via confocal imaging of immunofluorescence for HCN3 (green), TTC channels (red) and DAPI (blue). Results of these experiments revealed that HCN3⁺ cells (Fig. 4A, IgG control; C, HCN3 and DAPI) of the calyx co‐express TTC channel Cav3.2 (Fig. 4B, IgG control; Fig. 4D, Cav3.2 and DAPI; Fig. 4E, all merged), which was corroborated via high magnification cellular imaging (Fig. 4F, HCN3 and DAPI; Fig. 4G, Cav3.2 and DAPI; Fig. 4H, all merged). The specificity of these immunohistochemical analyses was substantiated by control experiments demonstrating that TTC channel immunostaining in the calyx was not detectable in the presence of blocking peptide (Supporting Information Fig. S2). These comparative analyses reveal that the cells of the porcine minor calyx express at least two ion channels known to underlie murine renal pacemaker activity, and elucidate a conserved ion channel expression between murine and porcine renal pacemaker tissues.

Figure 4.

HCN‐expressing cells of the porcine calyx co‐express T‐type calcium channels. (A‐H) The HCN+ cells of the porcine calyx pacemaker tissue were characterized via triple immunofluorescence for HCN3 (green), Cav3.2 (red) and DAPI (blue). Results of these experiments revealed HCN3⁺ cells (A, IgG; C, HCN3/DAPI) co‐express T‐type calcium channel isoform Cav3.2 (B, IgG control; D, Cav3.2/DAPI; E, HCN3/Cav3.2/DAPI merged). The co‐expression of Cav3.2 by HCN3⁺ cells of the calyx pacemaker was evident via single cell analyses (F, HCN3/DAPI; G, Cav3.2/DAPI; H, HCN3/Cav3.2/DAPI merged).

Human minor calyx pacemaker tissues contain HCN‐expressing cells that are integrated into the smooth muscle coat and co‐express TTC channels

Our discovery that HCN⁺/TTC⁺ cells are conserved in murine and porcine renal pacemaker tissues raises the possibility that this cell population may also localize to the human minor calyx, the renal pacemaker tissues of the human upper urinary tract (Longrigg, 1975, 1982). To test this hypothesis, protein expression analyses were performed on human minor calyx tissues (Fig. 5). Adult human urinary tract samples (n = 3 normal minor calyx tissues, from two different individuals) utilized in our study were first examined via H&E staining to observe the anatomic structures of the minor calyx. As can be seen in Fig. 5A, H&E histological analyses enabled the visualization of the minor calyx tissue structure, with the calyx smooth muscle localized between adjacent renal papillae. The calyx smooth muscle was demarcated by the characteristic pink‐red H&E staining of muscle, whereas the renal papillae were denoted by the presence of renal ductal structures. Both smooth muscle and papillary tissue layers were readily evident in high magnification analyses of the CKJ (Fig. 5B).

Figure 5.

HCN ⁺ cells localize to the calyx pacemaker tissue of the human upper urinary tract. Histochemical and immunofluorescence analyses were performed to determine whether HCN ⁺ cells localize to the minor calyx pacemaker tissue of the human upper urinary tract. (A,B) H&E analyses enabled the visualization of the human minor calyx anatomy, revealing the smooth muscle (sm) coat localized between adjacent papillae (pa) (A, low magnification). The smooth muscle layer exhibited the characteristic pink‐red staining of muscle, whereas the adjacent papillae were differentiated by the presence of renal tubular structures. Both smooth muscle and papillary tissue layers were evident in high magnification analyses (B) of the calyx‐kidney junction (CKJ). (C,D) The human calyx‐kidney junction was assayed to determine the extent of background fluorescence in the smooth muscle and kidney tissue layers. Green and red autofluorescence was readily evident in the papilla (pa), whereas the adjacent smooth muscle had low relative background fluorescence. (E‐J) The human minor calyx was assayed via immunofluorescence for HCN3 (green), SMA (red) and DAPI (blue). Results of these experiments revealed HCN3⁺ (E, IgG control; G, HCN3/DAPI) localized to the smooth muscle (F, IgG control; H, SMA/DAPI) of the human minor calyx at the CKJ, which could be observed at both low (I, HCN3/SMA/DAPI merged) and high magnification (J, HCN3/SMA/DAPI) (arrow in I points to region observed at high magnification in J).

Human minor calyx tissues were then analyzed to determine the extent of autofluorescence (Fig. 5C,D). Strikingly, we found that the human minor calyx exhibited an autofluorescence consistent with that observed in porcine renal pacemaker tissues. Green (Fig. 5C) and red (Fig. 5D) autofluorescence could be detected in tubular compartments of the renal papillae, whereas a relative autofluorescence was not detected in the adjacent calyx smooth muscle. Thus, immunofluorescent analyses of the human minor calyx were not precluded by high tissue autofluorescence.

We next assayed whether HCN⁺ cells localize to the human minor calyx (Fig. 5E‐J). For these studies determining the presence of HCN⁺ cells in the human minor calyx, we utilized an HCN3 antibody that has been validated in human samples as part of the Human Protein Atlas project (HPA; Ponten et al. 2008; Fagerberg et al. 2014; Uhlen et al. 2015). Prestige antibodies utilized in the HPA project undergo a stringent selection process that includes high‐throughput generation, immunohistochemical validation in different human cell lines and tissues, conformance with bioinformatic and published data, and verification that different antibodies raised against the same target give corresponding staining patterns. Human minor calyx tissues were assayed via immunofluorescence for HCN3 (green), SMA (red) and DAPI (blue). Low magnification analyses of the CKJ revealed that HCN3⁺ cells (Fig. 5E, IgG control; Fig. 5G, HCN3 and DAPI) were localized to minor calyx smooth muscle (Fig. 5F, IgG control; Fig. 5H, SMA and DAPI, Fig. 5I, HCN3, SMA and DAPI merged). High magnification analyses of the CKJ revealed that HCN3⁺ cells localized to the human CKJ are integrated within the minor calyx smooth muscle coat (Fig. 5J, HCN3, SMA and DAPI merged).

To confirm the presence of HCN⁺ cells in human renal pacemaker tissues, we next assayed two additional minor calyx samples from different individuals (Fig. 6). Calyx tissues were assayed via immunofluorescence for HCN3 (green), SMA (red) and DAPI (blue). Results of these experiments revealed that HCN3⁺ cells (Fig. 6A and G, HCN3 and DAPI) localized to the calyx smooth muscle (Fig. 6B and H, SMA and DAPI) of all the minor calyces assayed (Fig. 6C and I, HCN3, SMA and DAPI). High magnification analyses enabled the detection of HCN3⁺ cells (Fig. 6D and K, HCN3 and DAPI) integrated within the smooth muscle layer (Fig. 6E and L, SMA and DAPI) of the minor calyx (Fig. 6F and M, HCN3, SMA and DAPI merged). Thus, we have established that HCN⁺ cells localize to the smooth muscle of the human minor calyx in multiple independent tissue samples.

Figure 6.

Analyses of human urinary tissues from different individuals confirm that HCN ⁺ cells localize to the smooth muscle of the human minor calyx. (A‐M) Minor calyx tissues from different individuals (A‐F and G‐M) were assayed for the presence of HCN ⁺ cells via immunofluorescence for HCN3 (green), SMA (red) and DAPI (blue). Results of these experiments revealed that HCN3⁺ cells (A and G, HCN3/DAPI) localize to the smooth muscle (B and H, SMA/DAPI) of the human minor calyx tissues assayed (C and I, HCN3/SMA/DAPI merged, high magnification). Analyses at the cellular level (D‐F and K‐M) confirmed that HCN3⁺ cells (D and K, HCN3/DAPI) are integrated within the smooth muscle layer (E and L, SMA/DAPI) of the minor calyx (F and M, HCN3/SMA/DAPI merged).

Finally, we performed experiments to determine whether HCN⁺ cells of the human minor calyx co‐express TTC channels (Fig. 7). Calyx tissues were assayed via immunofluorescence for HCN3 (green), TTC channel isoform Cav3.2 (red) and DAPI (blue). Results of these experiments revealed that HCN3⁺ cells (Fig. 7A, IgG control; Fig. 7C, HCN3 and DAPI) of the human minor calyx pacemaker co‐express Cav3.2 (Fig. 7B, IgG control; Fig. 7D, Cav3.2 and DAPI; Fig. 7E, HCN3, Cav3.2, and DAPI merged). The co‐expression of TTC channels by HCN⁺ cells of the human minor calyx was confirmed at the single cell level via high magnification analyses (Fig. 7F, HCN3 and DAPI; Fig. 7G, Cav3.2 and DAPI; Fig. 7H, HCN3, Cav3.2 and DAPI merged). Collectively, these protein expression data reveal that HCN⁺ cells are localized to the smooth muscle of the human minor calyx, and that these HCN‐expressing cells co‐express T‐type calcium channels.

Figure 7.

HCN ⁺ cells of the human minor calyx co‐express TTC channels. (A‐E) Human minor calyx tissues were assayed via triple immunofluorescence for HCN3 (green), Cav3.2 (red) and DAPI (blue). Results of these experiments revealed HCN3⁺ cells (A, IgG control; C, HCN3/DAPI) of the human minor calyx co‐express TTC channel isoform Cav3.2 (B, IgG; D, Cav3.2/DAPI; E, all merged, high magnification). Analyses at the single cell level (F–H) confirmed that HCN3⁺ cells (F, HCN3/DAPI) co‐express TTC channel Cav3.2 (G, Cav3.2/DAPI; H, HCN3/Cav3.2/DAPI merged).

Discussion

Although pacemaker activity at the proximal end of the upper urinary tract has been appreciated for over 100 years, it has not been determined whether renal pacemaker tissues of different mammalian species exhibit an analogous expression of ion channels known to mediate pacemaker activity. In this study, we performed anatomical and protein expression analyses and revealed an ion channel expression pattern conserved in renal pacemaker tissues of small and large mammals. Specifically, we discovered that HCN and TTC channels that underlie murine renal pacemaker activity, as well as cardiac pacemaker activity, are expressed in minor calyx pacemaker tissues of the porcine and human upper urinary tract. Chromagenic and fluorescent immunohistochemistry analyzed at both low and high magnifications revealed that HCN channel expression is highly expressed and localized to the porcine minor calyx pacemaker, and that HCN3⁺ cells at this site are integrated within the smooth muscle coat and co‐express TTC channel Cav3.2. Moreover, H&E and immunofluorescent analyses revealed HCN3⁺/Cav3.2⁺ cells are also integrated within the smooth muscle coat of the human minor calyx pacemaker. The above data have elucidated a molecular signature of HCN and TTC channel expression that is conserved in renal pacemaker tissues of small and large mammalian species with distinct urinary tracts.

Additional studies will need to be performed to determine the exact role that HCN⁺/TTC⁺ cells play in renal pacemaker tissues of the multi‐calyceal urinary tract. HCN and TTC channels are important for various cellular processes including setting and stabilization of membrane potential (Mayer & Westbrook, 1983; Doan & Kunze, 1999; Meuth et al. 2006; Nolan et al. 2007), muscle tonicity (Kuo et al. 2010; Abd El‐Rahman et al. 2013), cellular excitability (Day et al. 2005; Li et al. 2007; Sui et al. 2007; Fenske et al. 2013), cell proliferation (Taylor et al. 2008; Oguri et al. 2010; Gray et al. 2013), and eliciting pacemaker excitation (Nilius & Carbone, 2014; Wahl‐Schott et al. 2014). Although our findings do not rule out any specific function for HCN⁺/TTC⁺ cells of the minor calyx, physiological and anatomical studies of the upper urinary tract support a role for HCN⁺ cells as renal pacemakers, akin to cardiac and murine renal pacemakers. Indeed, a conserved role for HCN⁺ cells as mammalian renal pacemakers is consistent with numerous studies that have discovered analogous renal pacemaker activity across a wide range of mammals. Studies of the unipapillate dog, cat and guinea pig first detected renal pacemaker excitation occurring via a gradual membrane depolarization after repolarization (Bozler, 1942). Subsequent studies have confirmed these findings (Irisawa & Kobayashi, 1962) and have identified corresponding renal pacemaker excitation in other mammals, including cells and muscle strips isolated from murine (Lang et al. 2007) and rabbit (Seki & Suzuki, 1990) renal pacemaker tissues, as well as in pacemaker tissues of the multicalyceal porcine (Morita et al. 1981, 1986) and sheep urinary tracts (Zawalinski et al. 1975). Notably, HCN channels drive a depolarizing inward cation current at negative membrane potentials that gradually depolarizes cells after repolarization, which in turn activates inward cation TTC channel conductance, further depolarizing the cell, a process termed diastolic depolarization in HCN⁺/TTC⁺ cardiac pacemakers (Hagiwara et al. 1988; Santoro et al. 1997; Ludwig et al. 1998; Masumiya et al. 1998; Biel et al. 2009; Ono & Iijima, 2010). The identification of renal pacemaker excitation consistent with HCN and TTC channel conductances in varied mammalian species, our previous findings that HCN⁺/TTC⁺ channels underlie murine renal pacemaker activity, the known role of HCN⁺/TTC⁺ cells as cardiac pacemakers, and our current discovery that HCN⁺/TTC⁺ cells are localized to porcine and human renal pacemaker tissues where peristaltic contractions initiate, raise the possibility that HCN⁺ cells are a conserved mammalian renal pacemaker population. Moreover, our discovery that HCN and TTC channel expression is conserved in porcine and human renal pacemaker tissues shows, for the first time, that the analogous renal pacemaker activity detected in both small and large mammals may be due to a common ion channel expression.

A role for HCN⁺/TTC⁺ cells as mammalian renal pacemakers is also in line with anatomical studies that have provided significant insight into the similarities between the renal pacemaker tissues of different mammalian species. Anatomical analyses by Gosling and Dixon first identified that the mammalian renal pacemaker has a specialized tissue structure (Gosling, 1970). Specifically, these studies and others have shown that renal pacemaker tissues of all assayed mammalian species, including the mouse, pig and human contain atypical smooth muscle cells that are morphologically distinct from the urinary tract smooth muscle (Gosling & Dixon, 1974; Dixon & Gosling, 1982; Klemm et al. 1999; Lang et al. 2007). Atypical smooth muscle cells are smaller in size and have a weaker Acta2 immunoreactivity than typical smooth muscle cells, and they are not grouped into tight cellular bundles. Strikingly, the reported morphology of atypical smooth muscle cells is consistent with the morphology of HCN⁺ cells localized to murine, porcine and human renal pacemaker tissues. As demonstrated previously in the mouse (Hurtado et al. 2010, 2014), and here in pig and humans, HCN⁺ cells of renal pacemaker tissues are smaller than smooth muscle cells, have a weaker Acta2 immunoreactivity, and exhibit a dispersed spatial association that does not consist of tight, continuous cellular contacts. Thus, it is possible that HCN⁺ cells are the atypical smooth muscle cells that have been identified across mammalian species, a hypothesis which has become increasingly recognized (Bohnenpoll & Kispert, 2014; Feeney & Rosenblum, 2014).

Our discovery of HCN and TTC channels in porcine and human renal pacemaker tissues has translational implications, as these ion channels play prominent roles in health and disease due to their important physiological functions. Indeed, recent studies have begun to corroborate a role for HCN⁺ cells as renal pacemakers required for establishing coordinated upper urinary tract peristalsis. Independent studies have confirmed the presence of HCN⁺ cells in the murine upper urinary tract, and have also shown that aberrant upper urinary tract peristalsis in a mouse model of human Pallister Hall syndrome is marked by a loss of the HCN⁺ pacemakers our group originally identified (Cain et al. 2011; Calejo et al. 2014). Notably, HCN⁺ pacemaker cell dysfunction has also been shown to underlie cardiac and neuronal disorders. Human HCN channel mutations impair cardiac pacemaker activity and are associated with cardiac arrhythmias (Baruscotti et al. 2010; Laish‐Farkash et al. 2010; Roubille & Tardif, 2013), whereas perturbation of TTC channels is seen in cases of human congenital heart block (Hu et al. 2004; Strandberg et al. 2013). In the nervous system, HCN and TTC channel defects have been implicated in the development of epilepsy (Chen et al. 2003; Ludwig et al. 2003; Liang et al. 2006; Eckle et al. 2014; Nava et al. 2014). Thus, our current findings and future studies into the role of HCN⁺/TTC⁺ cells in mammalian renal pacemaker tissues promise to provide much needed insight into upper urinary tract pathologies, such as congenital urinary tract obstructions.

Author contributions

This study was conceived, and experiments performed, by RH. Data were analyzed by RH and CSS. The manuscript was written by RH, with input from CSS.

Supporting information

Fig. S1. HCN3 immunofluorescent staining in porcine calyx pacemaker tissue is abolished in the presence of blocking peptide.

Fig. S2. Presence of blocking peptide results in a loss of Cav3.2 immunofluorescent staining in porcine calyx pacemaker tissues.