RENAL COLLECTING DUCT PHYSIOLOGY AND ......37 tubule (PT), the loop of Henle (the thin descending...

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RENAL COLLECTING DUCT PHYSIOLOGY AND PATHOPHYSIOLOGY

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2018-0192.R1

Manuscript Type: Mini Review

Date Submitted by the Author: 26-Jul-2018

Complete List of Authors: Lashhab, Rawad; University of Alberta, Dept of PhysiologyUllah, AKM Shahid; University of Alberta, Dept of PhysiologyCordat, Emmanuelle C.E.; University of Alberta, Dept of Physiology

Keyword: Kidney, nephron, collecting duct, epithelium, renal disease

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Biochemistry and Cell Biology

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2 RENAL COLLECTING DUCT PHYSIOLOGY AND PATHOPHYSIOLOGY

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5 Rawad Lashhab1,2, AKM Shahid Ullah1,2 and Emmanuelle Cordat2,3

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7 2Department of Physiology and Membrane Protein and Disease Research Group, University of Alberta,

8 Edmonton, AB, Canada, T6G2H7

9 1these authors have equally contributed to the preparation of this manuscript

10 3to whom correspondence should be addressed- Email: [email protected]

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12 Abstract

13 In the kidney, the collecting duct (CD) is composed of at least four cell types: principal, type-A intercalated

14 cells (IC), type-B IC and non-A, non-B IC. Although this heterogeneous composition has been recognized since

15 the end of the 19th century, the physiological role of the various cell types in the CD continues to be

16 deciphered as of today. Principal and IC are essential in ion/water balance and acid-base homeostasis,

17 respectively. However, recent research has revealed a striking interplay and overlap between the specific

18 functions of these cell types. This review summarizes the recent findings on CD cells and their role in multiple

19 pathophysiologies.

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21 Keywords: Kidney, collecting duct, intercalated cells, principal cells, acid-base balance, pH homeostasis

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23 1.The Functional Unit of the Kidney: the Nephron

24 Our everyday life and diet generates wastes that our bodies need to excrete. The human body relies on

25 two main organs to detoxify and excrete unwanted toxins, chemicals and metabolites: the liver and the

26 kidneys. The kidneys play an essential role in (i) ion, pH and water homeostasis, also contributing to hormonal

27 regulation of these processes, (ii) excretion of acids generated by our metabolism and (iii) conservation of key

28 molecules (amino acids, glucose, etc). With its complex structure encompassing the functional unit called

29 nephron, it provides a sophisticated machinery to specifically filter, excrete, secrete and reabsorb molecules

30 to/from the blood. Blood from the afferent arteriole that enters each renal corpuscle is filtered through

31 fenestrated glomerular endothelial cells, the basement membrane and podocytes foot processes prior to

32 entering the capsular space where it flows into the proximal tubule. Within 24 hours, an average of 180 liters

33 of plasma is filtered by the kidneys.

34 Each kidney contains about 1 million nephrons. Each nephron is composed of a sequence of tubular

35 segments that are defined by transitions in the epithelial cells underlying each segment (Kriz and Kaissling

36 2013, Chen et al. 2017, Park et al. 2018b). The various series of epithelial segments include the proximal

37 tubule (PT), the loop of Henle (the thin descending limb, thin ascending limb, thick ascending limb), the distal

38 convoluted tubule (DCT), the connecting tubule (CNT) and finally the collecting duct (CD). The type of cells,

39 their tight junction properties as well as the solute carriers (SLC) and channels expressed in these cells define

40 the function of each segment. Interestingly, in some sections of the nephron, the transition from one segment

41 to the next occurs in a gradual way, with for example overlapping expression of some key proteins such as the

42 epithelial sodium channel (ENaC) and the sodium/chloride cotransporter (NCC) in the DCT/CD transition.

43 The composition of the filtrate in the early part of the proximal tubule is similar to the plasma except

44 that it is devoid of blood cells and contains less proteins (Koeppen and Stanton 2007). As the filtrate flows

45 through the nephron, its composition is modified through reabsorption and excretion. The bulk reabsorption

46 of water, ions and solutes occurs in the PT. Unlike the thin descending limb, the thin ascending limb, the thick

47 ascending limb and the DCT all together contribute to sodium, chloride, calcium and magnesium reabsorption.

48 Lastly, the CNT and CD are the segments responsible for the fine tuning of urine composition and urine

49 acidification. The CD tightly regulates the movement of water, sodium, chloride, potassium, bicarbonate and

50 protons using a combination of both transcellular and paracellular pathways involving at least 3 different cell

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51 types: PCs, type-A IC (also called -IC) and type-B IC (also called -IC). This review focuses on the structure

52 and function of CD cells, specifically on principal and intercalated cells.

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54 2.CD cell types and key genes

55 A. Principal Cells

56 Various cells types of heterogeneous structural composition have been identified by electron

57 microscopy in the embryonic CD epithelium (Kloth et al. 1993). “Light PCs” and “dark IC” were distinguished

58 based on these morphological observations. In cortical rat (Hansen et al. 1980, Kim et al. 1999), mouse (Teng-

59 umnuay et al. 1996, Kim et al. 1999) and rabbit (LeFurgey and Tisher 1979) CD, two third of the cells were

60 found to be PCs. These cells are simple cuboidal epithelial cells that carry fewer organelles and mitochondria

61 than the dark IC. The mitochondria also appear to be smaller in size and randomly distributed in the

62 cytoplasm. Lysosomes, autophagic vacuoles, multivesicular bodies, both rough and smooth endoplasmic

63 reticulum are also evident by transmission electron microscopy (Madsen and Tisher 1986).

64 The major role of the PCs in the CD is to reabsorb sodium and water from primary urine and to excrete

65 potassium to the urine. They achieve this by the concomitant action of specific transporters including the

66 epithelial sodium channel ENaC, aquaporin 2 (AQP2), the renal outer medullary potassium channel (ROMK)

67 (Figure 1) and to a lesser extent the Ca2+ activated K+ channel (BK) (Madsen et al. 1988). In the PCs,

68 aldosterone and arginine vasopressin play a key role for regulation of ENaC, ROMK and AQP2 to facilitate

69 sodium, potassium and water transport, respectively (Pearce et al. 2015).

70 The amiloride-sensitive sodium channel ENaC is expressed at the apical membrane of the PC. Via the

71 action of aldosterone, arginine vasopressin and other hormones (Duc et al. 1994, Hager et al. 2001), high and

72 low sodium diets result in low and high apical expression of ENaC, respectively (Loffing et al. 2000). Its apical

73 abundance is tightly regulated via ubiquitination as detailed below.

74 Potassium secretion through the apical membrane of the PC is mediated by ROMK and is regulated by

75 mineralocorticoids. In rabbit isolated perfused CCD, mineralocorticoids stimulate potassium secretion and

76 sodium reabsorption (O’neil and Helman 1977, Schwartz and Burg 1978). The Na+/K+-ATPase activity is also

77 enhanced by mineralocorticoid stimulation in the CCD of rat (Mujais et al. 1984) and rabbit (Garg et al. 1981).

78 Apical expression of ROMK in the DCT, CNT or CD can be increased in a high potassium diet (Wade et al. 2011).

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79 Water molecules in the PC follow the lumen-to-interstitium osmotic gradient and their reabsorption is

80 mediated by the synchronized expression and activity of apical AQP2 and basolateral AQP3 and AQP4

81 (Ishibashi et al. 1997, Kim et al. 2005, Pearce et al. 2015)

82 B. Intercalated Cells

83 The other cell types found in the CD are IC or “dark” cells”. In comparison with the PCs, IC have a high

84 density of mitochondria, a dark cytoplasm, microprojections at the apical membrane, and they do not have a

85 central cilium (Schuster 1993). All IC are positive for carbonic anhydrase II (CA II) and V-H+-ATPase proteins

86 expression.

87 IC can be subdivided into 3 subtypes: type-A, type-B, and non-A, non-B IC. The location of the V-H+-

88 ATPase expression in addition to the expression of other key proteins define the IC subtype (Teng-umnuay et

89 al. 1996, Roy et al. 2015).

90 Type-A IC express V-H+-ATPase at the apical membrane and the kidney anion exchanger 1 (kAE1) at the

91 basolateral membrane. These cells significantly contribute to acid/base balance by secreting protons via the

92 apical V-H+-ATPase, and reclaiming bicarbonate via basolateral kAE1, both ions being generated from

93 hydrolysis of CO2 and water by the CA II (see section 3. B). On the other hand, type-B IC express V-H+-ATPase at

94 the basolateral membrane and pendrin at the apical membrane. In addition to contributing to acid/base

95 balance by secreting bicarbonate and reclaiming protons in case of alkalosis, type-B IC also contribute to

96 electrolyte homeostasis as they are involved in chloride reabsorption (see section 3. A) (Teng-umnuay et al.

97 1996, Roy et al. 2015).

98 Although IC can be morphologically, structurally and functionally distinguished from the PCs,

99 experimental evidence support that both cell types originate from the same precursor (Trepiccione et al.

100 2017). Immortalized type-B IC plated at a high density were able to convert to type-A IC and secrete acid

101 instead of alkali (van Adelsberg et al. 1994). This ability to convert from one cell type to the other was due to

102 the secretion of the extracellular matrix hensin protein by the type-B IC (Gao et al. 2010). In support of these

103 findings, a hensin knock-out mouse model displayed a predominant abundance of type-B IC, the absence of

104 type-A IC in the CD, and development of metabolic acidosis (Gao et al. 2010).

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105 Lastly, non-A, non-B IC express both V-H+-ATPase and pendrin at the apical membrane. The function of

106 non-A, non-B IC is still unclear. However, the fact that both V-H+-ATPase and pendrin are at the apical

107 membrane suggests that these cells are not involved in acid/base balance but instead may be involved in

108 electrolyte homeostasis. It is also thought that these cells may represent a transition state between the other

109 two types depending on diet and plasma pH (Roy et al. 2015). A very recent publication showed that the

110 mouse CD contains a third type of cells in addition to PC and IC. These cells have features characteristics of PC

111 and IC as they were positive for both AQP2 and V-H+-ATPase, indicating that they may represent a previously

112 un-identified transitional state between the two main cell types (Park et al. 2018b).

113 The distribution of the different IC types in the distal nephron varies among species. In mouse, type-A IC

114 make 40%, 60% and 100% of the IC in CNT, CCD and OMCD/IMCD, respectively. On the other hand, type-B and

115 non-A, non-B IC make 10% and 50%, 20% and 20%, and 0% of the IC in CNT, CCD and OMCD/IMCD,

116 respectively (Kim et al. 1999).

117 Recent years of research have demonstrated a clear interplay between PC and IC. Indeed, in a similar

118 finding to what was observed in RTA patients (Sebastian et al. 1976), mice knockout on the B1 subunit of the

119 V-H+-ATPase displayed a defective conservation of sodium and chloride due to altered function of ENaC and

120 decreased abundance of pendrin (Gueutin et al. 2013). Thus, a knockout in IC results in functional defects of

121 not only IC but also PC. These animals displayed elevated levels of urinary ATP and prostaglandin E2 (PGE2)

122 originating from type B-IC but acting on PCs in a paracrine process. Thus, these findings highlight that the

123 function of one cell type is linked to that of its neighbor cells in the CD.

124 C. Tight Junctions

125 Tight junctions are separating epithelial cells thereby controlling the nature and amount of molecules

126 that are transported between rather than through the cells. Their role in ion and water homeostasis is less

127 well-defined than the transcellular pathways. Within these junctions, claudins are dynamically participating in

128 the renal epithelial function, and specifically in the so-called “chloride shunt” (Hou 2016a). In mouse CD cells

129 that express claudin 3, 4, 6, 7, 8 and 10 (Kiuchi-Saishin et al. 2002, Li et al. 2004), claudin-4 and -8 form

130 heterodimers that are expressed in both principal and IC. Claudin-4 abundance is regulated by dietary NaCl

131 and aldosterone (Moellic et al. 2005). PC-specific claudin-4 or claudin-8 knockout mice developed

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132 hypotension, hypochloremia and metabolic alkalosis (Gong et al. 2014, 2015), thereby demonstrating that in

133 CD cells, the tight junction proteins claudin-4 and claudin-8 are involved in paracellular chloride reabsorption

134 and in ion homeostasis.

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136 3. Functions of the CD cells

137 A. Ion homeostasis and blood pressure

138 The extent of renal excretion and reabsorption of ions varies to maintain an appropriate extracellular

139 fluid volume and acid-base balance (Pearce et al. 2015). In the CD, IC and PC regulate the homeostasis of acid-

140 base, fluid and electrolytes, respectively (Gueutin et al. 2013, Kriz and Kaissling 2013). Interestingly, in this

141 segment, reabsorption of sodium is partially separated from that of chloride. In PC, sodium reabsorption

142 takes place via ENaC (Reeves and Andreoli 2008), which results in the generation of a lumen-negative

143 transepithelial potential (Figure 2). This potential generates the driving force for potassium secretion via the

144 apical channel ROMK and favors proton secretion from the IC (see next paragraph) (Pearce et al. 2015).

145 Variations in blood pressure or an increase in plasma pH trigger the release of various hormones

146 including aldosterone, insulin and angiotensin II, which in turn regulate the function of ENaC (Pao 2016, Wall

147 2017). Upon aldosterone stimulation, the serine-threonine kinase (SGK1) phosphorylates and thereby

148 inhibits Nedd4-2, an ubiquitin ligase that normally ubiquitinates ENaC to initiate its termination and

149 ultimately degradation (Kabra et al. 2008, Soundararajan et al. 2010). Thus, SGK1 activation results in

150 increased abundance of ENaC at the apical membrane and further sodium reabsorption (Bhalla et al. 2006).

151 SGK-1 has a small regulatory effect on ROMK as well (Lang et al. 2010). Aldosterone also triggers the activity

152 of basolateral Na+/K+-ATPase in PC (Verrey et al. 1987)

153 Chloride reabsorption takes place partially in a paracellular way through the tight junctions and

154 transcellularly via type-B IC (Gueutin et al. 2013, Hou 2016a). The transepithelial potential generated by

155 ENaC-mediated electrogenic sodium reabsorption is enough to promote lumen-to-blood flux of chloride ions,

156 despite the unfavorable chloride concentration gradient (Hou 2016b). Claudin-4 and claudin-8 form the

157 paracellular pathway for chloride permeation, which is regulated by a number of proteins including Cap1 and

158 Kelch-like 3 (KLHL3) (See section 4. C) (Gong et al. 2014, 2015).

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159 The transcellular chloride reabsorption pathway takes place through type-B IC and is coupled with

160 the electroneutral reabsorption of sodium in the mouse model (Figure 2). This process requires the

161 concomitant action of apical pendrin and SLC4A8, and the basolateral SLC4A9 (although the stoichiometry of

162 this transporter remains a matter of debate) and ClC-Kb in a process energized by the basolateral V-H+-

163 ATPase (Leviel et al. 2010, Gueutin et al. 2013). Bicarbonate apically excreted in exchange for chloride uptake

164 through pendrin is recycled back into the cells by SLC4A8 together with a sodium ion and in exchange for a

165 chloride ion. Basolaterally, the sodium and chloride ions are transported to the interstitium via SLC4A9

166 (together with bicarbonate ions) and ClC-Kb, respectively. This functional coupling of apical and basolateral

167 transporters/channels results in the net and electroneutral absorption of both sodium and chloride ions. This

168 process is amiloride-resistant, thiazide-sensitive, and regulated by the Nedd4-2 ubiquitin ligase (Nanami et al.

169 2018). Of note, although SLC4A8 has been represented at the apical membrane of type-B IC in figure 2, its

170 precise location remains to be confirmed and further in vitro studies confirming the location and contribution

171 of each transporter need to be performed.

172 In parallel to ion reabsorption, water is also passively reabsorbed in PC, in a process regulated by the

173 arginine vasopressin (Olesen and Fenton 2017). In the normal kidney, an increase in plasma arginine

174 vasopressin concentration results in the arginine vasopressin receptor 2 gene (AVPR2) activation, which

175 triggers a cAMP-dependent cytosolic signal to relocate intracellular vesicles containing AQP2 to the apical

176 membrane. The apical membrane then becomes more water permeable.

177 B. pH homeostasis

178 With our acid-generating Western diet, type-A IC is the predominant cell type that dictates the final

179 urinary pH. In these cells, CO2 diffuses into the type A-IC and is hydrolyzed via CA II in the cytosol producing

180 H2CO3, which in turn dissociates to proton and bicarbonate.

181 The bicarbonate ions are transported to the interstitial fluid in exchange for chloride via kAE1 at the

182 basolateral membrane. On the other hand, the protons are secreted to the lumen via the apical proton pump V-

183 H+ATPase and the H+K+-ATPase. Both the pH difference across the apical membrane and the potential

184 difference across the epithelium affect the function of the V-H+ATPase (Andersen et al. 1985). In the lumen,

185 the secreted protons bind either to ammonia (NH3+) and generate ammonium (NH4

+) or bind to phosphate ions

186 (HPO42-) to generate titratable acids that are excreted in the urine. The extent of urinary acidification is

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187 regulated by the ratio of type-A versus other CD cell types and by aldosterone (Wagner 2014). Any

188 malfunction of the 3 main contributors of pH acidification (CA II, kAE1 or V-H+ATPase) results in a disease

189 characterized by a low blood pH called distal renal tubular acidosis (dRTA) (See section 4.B.).

190 Various molecules are sensitive to intracellular and extracellular pH variations and affect IC’s

191 function and plasma pH. The soluble adenylate cyclase senses bicarbonate concentration and indirectly acts on

192 the apical V-H+ATPase (Gong et al. 2010). Another example is the G protein-coupled receptor 4 (GPR4) whose

193 deletion results in decreased renal acid excretion and a metabolic acidosis in mice (Sun et al. 2015). Additional

194 pH sensors identified in the kidney are reviewed elsewhere (Brown and Wagner 2012).

195 C. Innate immunity

196 IC also play a previously un-recognized role in defending renal epithelia against bacterial infections.

197 Uropathogenic Escherichia coli (UPEC) preferentially bind to CD cells and specifically to type-A IC. Therefore,

198 in addition to being involved in urine acidification, these cells also play a significant protective role in keeping

199 the urine sterile. They achieve this goal in two major ways. First, these cells secrete protons to the urine,

200 thereby creating an acidic environment unfavorable for bacterial growth (Paragas et al. 2014). Second, upon

201 infection by UPEC, these cells specifically secrete a bacteriostatic protein called neutrophil gelatinase-

202 associated lipocalin (NGAL) or lipocalin-2 (LCN2). This protein specifically interacts with enterochelin, a

203 protein secreted by gram negative bacteria, and thereby prevents iron’s transfer to the bacteria. Additionally,

204 LCN2 is necessary for Toll-like receptor 4 (TLR4) activation in IC, via the microRNA Let-7i (Sadio et al. 2018),

205 and initiation of innate immune response by IC. This process is also hormonally regulated through AVPR2

206 activation (Chassin et al. 2007). Therefore, the function of type-A IC is not restricted to acid secretion to the

207 urine but also encompasses immune defense against bacterial invasions.

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209 4. Diseases associated with mutated proteins in the CD & phenotype of mouse models

210 A. Liddle Syndrome

211 Liddle syndrome, an autosomal dominant inherited disease, was first reported by Grant Liddle and

212 his co-workers in a kindred from Alabama, US presenting some characteristics of hypertensive symptoms with

213 negligible aldosterone secretion (Liddle 1963). The clinical presentation of patients with this syndrome

214 includes hypertension, hypokalemia, metabolic alkalosis associated with low plasma renin and aldosterone

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215 (Warnock and Bubien 1994). A successful treatment approach by administering spironolactone and

216 triamterene (two potassium-sparing diuretic) provided the first hint that the cause of the disease could be

217 somewhere between the mineralocorticoid receptor and ENaC. In fact, an over-activity of the hetero-trimeric

218 ENaC due to germline mutations in the , or -subunits can be a cause of Liddle syndrome (Hansson et al.

219 1995, Schild et al. 1996). The cytosolic domain of the 3 ENaC subunits contains a conserved PY (proline

220 tyrosine) motif, which serves as the Nedd4-binding domain (Staub et al. 1996). This interaction results in the

221 channel’s ubiquitination and proteosomal degradation. Mutations that cause Liddle syndrome were found to

222 disrupt this binding motif, thereby resulting in prolonged and uncontrolled sodium reabsorption (Rotin et al.

223 1994, Schild et al. 1996, Staub et al. 1996).

224 One mouse model of Liddle’s syndrome was developed by knocking out the -subunit of ENaC. Under

225 a high salt diet, these mice displayed a high BP, metabolic alkalosis and hypokalemia, thereby recapitulating

226 the symptoms observed in patients (Pradervand et al. 1999). Mice lacking Nedd4-2 developed a similar

227 phenotype accompanied by increased ENaC expression (Huysse et al. 2012), supporting the important role of

228 the channel and this ubiquitin ligase in development of the disease.

229 B. Distal renal tubular acidosis

230 Type 1 or distal renal tubular acidosis (dRTA) results from a renal defect in acid secretion (Mohebbi and

231 Wagner 2018) and consequently in bicarbonate reabsorption in tubules of the distal nephron (Cordat and

232 Casey 2009). Mutations in the genes encoding either kAE1 (Bruce et al. 1997, Karet et al. 1998, Jarolim et al.

233 1998), the V-H+-ATPase (Karet et al. 1999) or CA II (Lewis et al. 1988) can result in dRTA. dRTA patients

234 carrying dominantly or recessively-inherited mutations develop renal stones, hypokalemia, hyperchloremia,

235 nephrocalcinosis, metabolic acidosis and a defective urine acidification in addition to facing difficulties to

236 thrive (Trepiccione et al. 2017). AE1 is a 14 transmembrane domains Cl-/HCO3- exchanger that is expressed in

237 erythrocytes (eAE1) and in the kidney (kAE1) (Arakawa et al. 2015). The kAE1 protein is a truncated version

238 of the eAE1 protein, as it lacks the first 65 amino-terminal residues of the erythroid protein. At this time, 23

239 point or frameshift mutations in the SLC4A1 gene encoding kAE1 protein are reported to cause dRTA, in a

240 homozygous, heterozygous or compound heterozygous state (Zhang et al. 2012, Fry et al. 2012, Cordat and

241 Reithmeier 2014, Park et al. 2018a). Investigations in Madin-Darby canine kidney (MDCK) cells showed that

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242 dRTA-causing kAE1 mutants were either non-functional or mis-trafficked to the apical membrane, the Golgi or

243 the endoplasmic reticulum (Devonald et al. 2003, Toye et al. 2004, Cordat et al. 2006)(Figure 3). Co-

244 expression of dominant dRTA mutants with the wild-type (WT) kAE1 protein, thereby mimicking the situation

245 found in patients with a dominant form of the disease, showed that the mutant affected the trafficking of the

246 WT protein in these cells (Cordat et al. 2006). In contrast, co-expression of recessive mutants with kAE1 WT,

247 as found in parents of the patients with recessive dRTA, showed that the WT protein rescued the mutant’s

248 trafficking. These findings provided a molecular mechanisms for development of dRTA.

249 However, recent in vivo findings challenged our understanding of dRTA pathophysiological

250 mechanisms. Indeed, when expressed in mouse inner medullary collecting duct (mIMCD3) or mouse cortical

251 collecting duct M1 cells, the dominant mutant kAE1 R607H (corresponding to the human dominant kAE1

252 R589H dRTA mutation) showed a normal function and proper targeting of the protein to the basolateral

253 membrane (Mumtaz et al. 2017). This mutant was previously reported to be retained in the endoplasmic

254 reticulum in MDCK cells (Toye et al. 2004, Cordat et al. 2006). Moreover, in a kAE1 R607H knock-in mouse

255 model that developed dRTA upon acid challenge, the protein was properly targeted to the basolateral

256 membrane, although its expression level was lower compared with wild type mice (Mumtaz et al. 2017). In

257 fact, these mice had a lower amount of V-H+-ATPase and were unable to relocate this protein to the apical

258 membrane upon acid challenge. In fact, the number of type-A IC in these mice was significantly lower in

259 comparison with the WT mice. A recent study investigating the interactome of the V-H+-ATPase identified the

260 nuclear receptor coactivator 7 (Ncoa7) as an interactor (Merkulova et al. 2015). A targeted deletion of this

261 protein in mice resulted in incomplete dRTA (Merkulova et al. 2018). These recent findings may be the first

262 step towards deciphering the functional link between basolateral kAE1 and apical targeting of the V-H+-

263 ATPase. Therefore, our understanding of the pathophysiology associated with dRTA remains obscure and

264 further studies will be necessary to fully understand the molecular mechanisms of this complex disease.

265 The CA II enzyme is found in the cytosol of the PT cells, loop of Henle and in the IC of the CD (Laing et

266 al. 2005). CA II converts CO2 and water into bicarbonate and protons in PT cells and IC. Accordingly, the lack of

267 or dysfunction of CA II results in an impaired bicarbonate reabsorption and acid secretion (Ring et al. 2005),

268 defined as Type 3 renal tubular acidosis. Patients with type 3 RTA have acidemia, alkaline urine, osteopetrosis,

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269 cerebral calcification and mental retardation. Beside the kidney, the tissues and organs affected in type 3 RTA

270 correlate with tissue-expression of the CA II. A recent study of CA II deficient mice showed that CA II also plays

271 a significant role in urine concentration (Krishnan et al. 2017). In addition to type 3 RTA, these mice had

272 polyuria and polydipsia without altered sodium or calcium reabsorption/excretion indicating that they had a

273 specific defect in water reabsorption.

274 C. Pseudohypoaldosteronism type II

275 Another disease that manifests in acid/base dysregulation is pseudohypoaldosteronism type II

276 (PHAII). This condition originates from defects in the DCT, CNT and CD but for the purpose of this review, we

277 will focus on the role of the CD. Patients with PHA II present symptoms of increased blood pressure,

278 hyperkalemia and hyperchloremic acidosis (Wilson et al. 2001). PHA II is either caused by a deletion in the

279 first intron or missense mutations in the genes encoding with-no-lysine-kinase 1 or 4 (WNK1 or WNK4),

280 respectively. Both deletion or mutations lead to an increase in WNK activity. WNK1/WNK4 are both

281 expressed in IMCD cells (Uawithya et al. 2008), with specific expression of the long WNK1 in IC (Webb et al.

282 2016). While WNK1 is mainly cytoplasmic, WNK4 is localized to the tight junction in the distal nephron

283 (Wilson et al. 2001). Both proteins are sensitive to intracellular chloride concentration [Cl-]i changes (Terker et

284 al. 2016). An increase in [Cl-]i inhibits WNK1/WNK4 activities. However, WNK4 is inhibited at a lower

285 concentration of [Cl-]i than WNK1. Elevated [Cl-]i inhibits WNK1 activity by inhibiting its auto-phosphorylation

286 (Piala et al. 2014). Overexpression of WNK1 and disease-causing WNK4 mutants in MDCK II cells increased

287 chloride permeability through phosphorylation of the tight junction protein claudin-4 (Ohta et al. 2006).

288 WNK4 also inhibits the activity of the sodium channel ENaC, therefore PHAII-causing WNK4 mutations result

289 in increased ENaC conductivity and un-regulated sodium reabsorption (Ring et al. 2007).

290 KLHL3 and cullin 3 (Cul3) are two additional proteins whose malfunction causes PHAII (Boyden et al.

291 2012, Louis-Dit-Picard et al. 2012). These two proteins form the “cullin-ring E3 ligase” (CRL) complex where

292 KLHL3 serves as a substrate adaptor for the Cullin-3-mediated ubiquitylation of several proteins, including

293 WNK1, WNK4 and claudin-8 (Gong et al. 2015, Sohara and Uchida 2016). KLHL3 mutations are either

294 dominant or recessive and impair KLHL3 interaction with the target protein, while Cul3 mutations are all

295 dominant and alter the structure and stability of the CRL complex. These mutations result in inappropriate

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296 sodium and chloride reabsorption in the CD by at least two pathways. One pathway involves the ubiquitylation

297 of claudin-8 by KLHL3 (Hou et al. 2010, Gong et al. 2015). Knocking down claudin-8 in immortalized mouse

298 IMCD cells resulted in the loss of claudin-4 localization to tight junctions and as a result a decrease in

299 paracellular chloride conductance. PHAII-causing mutations in KLHL3 disrupt its interaction with claudin-8,

300 thereby preventing claudin-8 ubiquitination and degradation, and causing an increase in paracellular chloride

301 flux. The second pathway involves the ubiquitylation of WNK proteins by KLHL3. In a mouse model knocked-in

302 with the PHAII-causing KLHL3 R528H dominant mutation, WNK1 and WNK4 expression level were increased

303 compared to control littermates due to a loss of interaction with the KLHL3 mutant and impaired WNK1 and 4

304 ubiquitylation (Susa et al. 2014). This disease illustrates the complex interplay between tight junction

305 properties and transcellular ion fluxes.

306 D. Pendred syndrome

307 Pendred syndrome is an autosomal recessive disease characterized by sensorineural deafness, non-

308 endemic goitre and in some cases iodide organification (Kopp and Bizhanova 2011). This disease was first

309 described by British practitioner Vaughan Pendred from his observation of the association of goitre and

310 deafness (Pendred 1896, Kopp and Bizhanova 2011). Pendred syndrome is the underlying cause for 10% of all

311 syndromic deafness with an estimated incidence rate of 7.5-10 in 100,000 population (Reardon et al. 1997).

312 Encoded by the SLC26A4 gene, pendrin is a transmembrane electroneutral exchanger for various

313 anions including bicarbonate and chloride (Wall 2016) and is responsible for the Pendred syndrome (Everett

314 and Green 1999). The gene is highly expressed in various organs including inner ear, thyroid gland (symptoms

315 of this disease include deafness and goitre) and in the kidney. In this organ, pendrin can increase the urinary

316 bicarbonate concentration in alkalosis, a process synchronized with reabsorption of NaCl and thus in

317 maintenance of blood pressure as detailed in section 3.A.

318 Pendrin is expressed at the apical surface of the type-B and in non-A-non-B IC in the CD (Royaux et al.

319 2001, Soleimani et al. 2001). During metabolic alkalosis, pendrin expression increases significantly and the

320 protein localizes predominantly at the apical membrane of mice CD cells (Wagner et al. 2002). In contrast, the

321 mice demonstrated a dwindled expression and cytosolic pendrin upon metabolic acidosis. Compared with

322 their WT counterparts, pendrin knockout mice fed an alkaline diet were not able to secrete bicarbonate into

323 the urine (Royaux et al. 2001). When fed a low NaCl diet, pendrin knockout mice demonstrated a lower blood

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324 pressure and higher plasma pH than WT mice, most likely due to a reduced chloride/bicarbonate exchange

325 activity (Verlander et al. 2003). The knockout mice also displayed a significantly reduced ENaC expression, a

326 finding that could explain the hypotension observed at steady-state (Kim et al. 2007). Interestingly however,

327 patients with Pendred syndrome as well as the pendrin knockout mice neither have abnormal renal function,

328 acid-base homeostasis nor fluid and electrolyte homeostasis at steady state, which suggests that other renal

329 chloride/bicarbonate exchangers are able to compensate the loss of pendrin (Royaux et al. 2001, Verlander et

330 al. 2003, Wall et al. 2004, Kopp and Bizhanova 2011).

331 E. Nephrogenic Diabetes Insipidus

332 First introduced in 1947, the term “nephrogenic diabetes insipidus’ (NDI) emerged in a study of 7

333 cases in one family where the condition was transmitted by mothers to their male progeny (Williams and

334 Henry 1947). This disease can be defined as the nephrogenic incompetence to concentrate urine in response to

335 the antidiuretic hormone arginine vasopressin. This results in defective permeability to water in the DCT or

336 CD, or an increase of the corticopapillary interstitial osmotic gradient or a combination of both (Valtin and

337 Schafer 1995). Therefore, NDI patients excrete a large volume of water through urine (water diuresis)

338 (Morello and Bichet 2001). Diuresis is also associated with increased fluid/water intake (polydipsia), and

339 could also lead to secondary issues like hypernatremia and dehydration.

340 About 90 % of patients with congenital NDI carry a mutation in the gene encoding AVPR2 , while the

341 remaining 10 % have a mutation in the gene encoding the water channel AQP2 (Bech et al. 2018). AQP3

342 knockout mice also developed NDI (Ma et al. 2000), but there is no NDI patient with a mutation in this gene

343 described at this time. The most common non-hereditary cause of NDI is chronic lithium therapy

344 (administered for psychiatric disorders including depressive or bipolar disorder) due to an inhibition of the

345 vasopressin-triggered cAMP signaling cascade (Bech et al. 2018).

346 Although no viable AQP2 knockout mouse model has been described, knocking-in an NDI-causing

347 AQP2 mutation (T126M) in mice confirmed the essential role of this channel in NDI (Yang et al. 2001). The

348 mutant pups developed normally until the first 2-3 days but then started failing to thrive and did not survive

349 past day 6 unless given supplemental fluids. When heterologously expressed in Xenopus oocytes or Chinese

350 hamster ovarian CHO cell line, the human ortholog of this mutant was functional but retained in the

351 endoplasmic reticulum unless chemical chaperones were provided. In the CD epithelial cells, AQP3 is

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352 expressed at the basolateral membrane. Deletion of this gene resulted in normally developed mice that

353 presented polyuria and a dilute urine (Ma et al. 2000). In these mice, expression of AQP1 and AQP4 was not

354 affected, however, AQP2 abundance was reduced significantly. Arginine vasopressin administration to these

355 animals revealed a mild urine-concentrating defect.

356 A mouse model carrying an NDI-causing AVPR2 mutation also displayed an inability to concentrate

357 its urine and a dilute basal urine. Similarly to the AQP2 knocked-in mice described above, these pups failed to

358 thrive and died within 2 weeks from birth from dehydration (Yun et al. 2000).

359 Over the past decade, considerable research efforts have aimed at identifying therapeutic strategies

360 to overcome the molecular defects causing NDI. Strategies have focused on bypassing or potentiating AVPR2

361 signaling and on increasing the abundance of apical AQP2 channel in principal cells as reviewed elsewhere

362 (Sands and Klein 2016).

363

364 5. Conclusions

365 The renal collecting duct is the site where the final urine composition is set. This occurs through fine

366 hormonal regulations, the removal or relocation of specific proteins from/to the apical membrane and the

367 dynamic regulation of the abundance of various cell types. As illustrated above, this segment of the kidney

368 plays essential roles in ion, water and acid-base homeostasis. Many pathologies are associated with

369 dysfunctional CD. However, despite our current understanding of the CD function, we are still uncovering

370 unsuspected features of these cells. The recent report of the role of type-B IC in blood pressure regulation

371 illustrates that there remains many gaps in our knowledge of the CD. There is more to discover, including the

372 respective role of electrogenic versus electroneutral ion reabsorption in PC and type-B IC, respectively. What

373 regulates one pathway versus the other in maintainance of blood pressure? What is the role of tight junctions

374 in this process? Are mutated tight junction proteins also causing abnormal blood pressure? Similarly, the role

375 of non-A, non-B IC remains obscure. The regulation of the CD cells interconversion is not well-understood

376 either. Further, the interplay between the various CD cell types needs further investigation, but a robust cell

377 model recapitulating all the features of CD cells is still lacking. Thus, further research is needed to decipher the

378 role of this kidney segment in health and diseases.

379

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380 Acknowledgements

381 This work was supported by funding from the Kidney Foundation of Canada (KFOC140013), the

382 Canadian Institutes of Health Research (MOP #142251), an innovation grant and bridge funding from the

383 Women and Children’s Health Research Institute (supported by the Stollery Children’s Hospital Foundation),

384 bridge funding from Alberta Innovates and the Canada Foundation for Innovation. R.L. and A.S.U received a

385 graduate studentship from the CREATE Program funded by the Natural Sciences and Engineering Research

386 Council (NSERC) and from the Department of Physiology and the Faculty of Medicine and Dentistry from the

387 University of Alberta.

388

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715

716

717 Figure legends

718 Figure 1: Types of transepithelial transport. As the plasma membrane is poorly permeable to ions and

719 water, two pathways are possible for transepithelial transport of molecules: the transcellular (green and blue

720 arrows and molecules) and paracellular (burgundy arrow) pathways. If molecules X or Y cross the epithelium

721 through the transcellular pathway, they need to cross a series of two membranes, the apical and the

722 basolateral membrane in addition to diffusing through the cytosol to reach the opposite membrane. Specific

723 channels and primary or secondary active transporters are expressed in these membranes to facilitate and

724 regulate the nature, direction and number of molecules crossing the plasma membrane. The localization of

725 these transporters is essential to drive molecules’ movement in one direction versus the other across the

726 epithelium (absorption versus secretion).

727 The second pathway facilitates the movement of a molecule Z in a paracellular way through tight

728 junctions (burgundy arrow). For this type of transport, molecules are passively moving between epithelial

729 cells down their electrochemical gradient, and only have to cross one “barrier”, the tight junctions (as opposed

730 to crossing apical and basolateral membranes in the transcellular pathway). Tight junctions are composed of

731 occludins, claudins, junction adherens molecules and tricellulins. The highly dynamic nature of tight junctions

732 allows a fast and specific regulation of the amount and the nature of molecules moving through this pathway.

733 Note that primary active transporters can be apically or basolaterally located and can carry one or more

734 substrates (in a co-transport or antiport mode of action).

735

736

737 Figure 2: Schematic diagram illustrating the two NaCl reabsorption pathways identified in CD cells. The

738 electrogenic pathway (shown on the left) includes the activity of PC and type-A IC. The activation and opening

739 of ENaC (1) results in a lumen-negative transepithelial potential. Apically reabsorbed sodium is actively

740 exported outside of the cells via the basolateral Na+/K+-ATPase (represented as “NKATPase”) (2). This

741 transepithelial potential generates a driving force for the apical secretion of potassium through KCC and ROMK

742 in PC and of protons through the V-H+-ATPase (represented as “HATPase”) and H+/K+-ATPase (represented as

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743 “HKATPase”) in type-A IC (3). This potential also favors paracellular fluxes of chloride ions through the tight

744 junctions, specifically via claudin-4 and 8 (not shown) (4). Water passively follows ion reabsorption through

745 apical AQP2 and basolateral AQP3/4.

746 The electroneutral pathway (shown on the right) includes the reabsorption of sodium and bicarbonate ions in

747 exchange for a chloride ion through SLC4A8. The bicarbonate is provided by the activity of apical pendrin

748 which exports bicarbonate in exchange of chloride reabsorption. At the basolateral membrane, chloride ions

749 are transported to the blood via ClC-Kb and sodium and bicarbonate via the cotransporter SLC4A9.

750

751 Figure 3: Schematic diagram illustrating the previous and the new model for SLC4A1-mediated dRTA.

752 Left, dRTA-causing SLC4A1 mutant proteins were described as either non-functional (1), intracellularly

753 retained (2) or apically mis-trafficked (3), based on MDCK cell studies. Right, recent in vivo findings showed

754 that a dominant dRTA-causing mutation did not cause mis-trafficking of the protein, but rather a lack of

755 relocation of the V-H+-ATPase to the apical membrane upon plasma acidification, possibly resulting in a

756 dramatic loss of type-A IC in the CD.

757

758

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DraftChannel

Cotransporter

BloodLumen

Z

Ion exchangers Channel

X

YYY

Y

XX

X

Cotransporter

Channel

Ion exchangers

Primary active transporters

ATP

ADP + Pi

Primary active transporters

Figure 1 Lashhab & Ullah

ATP

ADP + Pi

Transcellular secretion

Transcellular absorption

Paracellular absorption

Page 30 of 32



DraftA-IC

PC

AE1

NKATPase

HATPase

ROMK--

--

---

KCC

ENaC

BloodLumen

K+

K+

Na+

K+

Na+

AQP2 H2O-

AQP3/4H2O

K+

Cl-

Cl- Cl-

H+

HKATPase

H+

Cl-

HCO3-

SLC26A7

Cl-

HCO3-


Principal cell-mediated electrogenic NaCl reabsorption

B-IC HATPase

BloodLumen

Na+

Na+

H+

SLC4A9

HCO3-

Pendrin

2Cl-

Cl-

2HCO3-

HCO3-SLC4A8

B-IC cell-mediated, electroneutral NaCl reabsorption

1

3

2

3

4

4

ClC-KbCl-

Page 31 of 32



Draft

A-IC

HATPase

BloodLumen

K+

H+

HKATPase

H+ SLC26A7

Cl-

HCO3-


Previous model for SLC4A1-mediated distal RTA

BloodLumen

New model for SLC4A1-mediated distal RTA

A-IC AE1

HATPase

K+

HKATPase ?

H+

Cl-

HCO3-

Cl-

HCO3-Cl-

HCO3-

SLC26A7?

Cl-

HCO3-

Cell Death?

1

2

3

AE1

AE1

Page 32 of 32



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