Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 21, 2015). doi:10.1152/ajpregu.00568.2013

1

Plasticity of Renal Endocrine Function

2

Birgül Kurt, Armin Kurtz

3 4 5

Institute of Physiology, University of Regensburg, Regensburg, Germany

6 7 8 9 10 11 12 13 14 15 16

Correspondence to:

17

Birgül Kurt, Ph.D.

18

Institute of Physiology

19

University of Regensburg

20

D-93053 Regensburg

21

Germany

22

Phone: +49-9419432940

23

Fax: +49-9419434315

24

e-mail: [email protected] 1

Copyright © 2015 by the American Physiological Society.

25

Abstract

26

The kidneys are important endocrine organs. They secrete humoral factors such as

27

calcitriol, erythropoietin, klotho and renin into the circulation and they are therefore

28

essentially involved in the regulation of a variety of processes ranging from bone

29

formation to erythropoiesis. The endocrine functions are established by cells such as

30

proximal or distal tubular cells, renocortical interstitial cells or mural cells of afferent

31

arterioles. These endocrine cells are either fixed in number such as tubular cells

32

which individually and gradually up- or downregulate hormone production or they

33

belong to a pool of cells which display a recruitment behavior such as erythropoietin

34

and renin producing cells. In the latter case regulation of humoral function occurs via

35

(de)recruitment of active endocrine cells. As a consequence renin and erythropoietin

36

producing cells in the kidney show a high degree of plasticity by reversibly switching

37

between distinct cell states. In this review we will focus on the characteristics of renin

38

and of erythropoietin producing cells, especially on their origin and localization, their

39

reversible transformations and the mediators, that are responsible for transformation.

40

Finally, we will discuss a possible interconversion of renin and erythropoietin

41

expression.

42 43 44

Introduction

45

The kidneys fulfil a variety of functions such as elimination of waste products of

46

metabolism, regulation of fluid volume and electrolyte concentrations and acid-base

47

balance. Besides these excretory and homeostatic functions, the kidneys are

48

endocrine organs essentially involved in the regulation of bone mineralization, blood

49

pressure and erythropoiesis. Thereto, the kidneys produce and secrete the hormones

50

calcitriol, erythropoietin (EPO), klotho and renin.

51

Klotho, that exists in a membranous and in a secreted form, regulates calcium and

52

phosphate homeostasis

53

Membrane klotho functions mainly as a coreceptor for fibroblast growth factor (FGF)

54

23

55

(187)

56

its soluble form

(85, 178)

(67,

83,

129)

, thus contributing to bone mineralization.

, which causes phosphaturia

(85)

and decreases circulating calcitriol levels

and renal phosphate resorption, resulting in reduced plasma phosphate levels. In (66, 110)

, klotho mediates enhanced Ca2+ (re-)absorption in the kidney 2

(21, 23)

57

and in the intestine, by activating TRPV5 and TRPV6

58

mutations or deficiency of klotho result in hyperphosphatemia, hypercalcemia and

59

vascular calcification (64, 84, 95, 157).

60

Calcitriol

61

mineralization by regulating homeostasis of calcium and phosphate through acting on

62

the intestinal tract and the kidneys Besides its role in regulation of calcium and

63

phosphate homeostasis, calcitriol contributes to erythropoiesis

64

activity of monocytes and macrophages

65

lymphocytes and thus the adaptive immunity are suppressed by calcitriol

66

D deficiency results in osteomalacia

67

expression and thus modulates the activity of the renin-angiotensin-aldosterone

68

system (RAAS) (99).

69

The aspartyl protease renin is the key regulator enzyme of the RAAS, which controls

70

blood pressure and fluid-electrolyte balance

71

appetite and thirst (6, 43, 106, 116, 147). Renin cleaves the decapeptide angiotensin-I (ANG

72

I) from liver-derived angiotensinogen. ANG I is further cleaved by the angiotensin-

73

converting enzyme ACE to the octapeptide ANG II,(163) which binds to two distinct

74

receptors, the AT1 and the AT2 receptor.(17, 20) The majority of actions of ANG II are

75

mediated via the AT1 receptor. The AT1 receptor mediated signaling cascades result

76

in vasoconstriction and increase of extracellular fluid volume, an increase of renal

77

tubular sodium re-absorption and stimulation of aldosterone release.(4,

78

effects lead to an increase of blood pressure. The AT2 receptor is considered to

79

oppose the actions of ANG II via the AT1 receptor, by mediating vasodilation via

80

formation of NO, prostanoids and bradykinin.(1, 16, 17, 33, 72, 109, 184) Dysregulation of the

81

RAAS is involved in the pathogenesis of several hypertensive disorders

82

fibrotic degenerations (185).

83

The glycoprotein hormone erythropoietin (EPO) is the primary humoral regulator of

84

red blood cell formation in mammals

85

blood hemoglobin concentration.(70,

86

(EPO-R) on the surface of cells belonging to the committed erythroid cell lineage

87

in the bone marrow, activates predominantly the Jak/STAT signaling cascade in the

88

target cells

(34, 35, 138)

channels. Missense

the physiologically active form of vitamin D contributes to bone

(5, 48)

as well as the

(30)

. In contrast, proliferation and activity of T-

(59)

(30)

. Vitamin

and in addition, calcitriol inhibits renin

(56, 180)

by regulating salt transport, salt

20)

These

(28, 96)

and

(77-79, 81, 149)

182)

. It is essential to maintain normal

Binding of EPO to high affinity receptors

(31, 32, 60)

(13)

, resulting in survival, proliferation and differentiation of immature

3

(12, 41, 54, 76, 168)

89

erythroid precursors to mature erythrocytes

90

EPO such as in states of chronic kidney disease, results in anemia.(70, 182)

91

The aforementioned endocrine functions of the kidneys are provided by different cell

92

types (Figure 1). Klotho is expressed by distal tubule cells

93

activated in the mitochondria of proximal tubule cells by the action of 1-α-

94

hydroxylase.(127,

95

cortex.(7, 78, 113) In the adult kidney renin is produced by juxtaglomerular (JG) cells that

96

are located in the media layer of afferent arterioles at the entrance into the

97

glomerulus.(56, 151)

98

It is known that under specific circumstances cells can (reversibly) transform into

99

another cell type with different protein expression patterns and characteristics.(36, 82,

172)

. Insufficient production of

(118, 122)

and calcitriol is

EPO is expressed by interstitial fibroblast-like cells of the renal

100

139)

101

type and hence cell plasticity is known.(15, 24, 49, 101, 165) For example, if the RAAS is

102

challenged, additional cells transform into renin producing cells.(15,

103

recruitment phenomenon is also known for EPO producing cells in the kidney.

104

Moreover, during development of interstitial kidney fibrosis, EPO expressing cells

105

undergo a phenotype switch into myofibroblasts, that stop producing EPO, resulting

106

in the well known renal anemia. As in the case of renin expressing cells, also the

107

transformation of EPO expressing cells into another cell type is reversible, at least to

108

some

109

transformation of renin expressing into EPO producing cells, indicating a novel

110

aspect of renal endocrine plasticity.(89)

111

In this review we will focus on the characteristics of renin and EPO producing cells,

112

especially their origin, structure and localization, their reversible transformations and

113

the mediators, that are responsible for transformation. Finally, we will discuss a

114

possible interconversion of renin and of EPO expression.

Also for renal renin and EPO expressing cells transformation into another cell

extent.(165) Recently

first

evidence

was

provided

for

90)

an

A similar

endocrine

115 116 117

Plasticity of renin production

118 119

Development of renin cells

120

In the adult kidney renin cells are located in the distal parts of afferent arterioles just

121

at the entrance into the glomerulus

(8, 56, 151, 175)

4

, thus coining their name,

122

juxtaglomerular cells. These cells, which have a cuboid-like shape, are an integral

123

part of the media layer of afferent arterioles, thereby forming the walls of the vessels

124

entering the glomeruli. This typical juxtaglomerular position of renin cells in the adult

125

kidney is the final result of a highly dynamic spatiotemporal renin expression pattern

126

during nephrogenesis.(151) During embryonic kidney development, renin expression is

127

occasionally found already in the undifferentiated metanephric mesenchyme

128

state before vessel formation has begun. It is thought that renin expressing cells

129

derive from FoxD1-mesenchymal cells,(100) which are also precursors for vascular

130

smooth muscle cells, mesangial cells, pericytes and fibroblasts.(101) Once renal

131

vascularization has begun, renin expressing cells are found as wall cells of the

132

developing interlobar and arcuate arteries.(50,

133

maturation of the arterial tree, when cortical radial arteries branch off and spread out

134

from arcuate arteries and afferent arterioles spread out from cortical radial arteries,

135

renin expressing cells appear as mural cells covering and stabilizing the newly

136

formed vessels. In this context renin expression appears to be highest in the

137

youngest and immature preglomerular vessels. With ongoing vessel maturation renin

138

expression in these segments ceases and the cells acquire a smooth muscle like

139

phenotype. As a result, developmental renin expression has a wave-like character

140

flushing over the preglomerular arteriolar tree. At the end of development, in the adult

141

kidney only few renin expressing cells are left over at the classical juxtaglomerular

142

position.(8,

143

smooth muscle cells of afferent arterioles on the one side and the mesangial cells on

144

the other, especially the extraglomerular mesangial (EGM) lacis or Goormaghtigh

145

cells

146

physiological site of renal renin production are developmentally related to both

147

smooth muscle and EGM cells.

56, 151, 175)

75)

(102)

in a

With ongoing nephrogenesis and

These cells form the interface between the mural vascular

(167)

. It is near at hand therefore to speculate that the JG cells as the normal

148 149

Structure of juxtaglomerular renin producing cells

150

Juxtaglomerular renin producing cells are morphologically characterized by large

151

electron-dense vesicles that occupy up to 40% of the extranuclear cell volume

152

These vesicles belong to the family of lysosome related organelles (LRO).(177) As

153

lysosomes they have an acidic content

154

lysosomal integral membrane protein 2 (LIMP-2), lysosomal-associated membrane

(137)

5

(169)

.

and they contain proteins such as

(98, 152)

155

protein 1 (LAMP-1) and LAMP-2

which are important for directing proteins to

156

lysosomes

157

these lysosome related organelles. Within the vesicles prorenin is proteolytically

158

cleaved to enzymatically active renin which remains stored in the vesicles until it is

159

released by controlled exocytosis, which resembles the mode of compound

160

exocytosis

161

occasionally fuse with the plasma membrane to release their contents into the

162

extracellular space. Notably, the development of the prominent vesicles is dependent

163

on (pro)renin accumulation. If prorenin is not directed to those lysosome related

164

organelles, but instead is constitutively secreted, only very few and small vesicles are

165

found in JG cells

166

cells is the high density of gap junctions which connect renin producing cells with

167

neighboring smooth muscle, endothelial and mesangial cells (2, 57, 63, 176).

(148)

. At the level of the Golgi apparatus glycosylated prorenin is sorted to

(169)

. Hereby vesicles fuse intracellularly to huge vesicular networks which

(169)

. Another remarkable structural characteristic of juxtaglomerular

168 169

Markers and other genes conferring identity of renin producing cells

170

Gene expression profiling of renin cells

171

(aldo-keto reductase family 1, member 7), an enzyme involved in NADPH-dependent

172

oxidoreduction of carbonyl groups

173

expression in the adult kidney is restricted to the juxtaglomerular renin expressing

174

cells (14, 103). Another gene, that is enriched in the JG cells compared with total kidney

175

cortex, is the gap junction (GJ) protein connexin 40 (Cx40), which has already been

176

shown in ultrastructural and immunohistochemical studies to be highly expressed in

177

juxtaglomerular renin expressing cells. Additionally, Cx40 is expressed in endothelial

178

cells of the afferent arterioles and in mesangial cells. These two cell types, together

179

with renin producing cells, form a network of cells that are interconnected via Cx40

180

gap junctions.(2,

181

important for the correct positioning, as well as for the identity of renin expressing

182

cells in the kidney.(92,

183

genes in the JG renin cells such as RGS5 (regulator of G protein signaling 5) and

184

CRIP1 (cysteine-rich intestinal protein).(14) Their exact roles concerning the identity of

185

renin expressing cells is still elusive. Another factor, which has been discovered to be

186

important for renin cell identity, is the RNase III endonuclease Dicer.(162) Dicer is

187

responsible for the processing of a pre-miRNA to a mature miRNA, which are small

57, 63, 176)

179)

(136)

revealed strong enrichment of Akr1b7

(71)

. Immunohistochemistry confirmed, that Akr1b7

Obviously Cx40 and hence cell-to-cell communication is

The gene expression analysis identified other enriched

6

188

non-coding RNAs that regulate gene expression at the post-transcriptional level.

189

Renin cell specific deletion of Dicer results in an almost complete loss of renin

190

expressing cells and a thinner and smaller morphology of the residual renin cells.

191

Further analysis revealed the two miRNAs miR-330 and miR-125b-5p to be relevant

192

for the identity of renin expressing cells with opposite actions.(117)

193

In this context several candidate transcription factors have been suggested to be of

194

higher relevance for the identity of renin producing cells. One among these is the

195

transcription factor "recombination signal binding protein for immunoglobulin kappa J

196

region" (RBP-J), which is the main transcriptional mediator of the Notch signaling

197

pathway that regulates cell fate. Conditional deletion of RBP-J in the renin cell

198

lineage results in a strongly decreased number of renin expressing cells, thus

199

emphasizing the impact of this transcription factor for renin cell identity.(144) Besides

200

RBP-J, the transcription factor CREB (cAMP response element-binding protein) has

201

been found enriched at the corresponding binding site in renin-expressing cells.(14)

202

Indeed, the importance of the cAMP/CREB signaling pathway concerning the renin

203

cell identity has already been shown in several studies

204

effects via CREB, which acts in association with the histone acetyl transferases CBP

205

and p300. Renin cell specific knockouts of CBP and p300 resulted in a markedly

206

diminished number of renin expressing cells, indicating their importance for renin cell

207

identity.(51) Also renin cell specific deletion of Gsα, that mediates activation of

208

adenylate cyclase and thus cAMP formation, resulted in a nearly complete abolition

209

of renin expressing cells in the mouse kidney

210

more transcription factors have been found, that might determine the renin cell

211

phenotype such as KLF2 (krueppel-like transcription factor 2) and NR2F (nuclear

212

receptor subfamily 2).(14) Their exact contributions to the renin cell identity have to be

213

determined in further studies.

(51, 125)

. cAMP mediates its

(125)

. In addition to RBP-J and CREB,

214 215

Recruitment of renin expressing cells

216

Chronic challenges of the renin-angiotensin-aldosterone system such as reduced

217

renal perfusion pressure, salt restriction or pharmacological inhibition of the RAAS

218

result in an enhancement of renin expression. Elevated renin expression is caused

219

by an increased number of renin expressing cells

220

gradual increases of renin mRNA in existing renin producing cells.(104) Recruitment of 7

(9, 49, 52, 153, 166)

rather than by

221

renin expressing cells (Figure 2) occurs mainly via reversible metaplastic re-

222

transformation of EGM cells (known as hypertrophy of the juxtaglomerular apparatus)

223

and of smooth muscle cells along the media layer of afferent arterioles into renin

224

producing cells.(26, 101, 102, 131) In addition, also cell divisions of renin expressing EGM

225

has been reported, suggesting that recruitment of renin cells may also include limited

226

renin cell hyperplasia

227

from renin expressing precursors, can undergo the reversible metaplastic re-

228

transformation into the renin expressing endocrine state, when homeostasis is

229

threatened.(101) Obviously, those smooth muscle and mesangial cells, which had

230

already expressed renin during nephrogenesis, retain the memory to re-enact the

231

developmental program. The re-transformation of VSM cells and hence recruitment

232

of renin expressing cells along the afferent arterioles occurs in a retrograde direction

233

starting from the juxtaglomerular position back to interlobar arteries.(15,

234

same time, when VSM cells re-differentiate into renin expressing cells, genes that are

235

typically expressed in JG cells such as Akr1b7

236

the recruited renin cells, underlining the phenotype switch of VSM into renin

237

expressing cells. Parallel to the appearance of Akr1b7 and Cx40, as markers of the

238

renin cell phenotype, the VSM cell specific markers Cx45, α-smooth muscle actin

239

(αSMA), smoothelin and myosin heavy chain disappear due to the re-differentiation

240

from the contractile phenotype of VSM cells to the endocrine phenotype of renin

241

expressing cells.

242

recruitment of preglomerular smooth muscle cells into renin producing cells does not

243

occur in EGM cells which strongly express Cx40 but not α-smooth muscle actin

244

already in the resting renin negative state (91, 171, 189). This leads to the impression that

245

two quite different cell populations are operating under RAAS stress with the

246

common goal of producing renin.

(131)

. Notably, only VSM cells and mesangial cells that originate

(88, 136)

(14, 103)

and Cx40

(88)

50)

At the

are expressed by

This characteristic change of gene expression during

247 248

In vivo factors determining recruitment of renin expression

249

The number of renin cells is essentially regulated by the systemic and intrarenal

250

blood pressure, by the sodium balance of the organism and more directly by the

251

activity of the RAAS itself

252

pressure leads to recruitment of renin cells, whilst states of (non-renin dependent)

253

hypertension cause downregulation of the number of renin producing cells. These

(9, 52, 153, 166)

. Longer-lasting states of low arterial blood

8

254

effects are mediated by the activity of the sympathetic nervous system on the one

255

hand, which favors renin cell recruitment

256

other. How this intrarenal pressure related mechanism works to determine the

257

number of renin cells, is not understood in detail. It is conceivable that the macula

258

densa could play a mediator function in this context

259

cells express cyclooxygenase 2 (COX-2), and some lines of evidence suggest an

260

involvement of COX-2 and prostaglandin E2 (PGE2) which should favor recruitment

261

of renin cells. COX-2 and PGE2 are also thought to mediate renin cell recruitment in

262

states of sodium depletion

263

renin cells.

264

Genetic loss of function defects of the RAAS cause a massive overformation of renin

265

cells in the kidney

266

is independent of the affected component of the RAAS

267

cell hyperplasia is triggered by parameters dependent on the activity of the RAAS. In

268

line, also inhibitors of the RAAS such as direct renin inhibitors, ACE-inhibitors or AT1

269

receptor inhibitors cause a compensatory strong recruitment of renin cells in adult

270

kidneys of humans and laboratory animals

271

involves hypertrophy of the juxtaglomerular apparatus due to re-transformation of

272

EGM cells and re-transformation of smooth muscle cells of afferent arterioles. In

273

addition, limited proliferation of renin expressing cells can occur in this context. Re-

274

transformation of smooth muscle cells but not of extraglomerular mesangial cells

275

appears to be dependent on the availability of nitric oxide, in particular endothelium

276

derived nitric oxide (126).

(61)

and by an intrarenal mechanism on the

(58, 120, 130)

. The macula densa

(40)

, whilst sodium overload downregulates the number of

(53, 170, 174)

.The development of this special form of cell hyperplasia (105)

. This suggests that renin

(90)

. Recruitment of renin cells thereby

277 278

Intracellular signaling pathways involved in renin cell recruitment

279

Some genes/factors that confer or contribute to the identity of adult JG renin

280

expressing cells, have also strong impact on the ability for recruitment of renin

281

expressing cells. The enzyme Akr1b7 has been shown to be co-regulated with renin,

282

meaning that recruited renin expressing cells also express Akr1b7. Since identity and

283

plasticity of renin expressing cells in Akr1b7 knockout mice are not affected

284

suggested that Akr1b7 plays no important role for the integrity and plasticity of renin

285

expressing cells. Cell-to-cell communication via the gap junction protein Cx40, that

286

couples renin expressing cells to each other, to endothelial and to mesangial cells, is 9

(103)

, it is

287

indispensible for the correct positioning of adult JG cells as well as for the ability to

288

recruite when the RAAS is challenged. Even during prolonged stimulation of renin

289

expression/secretion by severe sodium depletion, Cx40 deficient renin expressing

290

cells are not recruited in preglomerular arterioles. Notably, these mutant dislocated

291

renin cells escape the physiologic negative feedback control by pressure.(92,

292

Renin cell specific deletion of Dicer, the RNase III endonuclease that produces

293

microRNAs, results in a decreased number of renin expressing cells, showing the

294

importance of post-transcriptional control for the maintenance of renin expressing

295

cells. It is suggested, that two miRNAs balance the endocrine/VSM cell phenotype of

296

the JG cells: under normal conditions, miR-125b-5p is expressed in VSM and in JG

297

cells, to ensure their contractility. However, when homeostasis is threatened, miR-

298

125b-5p expression decreases in the VSM cells thereby allowing them to regain the

299

endocrine phenotype. But in the JG cells, miR-125b-5p is still expressed.

300

Additionally, miR-330 is expressed in the JG cells, which inhibits the expression of

301

vascular smooth muscle genes in favor of an endocrine phenotype. Besides the

302

before mentioned genes two transcription factors have been identified, which are

303

crucial for identity as well as for recruitment of renin expressing cells: CREB and

304

RBP-J. In absence or impaired activation of these transcription factors in the renin

305

cell lineage, recruitment of renin expressing cells due to homeostatic challenge is lost

306

(19, 135)

179)

.

307 308 309

Plasticity of EPO production

310 311

Development of EPO cells

312

During embryonic development EPO is predominantly produced by the liver, mainly

313

by hepatocytes surrounding the central veins.(79) Besides hepatocytes, also

314

nonparenchymal perisinusoidal cells, identified as Ito cells, have been shown to be

315

EPO producers.(111,

316

during late gestation in humans or at around and shortly after birth in rodents EPO

317

expression switches from the liver to the kidneys as main production site,(38, 121, 188)

318

with the kidneys accounting for about 85-90% of serum EPO levels during normal

319

erythropoiesis.(46,

77)

154)

With ongoing maturation of the kidney cortex which occurs

In adults, the liver retains its ability to produce EPO to some 10

(73, 119, 128)

320

extent in response to challenge

but cannot compensate for the loss of

321

kidney-derived EPO.(68, 173) In the kidneys, EPO is produced by peritubular fibroblast-

322

like interstitial cells. So far only data for the distribution of EPO producing cells in

323

postnatal kidneys are available. They show that in the unstressed kidney EPO

324

expressing cells are located in the deep renal cortex (predominantly juxtamedullary

325

region) and outer medulla.(7, 113, 133) Notably, in the continuously hypoxic area of the

326

inner medulla no EPO expressing cells are found

327

cells is not fully clear. According to their fibroblast-like character a descent from

328

FoxD1 positive cells of the renal mesenchyme would be plausible

329

some neuronal marker proteins have been detected in EPO producing cells, also a

330

descent from the neural crest has been discussed

331

lineage with myelin protein zero-Cre (P0-Cre) in the kidney revealed that almost 90%

332

of the fibroblast-like EPO expressing cells belonged to this lineage.(3) The

333

spatiotemporal development of EPO producing cells in the developing kidney has not

334

yet been investigated in detail. There is, however, evidence that in the adult kidney

335

potential EPO producing cells exist, that have not produced EPO before, but can be

336

activated in the adult kidney for the first time.(186) This suggests the existence of a

337

silent reserve pool of cells capable to produce EPO.

(97)

. The origin of EPO expressing (62, 100)

. Since also

(128)

. Tracing of the neural crest

338 339

Structure of EPO cells

340

EPO producing cells in the kidney do not display special morphologic characteristics.

341

They appear as elongated cells with several extensions that are in close

342

neighborhood to basolateral surfaces of tubuli

343

in close contact to capillary endothelial cells

344

EPO producing cells might be endothelial cells (94).

(7, 134)

. Similar to pericytes the cells are

(7)

, leading to the early suggestion that

345 346

Markers and other genes conferring identity of EPO cells

347

The majority of EPO producing cells expresses CD73 (ecto-5´-nucleotidase)

348

128, 133, 134)

349

typically being expressed by fibroblasts.(25,

350

staining of the renal EPO producing cells for the neural markers such as MAP2

351

(microtubule-associated protein 2) and NF-L (neurofilament light polypeptide),

352

suggesting that the EPO producing cells originate from the neural crest.(128)

and PDGFR-β (the ß-receptor for platelet-derived growth factor)

11

47, 164)

(7, 45, 113,

(134)

, both

Another study showed positive

353

EPO secretion is directly linked to transcription of the EPO gene via the transcription

354

factor HIF-2 (hypoxia-inducible factor 2).(55,

355

transcription factors (HIFs) belong to the PAS (Per-ARNT-Sim) family of

356

heterodimeric basic helix-loop-helix transcription factors, that consist of the

357

constitutively expressed HIF-1β (ARNT) subunit, and one of the either O2 labile HIF-α

358

subunits HIF-1α, HIF-2α and HIF-3α.(114,

359

enhancer sequences of target genes, with HIF-1 inducing transcription of genes

360

involved in, for example, anaerobic glycolysis,(115) and HIF-2 as the main regulator of

361

EPO production.(55,

362

constructs of different length, it turned out that EPO expression in liver and kidney

363

are differently regulated. The same conclusion was reached by the expression of

364

gene constructs incorporating different sections of the EPO gene and its flanking

365

sequence in cell lines, The regulatory elements for hypoxic induction in the liver are

366

between 0.4 kb upstream and 0.7 kb downstream of the EPO gene coding

367

sequences, while an essential regulator element for renal EPO expression lies

368

between 12 and 8 kb in the 5´-region.(160,

369

factors GATA-2 and NF-κB are involved in the control of EPO expression. It was

370

shown in this context, that GATA-2 binds to a GATA element, that is located at -30 bp

371

of the 5´-promoter region, thereby inhibiting the promoter activity and hence the

372

expression of the EPO gene.(65) In another in vitro study, besides GATA-2 also NF-κB

373

has been shown, to inhibit the promoter activity of the EPO gene and hence EPO

374

production, thus providing a mechanism for downregulation or switch-off of EPO

375

expression during inflammatory diseases.(93) It was furthermore found in vivo that

376

inhibition of the EPO promoter via GATA-2 is essential for tissue-specific EPO gene

377

expression in the way that GATA-2 constitutively represses ectopic EPO expression

378

such as in distal tubules and collecting ducts of the kidney.(128) Regarding the switch

379

of EPO production from the liver to the kidney, the possibility has to be considered,

380

that the different regulation of EPO expression could result from trans-acting

381

elements, such as GATA factors, that are themselves expressed in a defined

382

temporal-spatial pattern.

140, 155)

140,

115, 159, 183)

156)

The hypoxia-inducible

The HIFs bind to promoter or

By generating transgenic mice carrying EPO gene

383 384

133,

Recruitment of EPO expressing cells

12

161)

Besides HIF-2, also the transcription

385

During normoxia, renal fibroblasts, hepatocytes and hepatic Ito cells express EPO,

386

but these cells are only few in number.(37,

387

hypoxia, with strongly increased expression of EPO mRNA. Kidneys respond to

388

insufficient oxygen supply by recruiting additional EPO producing fibroblasts (Figure

389

3). Notably, the number of EPO producing cells increases almost exponentially with

390

the severity of hypoxia in the kidneys

391

exclusively in the renal cortical labyrinth, with recruitment of EPO expressing cells -

392

depending on the degree of hypoxia- from the juxtamedullary cortical areas up to the

393

midcortical zone and finally to the superficial cortex.(37) It was also found in this

394

context that during hypoxia stimulation the amount of EPO mRNA expression per cell

395

was not markedly changed, suggesting that elevated EPO expression during hypoxia

396

mainly results from a recruitment of EPO producing interstitial cells.(37,

80)

397

phenomenon is very similar to that seen for recruitment of renin expression

(104)

398

not known if the recruitment of EPO producing cells requires de novo formation of

399

cells, or leads to a transformation of preexisting cells into EPO producers, or is

400

merely due to a switch-on of EPO gene transcription in preexisting silent cells. Since

401

massive recruitment of EPO expressing cells induced by acute hypoxia or

402

pharmaceutical prolyl-hydroxylase inhibitors occurs in very few hours

403

novo formation of EPO producing cells appears to be a less likely explanation. There

404

is in fact evidence for a reserve pool of EPO producing cells, that even may be

405

activated in the adult kidney for the first time

406

expression in the liver is to some extent different from that in the kidney. Common is

407

the fact that in hypoxia also in the liver a limited recruitment of EPO expressing

408

hepatocytes and Ito cells occurs. However, above a certain degree of hypoxia, higher

409

amounts of EPO are not achieved by a further recruitment of cells, but rather by an

410

elevated EPO production per cell.(79)

79, 80)

Both liver and kidneys respond to

(37, 80)

. EPO producing cells were found almost

This .It is

(37, 133)

, a de

(186)

. Hypoxia-related regulation of EPO

411 412

In vivo factors determining recruitment of EPO expression

413

The number of EPO expressing cells in the healthy kidneys is essentially determined

414

by the arterial oxygen tension, the oxygen carrying capacity of the blood and the

415

oxygen affinity of hemoglobin.(182) Finally, all these parameters determine the

416

essential control factor of EPO expression, namely the pericellular oxygen tension in

417

the interstitium of the renal cortex. Low oxygen tension in the arterial blood, low 13

418

hemoglobin concentrations and an increased oxygen affinity of hemoglobin lower

419

tissue oxygen tensions and thus cause recruitment of EPO producing cells. Notably,

420

renal perfusion (pressure) is not a physiological regulator of EPO cell recruitment

421

132)

422

oxygen consumption, because the energy consuming workload of kidney tubules is

423

determined by renal perfusion and glomerular filtration. As a consequence tissue

424

oxygen tensions in the renal cortex remain stable even if renal perfusion varies, at

425

least within the physiological range.

426

Prolyl-hydroxylase inhibitors (PHD-I) which are currently developed for therapeutic

427

use, also cause a strong recruitment of EPO producing cells (133).

428

De-recruitment of EPO producing cells is frequently seen during chronic kidney

429

disease, particularly in states of interstitial kidney fibrosis. Reasons for this are that

430

potential EPO producers have irreversibly transformed into cell types not capable to

431

express EPO.(3, 165) Another possibility is that potential EPO producers do not receive

432

the proper signal to turn on EPO expression (10).

(42,

. Changes of renal blood flow lead to parallel changes of oxygen delivery and

433 434

Intracellular signaling pathways involved in EPO cell recruitment

435

An essential process for the recruitment of renal EPO producing cells in response to

436

tissue hypoxia is stabilization of the transcription factor HIF-2

437

currently accepted concept of oxygen sensing for the control of gene expression

438

Whether HIF-2 alone is sufficient for recruitment of EPO producing cells is yet

439

unknown. HIF-2 certainly acts as an enhancer of EPO gene transcription also in the

440

kidney. Renal EPO gene regulation is still less understood than the well

441

characterized regulation of EPO gene transcription in hepatocytes

442

regulatory elements for hypoxic induction in the liver are between 0.4 kb upstream

443

and 0.7 kb downstream of the EPO gene coding sequences, while an essential

444

regulator element for renal EPO expression lies between 12 and 8 kb in the 5´-region

445

(160, 161)

446

addition to render EPO gene transcription susceptible in recruited renal cells, is also

447

unknown. This lack of knowledge is mainly due to the lack of suitable in vitro models,

448

to study the regulation of renal EPO gene expression at the cellular level. Although

449

successful isolation and culture of reno-cortical interstitial fibroblasts has been

450

described, these cells stop producing EPO under cell culture conditions (69, 134, 158).

(133)

according to the (27)

.

(140)

. The

. Whether HIF-2 related or other hypoxia induced events are required in

14

451

In most forms of chronic renal disease the patients suffer from anemia, which results

452

from a relative deficiency of renal EPO production.(18, 29, 39, 124) Chronic kidney disease

453

finally results in fibrosis, with the severity of renal anemia depending on the

454

progression of fibrosis.(124,

455

involve proliferation and increased size of fibroblasts plus altered expression profiles

456

of these cells.(112) Interstitial fibroblasts, especially those in the cortical labyrinth,

457

express substantially less CD73, but express instead desmin and αSMA, which are

458

markers of myofibroblasts.(3, 112) At the same time the number of EPO producing cells

459

declines due to renal injury. Recently published data suggest that most of the

460

myofibroblasts in the diseased kidneys result from a transformation of EPO producing

461

cells into non-EPO producing myofibroblasts.(165) It is known that inflammatory

462

cytokines such as TNF-α inhibit EPO expression

463

signaling pathway is responsible for myofibroblast transformation

464

involvement in transformation of EPO producing fibroblasts into non-EPO producing

465

myofibroblasts has been investigated. It was described that NF-κB signaling is

466

responsible for repression of EPO expression and that the Smad signaling pathway

467

promotes the transformation of EPO expressing cells.(165) Notably, there appears to

468

exist a reversible transition state between EPO producing cells and myofibroblasts

469

(165)

470

turn on the EPO gene in response to episodes of acute hypoxia, even if the daily

471

production of EPO is inappropriately low to maintain normal blood cell counts (11, 22).

143)

Several studies have shown, that interstitial changes

(44, 93, 123)

and that the TGFβ/Smad (74, 150)

. Their

. Those cells which still exist in chronically diseased kidneys are probably able to

472 473 474

Interconversion of renin and EPO producing cells

475 476

Recent evidence suggests that the relation between renin and EPO producing cells

477

might be even closer than previously thought and that at least renin producing cells

478

can convert into EPO producing cells under certain conditions. This surprising

479

observation was made in experiments in which purpose hypoxia-inducible

480

transcription factors (HIFs) were stabilized

481

from proteasomal degradation. Such a stabilization of HIFs in renin cells was

482

achieved by deletion of the von Hippel-Lindau protein (pVHL) from cells expressing

483

Cre under the control of the endogenous renin-1d promoter and their descendants

(114)

15

in the renin cell lineage by protection

484

(101)

485

renin cell lineage in the preglomerular vessel walls and JG cells

486

renin cells utilize HIF-2 rather than HIF-1 as main hypoxia inducible transcription

487

factor. A similar preferential use of HIF-2 has already been described for interstitial

488

fibroblasts, endothelial and mesangial cells of the kidneys

. It turned out that this maneuver increased HIF-2 but not HIF-1 protein in the

(133, 145)

(89)

, suggesting that

(133, 145, 146)

, whereas

489

tubular kidney cells preferentially utilize HIF-1

490

concomitant stabilization of HIF-2 in the renin cell lineage suppressed renin

491

expression in the JG area

492

agranular in the pVHL deleted state. This shift of the cell morphology was

493

accompanied by the disappearance of marker proteins for renin cells such as Akr1b7

494

and Cx40 and by the appearance of novel markers such as collagen I, CD73 and

495

PDGFR-ß

496

Strikingly, these cells now expressed EPO (Figure 4), leading to increased plasma

497

EPO concentrations and to polycythemia

498

factor shifted JG cells from a renin producer to a cell type resembling interstitial EPO

499

producing cells, suggesting a closer relationship between renin and EPO producing

500

cells of the kidney (86) (Figure 5). It has been reported that deletion of pVHL in several

501

cell types such as hepatocytes, glial cells and osteoblasts induces EPO expression

502

(141, 142, 181)

503

expression only in those tissues which physiologically produce EPO in response to

504

hypoxia or anemia

505

which is preferentially upregulated by pVHL deletion in these cells

506

HIF-2 is considered as main physiological controller of EPO gene expression also in

507

the kidney

508

of JG renin cells is not an artefact of genetically engineered mice, but may have

509

physiological implications. To clarify the physiological impact of this shift and to

510

define the (patho-) physiological conditions under which it occurs will be task for

511

future experiments. It will be also of interest to see if at all and whether and how EPO

512

producing cells in the kidney can be shifted to renin producing cells. Another issue

513

that needs clarification is to find out whether renin and EPO expression are mutually

514

exclusive or if an intermediate state of a renal endocrine cell exists capable to

515

produce both renin and EPO in substantial amounts.

(86)

. Notably, deletion of pVHL and

(89)

. The typical juxtaglomerular cells appeared flat and

suggesting that JG cells had transformed into a fibroblast-like cell. (89)

. Apparently, active HIF-2 transcription

. It has been inferred from those data that deletion of pVHL leads to EPO (37, 79, 107, 108, 111, 113)

. Notably, it is HIF-2 and not HIF-1 protein (140, 142, 181)

. In line,

(55, 133, 156)

. Summing up all these findings suggests that the endocrine shift

516 16

517 518

Acknowledgements

519

The authors’ work is financially supported by the German Research Foundation

520

(DFG; Ku 859/15-1 and SFB 699).

521 522 523

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524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563

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1017

Figures/Legends

1018 1019

Figure 1

1020

Schematic overview of the adult kidney cortex with different cell types expressing

1021

calcitriol, klotho, renin and EPO. Calcitriol is produced by proximal tubular cells (PT), and

1022

klotho by distal tubular cells (DT). Renin is produced by juxtaglomerular epithelioid cells

1023

(indicated by arrowheads), which are located in the media layer of afferent arterioles (aff.

1024

art.) at the entrance into the glomerulus (G). EPO is produced by interstitial fibroblast-like

1025

cells (indicated by arrows), which are located between proximal tubules. cTAL cortical thick

1026

ascending limb; CCD cortical collecting duct; cort. rad. art. cortical radial artery.

1027

Figure 2

1028

Schematic overview of the adult kidney cortex with recruited renin expressing cells. A)

1029

Arrowheads indicate recruited renin expressing cells along the afferent arterioles.

1030

Hypertrophy of the juxtaglomerular apparatus due to re-transformation of extraglomerular

1031

mesangial cells into renin expressing cells is indicated by arrows. G, glomerulus; cort. rad.

1032

art. cortical radial artery. B) Graph shows the inverse relationship between blood pressure

1033

and the number of renin expressing cells (modified from (104)).

1034

Figure 3

1035

Schematic overview of the adult kidney cortex with recruited EPO expressing cells. A)

1036

Arrowheads indicate recruited EPO expressing cells, which are located between proximal

1037

tubules (see brush border) in the cortical labyrinth. G, glomerulus; cort. rad. art. cortical radial

1038

artery. B) Graph shows the inverse relationship between inspiratory O2 concentration and the

1039

number of EPO expressing cells (modified from (37)).

1040

Figure 4

1041

Schematic overview of the endocrine transformation of juxtaglomerular (JG) cells due

1042

to pVHL deficiency. Renin producing cells (indicated by arrowheads) in a normal adult

1043

kidney are located in the media layer of afferent arterioles (aff. art.) at the entrance into the

1044

glomerulus (G). Deletion of pVHL in juxtaglomerular cells results in functional and

1045

morphological changes. pVHL deficient juxtaglomerular cells are thin and elongated, and

1046

they do not express renin. Instead, they express EPO now (indicated by arrows). Renin

1047

expressing cells in mutant kidneys are reduced in number, and the few remaining renin

1048

expressing cells are mostly found at the branching sites of afferent arterioles from cortical

1049

radial arteries (cort. rad. art). (Scheme derived from (87)). 27

1050

Figure 5

1051

Sketch summarizing the phenotype shift of juxtaglomerular cells induced by HIF-2.

1052

Renin expressing cells in a normal adult kidney have multiple renin storage vesicles (V),

1053

which lead to their typical cuboid-like shape. pVHL deletion in the renin cell lineage results in

1054

accumulation of HIF-2 protein in juxtaglomerular cells, that is responsible for the

1055

transformation from a renin producing into an EPO producing cell type with an elongated and

1056

agranular cell body. This endocrine shift of the cell phenotype is accompanied by the

1057

disappearance of marker proteins for renin cells such as Akr1b7 and Cx40 and by the

1058

appearance of novel markers such as collagen I, CD73 and PDGFR-ß, suggesting that JG

1059

cells had transformed into a fibroblast-like cell. If EPO producing cells in the kidney can be

1060

shifted to renin producing cells, will be task for future experiments.

1061 1062 1063 1064

28