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Expressão e regulação da
Na+,K+-ATPase em células
renais envelhecidas
Elisabete SilvaElisabete Silva
Porto 2011
Expressão e regulação da Na+,K+-ATPase em células
renais envelhecidas
Expression and regulation of Na+,K+-ATPase in aged
renal cells
Elisabete Silva
Orientador: Doutor Patrício Soares da Silva
Porto 2011
I
Dissertação de candidatura ao grau de Doutor em Biomedicina apresentada à Faculdade
de Medicina da Universidade do Porto.
Presidente do Júri:
Doutor José Agostinho Marques Lopes.
Vogais:
Doutor Carlos Alberto Fontes Ribeiro, Faculdade de Medicina, Universidade de
Coimbra;
Doutor Manuel Jesus Falcão Pestana Vasconcelos, Faculdade de Medicina,
Universidade do Porto;
Doutor Patrício Manuel Vieira Araújo Soares da Silva, Faculdade de Medicina,
Universidade do Porto;
Doutor António Albino Coelho Marques Abrantes Teixeira, Faculdade de Medicina,
Universidade do Porto;
Doutor Henrique Fernando Silva Luz Rodrigues, Faculdade de Medicina, Universidade
de Lisboa;
Doutor Fernando José Magro Dias, Faculdade de Medicina, Universidade do Porto.
Arguentes:
Doutor Carlos Alberto Fontes Ribeiro, Faculdade de Medicina, Universidade de
Coimbra;
Doutor Henrique Fernando Silva Luz Rodrigues, Faculdade de Medicina, Universidade
de Lisboa.
II
Art.º, 48º, §3º - “A Faculdade não responde pelas doutrinas expendidas na dissertação.”
(Regulamento da Faculdade de Medicina da Universidade do Porto – Decreto-Lei
nº19337 de 29 de Janeiro de 1931).
III
A candidata realizou o trabalho experimental com o apoio de uma bolsa de investigação
com a referência SFRH/BD/22876/2005, financiada pelo POPH-QREN-Tipologia 4.1-
Formação Avançada, comparticipado pelo Fundo Social Europeu e por fundos nacionais
do MCTES.
IV
Corpo Catedrático da Faculdade de Medicina da Universidade do Porto
Professores Catedráticos Efectivos
Doutor Manuel Alberto Coimbra Sobrinho Simões Doutor Jorge Manuel Mergulhão Castro Tavares Doutora Maria Amélia Duarte Ferreira Doutor José Agostinho Marques Lopes Doutor Patrício Manuel Vieira Araújo Soares da Silva Doutor Daniel Filipe de Lima Moura Doutor Alberto Manuel Barros da Silva Doutor José Manuel Lopes Teixeira Amarante Doutor José Henrique Dias Pinto de Barros Doutora Maria Fátima Machado Henriques Carneiro Doutora Isabel Maria Amorim Pereira Ramos Doutora Deolinda Maria Valente Alves Lima Teixeira Doutora Maria Dulce Cordeiro Madeira Doutor Altamiro Manuel Rodrigues Costa Pereira Doutor Rui Manuel Almeida Mota Cardoso Doutor António Carlos Freitas Ribeiro Saraiva Doutor Álvaro Jerónimo Leal Machado de Aguiar Doutor José Luís Medina Vieira Doutor José Carlos Neves da Cunha Areias Doutor Manuel Jesus Falcão Pestana Vasconcelos Doutor João Francisco Montenegro Andrade Lima Bernardes Doutora Maria Leonor Martins Soares David Doutor Rui Manuel Lopes Nunes Doutor José Eduardo Torres Eckenroth Guimarães Doutor Francisco Fernando Rocha Gonçalves Doutor José Manuel Pereira Dias de Castro Lopes Doutor Manuel António Caldeira Pais Clemente Doutor Abel Vitorino Trigo Cabral
V
Professores Catedráticos Jubilados ou Aposentados
Doutor Abel José Sampaio da Costa Tavares Doutor Alexandre Alberto Guerra Sousa Pinto Doutor Amândio Gomes Sampaio Tavares Doutor António Augusto Lopes Vaz Doutor António Carvalho Almeida Coimbra Doutor António Fernandes da Fonseca Doutor António Fernandes Oliveira Barbosa Ribeiro Braga Doutor António Germano Pina Silva Leal Doutor António José Pacheco Palha Doutor António Luís Tomé da Rocha Ribeiro Doutor António Manuel Sampaio de Araújo Teixeira Doutor Belmiro dos Santos Patrício Doutor Cândido Alves Hipólito Reis Doutor Carlos Rodrigo Magalhães Ramalhão Doutor Cassiano Pena de Abreu e Lima Doutor Daniel Santos Pinto Serrão Doutor Eduardo Jorge Cunha Rodrigues Pereira Doutor Fernando de Carvalho Cerqueira Magro Ferreira Doutor Fernando Tavarela Veloso Doutor Francisco de Sousa Lé Doutor Henrique José Ferreira Gonçalves Lecour de Menezes Doutor Joaquim Germano Pinto Machado Correia da Silva Doutor José Augusto Fleming Torrinha Doutor José Carvalho de Oliveira Doutor José Fernando Barros Castro Correia Doutor José Manuel Costa Mesquita Guimarães Doutor Levi Eugénio Ribeiro Guerra Doutor Luís Alberto Martins Gomes de Almeida Doutor Manuel Augusto Cardoso de Oliveira Doutor Manuel Machado Rodrigues Gomes Doutor Manuel Maria Paula Barbosa Doutor Manuel Teixeira Amarante Júnior Doutora Maria da Conceição Fernandes Marques Magalhães Doutora Maria Isabel Amorim Azevedo Doutor Valdemar Miguel Botelho dos Santos Cardoso Doutor Walter Friedrich Alfred Osswald Professores Catedráticos Eméritos
Doutor Mário José Cerqueira Gomes Braga Doutor Serafim Correia Pinto Guimarães
VI
“Looking for the answer.
You hunt it,
you catch it,
you fool yourself;
the answer,
is always,
a step ahead.”
Jens Christian Skou
VII
VIII
Ao Professor Doutor Patrício Soares da Silva
IX
X
Aos meus pais e irmão
XI
XII
Ao Eduardo e Henrique
XIII
XIX
Agradecimentos
Ao Professor Doutor Patrício Soares da Silva, por me ter integrado num excepcional
grupo de trabalho e pelos excelentes ensinamentos e orientação científica que
permitiram a realização dos trabalhos experimentais conducentes aos artigos publicados
ao longo dos últimos anos e à realização desta tese. Um obrigado especial por me ter
ajudado a crescer como cientista.
Ao Professor Doutor Serafim Guimarães, director do Instituto de Farmacologia e
Terapêutica à data da minha chegada, pela simpatia e por me ter acolhido nesta
instituição.
Ao Professor Doutor Daniel Moura, pelas interessantes conversas farmacológicas e
biológicas, pelo apoio incondicional e interesse demonstrado pelo meu trabalho.
Ao Doutor Pedro Gomes, pelos conhecimentos laboratoriais de electrofisiologia e pela
colaboração e espírito crítico permanente.
À Professora Doutora Maria Augusta Vieira Coelho, pela amizade e apoio.
À Engenheira Paula Serrão e à Engenheira Joana Afonso, pelo acompanhamento técnico
e científico e pela amizade e boa disposição permanente.
A todos os elementos do meu grupo, pelo bom ambiente e companheirismo. Agradeço
igualmente todo o apoio e colaboração no trabalho laboratorial.
A todos os professores, investigadores e funcionários do Instituto de Farmacologia e
Terapêutica e do Serviço de Bioquímica, pela simpatia e convívio saudável.
XV
Às investigadoras do Serviço de Nefrologia do Hospital de São João com quem
colaborei. Obrigada pela amizade e boa disposição permanente.
À Professora Doutora Leonor David, pelo apoio fundamental nos primeiros passos na
investigação científica.
Às minhas amigas do tempo de faculdade Ana Raquel Figueiredo e Sónia Moutinho,
pela amizade, cumplicidade e paciência.
Aos meus amigos porque sei que independentemente do tempo que estamos juntos estão
sempre presentes.
À minha família, pelo apoio incondicional. Em especial aos meus pais por estimularem
em mim a curiosidade e o gosto por aprender e pelos inúmeros sacrifícios pessoais que
fizeram para eu poder chegar até aqui.
Ao meu irmão André, por estar sempre presente com alegria contagiante.
Ao Eduardo, pela cumplicidade, amizade, companheirismo e paciência. Por me ajudar a
manter o ânimo e não me deixar esquecer que existe uma vida LÁ FORA.
Ao Henrique, por todos os dias me fazer sorrir.
XVI
Ao abrigo do Art.º 8º do Decreto-Lei nº388/70 fazem parte desta dissertação as
seguintes publicações:
Silva E, Gomes P, Soares-da-Silva P: Overexpression of Na+,K
+-ATPase parallels the
increase in sodium transport and potassium recycling in an in vitro model of proximal
tubule cellular ageing. Journal of Membrane Biology. 2006, 212: 163-175.
Silva E and Soares-da-Silva P: Reactive oxygen species and the regulation of renal
Na+,K
+-ATPase in opossum kidney cells. American Journal of Physiology –
Regulatory, Integrative and Comparative Physiology. 2007, 293:1764-1770.
Silva E and Soares-da-Silva P: Protein cytoskeleton and overexpression of Na+,K
+-
ATPase in opossum kidney cells. Journal of Cellular Physiology. 2009, 221:318-324.
Gomes P, Simão S, Silva E, Pinto V, Amaral JS, Afonso J, Serrão MP, Pinho MJ,
Soares-da-Silva P: Aging increases oxidative stress and renal expression of oxidant and
antioxidant enzymes that are associated with an increased trend in systolic blood
pressure. Oxidative Medicine and Cellular Longevity. 2009, 2: 155-162.
Silva E, Pinto V, Simão S, Serrão MP, Afonso J, Amaral J, Pinho MJ, Gomes P,
Soares-da-Silva P: Renal aging in WKY rats: Changes in Na+,K
+-ATPase function and
oxidative stress. Experimental Gerontology. 2010, 45: 977-983.
Silva E and Soares-da-Silva P: Long-term regulation of Na+,K
+-ATPase in opossum
kidney cells by ouabain. Journal of Cellular Physiology.2010, DOI: 10.1002/jcp.22575.
Silva E, Serrão MP, Soares-da-Silva P: Age-dependent effect of ouabain on renal
Na+,K
+-ATPase. Submitted for publication.
XVII
XVIII
Abbreviations
AP – adaptor protein
ATP – adenosine triphosphate
cAMP – cyclic adenosine monophosphate
ERK1/2 – extracellular signal-related kinase 1/2
GPCR – G-proteins coupled receptor
H2O2 – hydrogen peroxide
K+ – potassium ions
MAPK – mitogen-activated protein kinase
Na+ – sodium ions
NOX – nicotinamide adenine dinucleotide phosphate-oxidase
O2·- – superoxide anion
OK – opossum kidney
PI-3K – phosphoinositide 3-kinase
PKA –protein kinase A
PKC – protein kinase C
PLC – phospholipase C
PTP – protein tyrosine phosphatases
RNS – reactive nitrogen species
ROS – reactive oxygen species
SOD – superoxide dismutase
TEMPOL – 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy
WKY – Wistar Kyoto
XIX
XX
Index
Index ........................................................................................................................................... XXI
Introduction and Goals .................................................................................................................. 1
Introduction ............................................................................................................................... 3
Aim of the study ...................................................................................................................... 17
Part A .......................................................................................................................................... 19
Chapter I – Evaluation of Na+,K
+-ATPase function in serially passaged renal cells. ................. 21
Chapter II – Characterization of redox balance in aged renal cells and role of ROS in Na+,K
+-
ATPase regulation. ...................................................................................................................... 37
Chapter III – Na+,K
+-ATPase regulation by protein cytoskeleton: role of ankyrins. .................. 47
Chapter IV – Ouabain signalling in aged renal cells. .................................................................. 57
Part B ........................................................................................................................................... 67
Chapter I – Na+,K
+-ATPase function in renal cortex and medulla of aged WKY rats. .............. 69
Chapter II – Ageing and ouabain-mediated regulation of renal Na+,K
+-ATPase in WKY rats. . 87
Discussion and Conclusions ........................................................................................................ 97
Bibliography .............................................................................................................................. 113
Summary ................................................................................................................................... 125
Summary ............................................................................................................................... 127
Sumário ................................................................................................................................. 129
XXI
XXII
Introduction and Goals
1
2
Introduction
Na+,K
+-ATPase actions as ion transporter and functional
receptor
Na+,K
+-ATPase
Na+,K
+-ATPase was discovered by Skou in 1957 (Skou 1957). It is an integral
membrane protein that catalyses an adenosine triphosphate (ATP)-dependent transport
of three sodium ions (Na+) out and two potassium ions (K
+) into the cell per pumping
cycle, generating a Na+ gradient across the cell. The resulting gradient drives numerous
processes, such as the transport of glucose into intestinal and renal epithelial cells
through a Na+-glucose cotransporter, as well as the transport of other nutrients, such as
amino acids and ions (Aperia 2001; Feraille and Doucet 2001). Na+,K
+-ATPase also
generates the resting potential of cells and is therefore particularly important for
neuronal and muscle functions (Therien and Blostein 2000).
Na+,K
+-ATPase is a member of a family of integral membrane proteins called P-
type ATPases. The formation of a transiently phosphorylated aspartate residue during
the catalytic cycle is a hallmark of these family members. Na+,K
+-ATPase is composed
of two main non-covalent bound subunits, α and β (Figure 1) (Feraille and Doucet
2001; Xie and Cai 2003). The α-subunit (about 112 kDa) is the catalytic subunit
containing the binding site for Na+, K
+, ATP, steroid hormones and phosphorylation
sites for protein kinase A (PKA) and protein kinase C (PKC) (Schwartz et al. 1988;
Bertorello et al. 1991; Ewart and Klip 1995; Aperia 2001). The glycosylated β-subunit
is involved in enzyme maturation, localization to the plasma membrane and
stabilization of the K+-occluded intermediate (Geering 2008). A third subunit, the γ-
3
subunit, has been recently described to bind α and β complex in some tissues, such as
heart, kidney and brain (Figure 1) (Sweadner and Rael 2000). The γ-subunit belongs to
the FXYD proteins, a group of structurally similar polypeptides expressed in a tissue-
specific manner, and modulates cation binding affinity to Na+,K+-ATPase (Crambert
and Geering 2003; Geering et al. 2003; Geering 2006).
N
N
N
C C
CBinding site for Ouabain and K+
Large citoplasmaticloop / Binding site for Na+ and phosphate
α-SUBUNIT
β-SUBUNIT
γ-SUBUNIT
CYTOPLASM
1 2 5 6 7 8 9 103 4
Figure 1 – Schematic representation of Na+,K+-ATPase. Na+,K+-ATPase is
composed of a catalytic α-subunit (green) and a glycosilated β-subunit (red) and in some tissues
a γ-subunit (orange) that belongs to the FXYD protein family.
There are 4 known isoforms of the α-subunit: α1, α2, α3 and α4, all with a unique
tissue distribution. The α1-isoform is expressed ubiquitously (Blanco and Mercer 1998),
and it is the major isoform expressed in the kidney (Kaplan 2002); the α2-isoform is
predominantly expressed in the brain (Urayama et al. 1989), heart (Zahler et al. 1992),
vasculature (Zhang et al. 2005), skeletal muscle (Hundal et al. 1992) and adipocytes
(Lytton et al. 1985); the α3-isoform is mostly abundant in neuronal tissues (Urayama et
al. 1989) and in the heart of some species (Zahler et al. 1992), such as humans; and the
4
α4-isoform is essentially limited to the testis and is specifically expressed at the
spermatogonia where it regulates sperm motility (Shamraj and Lingrel 1994; Woo et al.
2000).
The β-subunit has 3 known isoforms: β1, β2 and β3. Detection of the tissue
distribution of the β-subunit isoforms has been more difficult due to the lack of specific
antibodies. However, antibody sensitivity has been improved by deglycosylation the β-
subunit. Current knowledge is that the β1-isoform is expressed in most tissues, including
the kidney (Vagin et al. 2007); the β2-isoform expression is largely localized to neuronal
tissues (Shyjan et al. 1990; Avila et al. 1998); and the β3-isoform is mainly expressed in
the rat lung and testis and is also present in the liver, skeletal muscle (Arystarkhova and
Sweadner 1997), neurons (LaCroix-Fralish et al. 2009) and peripheral blood cells
(Chiampanichayakul et al. 2006).
The tissue specific distribution of α and β subunits indicates that each
combination exhibits unique cellular functions. The α/β Na+,K
+-ATPase heterodimer is
under the control of a wide range of cell-specific regulatory mechanisms.
Mechanisms of Na+,K
+-ATPase regulation
The most direct regulation of Na+,K
+-ATPase is achieved through its substrates
Na+, K
+ and ATP. The major contributor to stimulate Na
+,K
+-ATPase activity is Na
+,
since ATP is saturable in most cell types (Haber et al. 1987). At the prevailing
intracellular concentrations of Na+ and K
+, Na
+,K
+-ATPase is working sub-maximally.
Thus, as intracellular Na+ rises, for example as a consequence of increased uptake via
Na+/H
+ exchanger, Na
+,K
+-ATPase is able to rapidly expel Na
+ and to lower
intracellular Na+ to a steady-state value. There is a growing body of evidence that low
5
K+ is also capable of stimulating membrane Na
+,K
+-ATPase due to reactive oxygen
species (ROS)-mediated stimulation of α-subunit transcription (Zhou et al. 2003).
Interaction of Na+,K
+-ATPase with several cytoskeleton proteins has been well
documented and is known to regulate Na+,K
+-ATPase function (Therien and Blostein
2000; Aperia 2001). Specific cytoskeletal proteins that interact with Na+,K
+-ATPase
include ankyrins, spectrins, adducins, actin and moesin (Nelson and Veshnock 1987;
Devarajan et al. 1994; Tripodi et al. 1996; Cantiello 1997; Zhang et al. 1998; Kraemer
et al. 2003). The main outcome of these interactions is believed to be the correct
assembly, delivery and stabilization of Na+,K
+-ATPase into the appropriate membrane
compartment. Furthermore, regulation of Na+,K
+-ATPase activity by cytoskeleton
proteins has also been reported. Some known polymorphisms of adducins and short
filaments of actin have been shown to stimulate Na+,K
+-ATPase activity by increasing
the affinity for ATP (Ferrandi et al. 1999) or by activating a pathway mediated by cyclic
adenosine monophosphate (cAMP)-dependent protein kinase (Cantiello 1995; Cantiello
1997), respectively.
Catecholamines and peptide hormones also play a role in Na+,K
+-ATPase
regulation. In the kidney Na+,K
+-ATPase is inhibited by many hormones that have a
natriuretic effect, including dopamine, atrial natriuretic peptide and parathyroid
hormone, and is stimulated by hormones that have an antinatriuretic effect, including
angiotensin II and noradrenaline (Therien and Blostein 2000; Aperia 2001; Feraille and
Doucet 2001).
The regulatory effect of dopamine on renal Na+,K
+-ATPase is exerted by non-
neuronal dopamine. Locally produced dopamine can act directly on proximal tubules in
an autocrinic fashion or on more distal tubular segments in a paracrinic fashion
(Hubbard and Henderson 1995; Hussain and Lokhandwala 1998; Brismar et al. 2000;
6
Jose et al. 2000). Most natriuretic effects are exerted via the activation of one class of
dopamine receptors, the D1-like receptors (D1 and D5). The D1-like receptors couple to
Gs-proteins and activate the adenylate cyclase-cAMP-PKA signaling pathway. In the
kidney and other tissues D1-like receptors can also couple to Gq/11 and activate the
phospholipase C (PLC)-diacylglycerol-PKC pathway. Both a cross-talk between the
PKA and PKC signaling pathways or an activation of the PLC-PKC pathway by PKA
have been described (Hussain and Lokhandwala 1998; Aperia 2000; Brismar et al.
2000; Jose et al. 2000; Gomes and Soares-da-Silva 2002b). Thus, activation of plasma
membrane D1-like receptors stimulates a tissue specific signalling cascade that leads to
the activation of the PKC δ-isoform. PKC δ-isoform phosphorylates the α1-subunit of
Na+,K
+-ATPase producing a conformational change of amino-terminal, which through
interaction with other domains of the α1-subunit of Na+,K
+-ATPase exposes the binding
domains for phosphoinositide 3-kinase (PI-3K) and adaptor protein (AP)-2. Binding of
these proteins induces the activation of Na+,K
+-ATPase endocytosis in the proximal
tubules (Efendiev et al. 2003; Pedemonte et al. 2005; Cinelli et al. 2008).
Locally produced angiotensin II exerts an antinatriuretic effect via the activation
of angiotensin II type 1 (AT1)-receptors. AT1-receptors are predominantly coupled to G-
proteins and signal through phospholipases, inositol-phosphatases, calcium channels
and serine/threonine and tyrosine kinases. Activation of plasma AT1-receptors
stimulates a tissue-specific signaling cascade that leads to the activation of the PKC β-
isoform. The PKC β-isoform phosphorylates the α1-subunit of Na+,K
+-ATPase
producing a conformational change that increases the interaction between the α1-subunit
of Na+,K
+-ATPase and AP-1, which results in the recruitment of the enzyme to the
plasma membrane (Efendiev et al. 2000; Efendiev et al. 2003).
7
Cardiotonic steroids have also been shown to be able to regulate Na+,K
+-
ATPase. This group of molecules binds specifically to the α-subunit of Na+,K
+-ATPase,
inhibit enzyme activity and also initiates cell specific signalling pathways (Xie and
Askari 2002; Xie and Cai 2003). Several exogenous cardiotonic steroids have been
identified, including plant-derived digitalis drugs such as digoxin and ouabain, and
vertebrate-derived aglycones such as bufalin and marinobufagenin (Figure 2).
Exogenous cardiotonic steroids have long been used in the treatment of congestive heart
failure. However, only more recently have endogenous cardiac glycosides been
identified in mammals (Hamlyn et al. 1991; Bagrov et al. 1995; Schoner 2002; Yoshika
et al. 2007). Structurally they consist of a cholesterol core conjugated to either a lactone
or pyrone ring and contain various combinations of hydroxyl, sulfate or carbohydrate
groups (Doris and Bagrov 1998) (Figure 2). Endogenous cardiotonic steroids are
synthesized in the adrenal glands and brain (Schoner and Scheiner-Bobis 2007b) and
their secretion is regulated by multiple stimuli including angiotensin II and
noradrenaline (Xie and Cai 2003). Currently, they are believed to have an important
physiological role not only in the control of blood pressure but also in the control of
cellular functions due to the activation of cellular signalling pathways (Hamlyn et al.
1991; Tian et al. 2009; Jaitovich and Bertorello 2010).
R
OH
OH
OH
OH
O
CH3
CH2
OH
O
O
OuabainR = Rhamnose
OH CH3
O
O
DigoxinR = 3 Digitoxoses
H
R
OH
O
CH3
H
H
OH CH3
O
Marinobufagenin
OH
CH3
O
O
Figure 2 – Chemical structures of cardiotonic steroids. Ouabain, digoxin and
marinobufagenin.
8
Na+,K
+-ATPase/Src complex as a functional receptor
In recent years, several studies have indicated that apart from its transport
function, Na+,K
+-ATPase can also act as a signal transducer (Xie and Cai 2003; Nesher
et al. 2007; Schoner and Scheiner-Bobis 2007a; Schoner and Scheiner-Bobis 2007b). A
pool of Na+,K
+ATPase localized in specialized microdomains of the plasma membrane
interacts with Src protein keeping it in an inactive state. Binding of endogenous
cardiotonic steroids to the complex activates the Na+,K
+-ATPase associated Src.
Subsequently, the activated Src transactivates other tyrosine kinases and together recruit
and further phosphorylate multiple proteins, which can result in the activation of
mitogen-activated protein kinase (MAPK) and PI-3K pathways and in the generation of
second messengers such as mitochondrial ROS and PLC (Xie and Cai 2003; Wang et al.
2004a; Li and Xie 2009). Activation of these pathways is achieved by circulating
endogenous cardiotonic steroids in the nanomolar concentration range and seems to be
independent of its effects on Na+,K
+-ATPase-mediated ion transport (Li and Xie 2009).
Signalling pathways activated by endogenous cardiotonic steroids alter cellular
functions and cell growth in a cell-specific manner (Haas et al. 2000; Haas et al. 2002;
Xie and Cai 2003; Tian et al. 2009). Altered plasma levels of endogenous cardiotonic
steroids have been associated with the development of several conditions such as Na+
imbalance, chronic renal failure, hypertension and congestive heart failure (Bagrov et al.
2009; Jaitovich and Bertorello 2010).
The kidney and Na+,K
+-ATPase regulation
In an adult organism the kidney plays an important role in the regulation of
blood pressure, nutrient and electrolyte reabsorption and drug and metabolite excretion.
This is achieved due to the presence of specialized proteins that are distributed into
specific domains of the apical or basolateral membrane of the distinct nephron segments
9
(Abdolzade-Bavil et al. 2004). One of the most important renal transporter, located at
the basolateral membrane of all nephron segments, is Na+,K
+-ATPase (Jaitovich and
Bertorello 2010). In proximal tubules, the activity of Na+,K
+-ATPase plays an essential
role in the bulk reabsorption of Na+ and K
+ and in the maintenance of the gradients of
Na+ and K
+ across the plasma membrane (Feraille and Doucet 2001).
Despite the bulk of Na+ and K
+ being reabsorbed in the proximal tubules, the
final adjustment is made in the distal tubules and the collecting ducts where Na+,K
+-
ATPase also plays a crucial role. Undoubtedly, understanding the mechanisms involved
in the regulation of Na+,K
+-ATPase throughout the nephron is of major importance.
The majority of studies have addressed Na+,K
+-ATPase regulation due to
phosphorylation of the α1-subunit mediated by PKC and PKA (Pedemonte et al. 1997;
Gomes and Soares-da-Silva 2002a; Pierre et al. 2002) through the activation of a
cascade of intracellular mechanisms by hormones such as dopamine, noradrenaline, and
insulin, growth factors and more directly by ionic distribution across the membrane
(Efendiev et al. 2000; Therien and Blostein 2000; Feraille and Doucet 2001). Changes
in Na+,K
+-ATPase and in oxidative stress have been associated with the development of
conditions such as hypertension, obesity-associated hypertension and diabetes as well as
during the ageing process. However, little is known about a possible role of ROS-
mediated regulation of Na+,K
+-ATPase.
Oxidative stress and oxidant signalling
ROS: dual role in the organism
Free radicals can be defined as molecules or molecular fragments containing one
or more unpaired electrons in atomic or molecular orbitals. The unpaired electron(s)
generally give(s) reactivity to the free radical. Until the publication of Gershman's free
10
radical theory of oxygen toxicity in 1954, the toxicity of ROS was practically unknown
(Gerschman et al. 1954). Two years latter, Denham Harman explored Gershman´s
theory and proposed the concept of free radicals playing a role in the ageing process
(Harman 1956). According to the free radical theory of ageing, overproduction of ROS
results in oxidative stress, a deleterious process that can be an important mediator of
damage to cell structures, including lipids, membranes, proteins and DNA, leading to
cellular dysfunction and eventually cell death (Harman 1956).
Presently, ROS as well as reactive nitrogen species (RNS) are known normal
products of cell metabolism used in various physiological functions and recognised for
playing a dual role as both harmful and beneficial to the organism (Valko et al. 2006;
Valko et al. 2007). Produced in low/moderate concentrations ROS and RNS play a role
in cellular responses to noxia, in the defence against infectious agents, in cellular
signalling pathways and in the induction of a mitogenic response (Finkel and Holbrook
2000; Gill and Wilcox 2006; McCubrey et al. 2006; Valko et al. 2007).
ROS encompass a series of oxygen intermediates that include the superoxide
anion (O2·-), hydrogen peroxide (H2O2), the hydroxyl radical and hypochlorous acid. In
the organism they can be produced by xanthine oxidase, nicotinamide adenine
dinucleotide phosphate-oxidase (NOX), mitochondrial oxidative phosphorylation,
lypoxygenase, cytochrome P450 mono-oxygenase and heme-oxygenase 1. Despite the
existence of several sources of ROS, NOX appears to be especially important for the
redox signal (Lassegue et al. 2001; Gill and Wilcox 2006). NOX activity is influenced
by diverse stimuli such as G-protein coupled receptor agonists, cytokines, growth
factors and ischemia-reperfusion (Figure 3) (Cave et al. 2006; Dworakowski et al.
2006; Gill and Wilcox 2006). NOX-dependent ROS production follows a rapid kinetic
of activation and inactivation which allows a tight regulation of intracellular ROS levels
11
within the short time required for signal transduction (Lassegue et al. 2001; Wilcox
2005; Beltowski et al. 2006; Marciniak et al. 2006).
Oxidant signalling
Transduction of the chemical ROS signal into biological relevant events can
occur through a stable sulfenic acid modification of cysteine residues in selected
proteins, resulting in protein function alterations (Finkel 2003; Cave et al. 2006;
McCubrey et al. 2006). Once oxidized, proteins can undergo spontaneous or enzymatic
reduction back to the initial conformation. This mechanism represents a form of signal
transduction similar to phosphorylation. A large number of proteins have been
identified as specific targets of reversible oxidation, including structural proteins,
transcription factors, membrane receptors, ion channels, protein kinases and protein
phosphatases (Figure 3) (Cave et al. 2006; Bedard and Krause 2007). Protein tyrosine
phosphatases (PTP) are probably the most well studied, since they control the
phosphorylation status of numerous signal transducing proteins (Meng et al. 2002;
Finkel 2003). ROS-induced oxidation of PTP decreases phosphatase activity by altering
the tyrosine/phosphatase balance and thereby influencing signal transduction. This
mechanism constitutes an indirect way of ROS-mediated activation of the MAPK
signalling pathway. However, a direct mechanism of activation of these pathways is
also possible through ROS-induced activation of membrane receptors, such as
endothelial growth factor receptor and platelet-derived growth factor receptor
(McCubrey et al. 2006).
The loss of redox homeostasis and increased cellular levels of ROS have been
related to development of ageing associated conditions such as brain dysfunction,
cancer, diabetes and cardiovascular and renal diseases (Makino et al. 2003; Touyz and
Schiffrin 2004; Wilcox 2005; Bedard and Krause 2007; Valko et al. 2007).
12
FAD/NADPH binding domains
N
C
O2-
Growth factors
CytokinesGPCR agonists
Ischaemia-reperfusion
CYTOPLASM
ROSTranscription factors
Membrane receptorsIon channels
Protein kinases/phosphatases
Figure 3 – Schematic representation of known activators of NOX isoforms and
downstream effects of NOX-derived ROS. Diverse stimuli activate NOX isoforms including
G-protein coupled receptor (GPCR) agonists, cytokines, growth factors and ischemia-
reperfusion. NOX-derived ROS may influence several signalling pathways through changes in
the activity of structural proteins, transcription factors, membrane receptors, ion channels and
protein kinases/phosphatases.
Studies on ageing
Ageing is a natural, complex and multifactorial biological process characterized
by a progressive deterioration in physiological functions and metabolic processes
associated with increased risk of contracting age-associated diseases and death (Paradies
et al. 2010) during the adult period of life. Changes in phenotype due to ageing occur in
all individuals in a population, while age-associated diseases affect only a subset. Thus,
13
it can be argued whether the process of ageing is itself a disease. Nevertheless both have
an impact on life span.
According to the Portuguese National Institute of Statistics, between 2006 and
2008, the Portuguese average life expectancy was 78.7 years. Consequently, improving
our understanding of the biological changes that occur during ageing and ageing-
associated conditions became a major health issue in modern society. However, several
obstacles arise when ageing is studied. In addition to ethical problems, one of the major
obstacles is the long study period required to reach the end-point in human populations.
The development of experimental models with short-living organisms has been
proven useful in understanding several mechanisms of ageing and disease. This is
possible because ageing in organisms such as yeast, the nematode Caenorhabditis
elegans, the fruit fly Drosophila melanogaster, the mouse Mus musculus and the rat
Rattus norvegicus is regulated by specific conserved genes that have human homologs
(Guarente and Kenyon 2000; Browner et al. 2004). Normal somatic cells that are
serially-cultured have also been used as a model to study development and ageing
(Porter et al. 1997; Huang et al. 2001; Wang et al. 2004b; Vacanti et al. 2005; Phipps et
al. 2007). However, these studies are restricted to specific age-related conditions
whereas ageing is a complex process difficult to address and mimic in in vitro cultures.
Nevertheless, these models have already contributed towards a better understanding of
the mechanisms responsible for the reduced ability of older osteoblasts to form bone
(Huang et al. 2001), for the age-dependent increase in the density of calcium channels
and neuronal death (Porter et al. 1997) and for the age-related macular degeneration
(Wang et al. 2004b).
Current knowledge is that alterations in insulin metabolism, redox balance and
in the levels of oxidative damage seem to play a role in the regulation of the life span of
14
organisms (Browner et al. 2004). At the cellular level it appears that ageing results from
alterations that encompass nutrient sensing and mitogen-activated, stress-responsive and
DNA damage-dependent signaling pathways (Finkel and Holbrook 2000; Browner et al.
2004; Blagosklonny et al. 2009). Further research in animal and cellular models is
required to uncover the cellular and molecular changes which give rise to ageing and
ageing-associated conditions.
15
16
Aim of the study
The first goal of this study was to develop and characterize an in vitro model of
cellular ageing that could be useful in the study of Na+,K
+-ATPase regulatory
mechanisms. The second goal was to evaluate Na+,K
+-ATPase regulation by ROS, the
cytoskeleton and ouabain in cells aged in vitro and identify possible age-induced
alterations in Na+,K
+-ATPase regulatory pathways. The third goal was to evaluate
Na+,K
+-ATPase function in the kidney of Wistar Kyoto (WKY) rats during ageing.
For this purpose the following questions were raised:
Part A
Chapter I and III: Can serially-passaged opossum kidney (OK) cells be used as
a model of cellular ageing? Is Na+,K
+-ATPase activity affected by cellular ageing?
Chapter I and II: Is the cellular redox balance altered in aged cells? Do
changes in cellular redox balance influence Na+,K
+-ATPase activity?
Chapter III: Is the membrane-based cytoskeleton affected by cellular ageing?
Does it play a role in the regulation of Na+,K
+-ATPase?
Chapter I and IV: Are ouabain-mediated signalling pathways activated in low-
and high-passaged cells? How can cellular ageing influence ouabain long-term
regulation of Na+,K
+-ATPase?
Part B
Chapter I: Is Na+,K
+-ATPase function altered in the kidney of aged WKY rats?
Is there a correlation between redox balance and Na+,K
+-ATPase function in vivo?
Chapter II: How does ageing affects in vivo ouabain-mediated regulation of
renal Na+,K
+-ATPase?
17
18
Part A
19
20
Chapter I – Evaluation of Na+,K+-ATPase function in
serially passaged renal cells.
“Overexpression of Na+,K+-ATPase parallels the increase in sodium transport
and potassium recycling in an in vitro model of proximal tubule cellular ageing.“
Journal of Membrane Biology. 2006, 212: 163-175.
21
22
Chapter II – Characterization of redox balance in aged
renal cells and role of ROS in Na+,K+-ATPase
regulation.
“Reactive oxygen species and the regulation of renal Na+,K+-ATPase in
opussum kidney cells.” American Journal of Physiology – Regulatory,
Integrative and Comparative Physiology. 2007, 293: 1764-1770.
37
38
Chapter III – Na+,K+-ATPase regulation by protein
cytoskeleton: role of ankyrins.
“Protein cytoskeleton and overexpression of Na+,K+-ATPase in opossum kidney
cells.“ Journal of Cellular Physiology. 2009, 221: 318-324.
47
48
Chapter IV – Ouabain signalling in aged renal cells.
“Long-term regulation of Na+,K
+-ATPase in opossum kidney cells by ouabain.
Journal of Cellular Physiology. 2010, DOI: 10.1002/jcp.22575.
57
58
Part B
67
68
Chapter I – Na+,K
+-ATPase function in renal cortex
and medulla of aged WKY rats.
“Renal aging in WKY rats: Changes in Na+,K
+-ATPase function and oxidative
stress.” Experimental Gerontology. 2010, 45: 977-983.
“Aging increases oxidative stress and renal expression of oxidant and
antioxidant enzymes that are associated with an increased trend in systolic blood
pressure.” Oxidative Medicine and Cellular Longevity. 2009, 2: 155-162.
69
70
Chapter II – Ageing and ouabain-mediated regulation
of renal Na+,K+-ATPase in WKY rats.
“Age-dependent effect of ouabain on renal Na+,K+-ATPase.” Submitted for
publication.
87
88
Discussion and Conclusions
97
98
Discussion and Conclusions
It is well known that ageing is associated with the development of several
conditions such as neurodegenerative diseases, hypertension, atherosclerosis, diabetes,
obesity and cancer (Harman 2001). Moreover, it is generally accepted that a redox
imbalance is implicated in their pathogenesis. Being the kidney an organ that is severely
affected in many of the age-associated conditions, it is possible that changes in ROS
levels could play a role in the regulation of renal transporters. It was known that an
increase in O2·- production in the renal medulla decreased urinary Na+ and water
excretion due to O2·--induced stimulation of the Na+/K+/2Cl- co-transporter (Graier et al.
1998; Kourie 1998; Zou et al. 2001; Majid and Nishiyama 2002; Juncos and Garvin
2005). Furthermore, it was also known that low extracellular K+ enhances Na+,K+-
ATPase protein content and membrane expression by a ROS-mediated stimulation of
the α1- and β1-subunit promoters (Zhou et al. 2003). However, there were not much
additional data on this subject. Therefore, the first part of this study focused on the
establishment of an in vitro model of renal cellular ageing and on the study of Na+,K+-
ATPase regulation in this model. In the second part of this study renal Na+,K+-ATPase
function was evaluated in an in vivo model of ageing.
Normal somatic cells that are serially-cultured have been used as a model for
studying development and ageing due to the existence of some correlations between
ageing cells in culture and cellular ageing. Some examples are: porcine mesenchymal
stem cells in culture that have been used to study multiple differentiation cascades in the
context of cellular ageing (Vacanti et al. 2005); normal human fibroblasts that have
been extensively used to identify genes linked with ageing (Phipps et al. 2007); and
human keratinocytes that have been used to study processes of differentiation and
99
apoptosis (Norsgaard et al. 1996). Serially-passaged cells present progressive and
cumulative changes that ultimately lead to an irreversible cessation of cell proliferation
followed by cell death. These changes have been considered as indicative of cellular
ageing in vitro (Rattan 1991; Hayflick 1992). Macieira-Coelho (Macieira-Coelho 1988)
reviewed the overlapping events of in vitro and in vivo cellular ageing and identified
among others the loss of division potential, the alteration of chromatin, the loss of the
capacity to migrate, the increase of cell size, volume, and protein content and the
decrease of the mitogenic response to growth factors. Therefore, it was fundamental to
address the question of whether prolonged cell passaging of a renal epithelial cell line
could provide a model system to study Na+,K
+-ATPase regulation in the context of
cellular ageing.
Most studies using prolonged cell passaging as an in vitro model of cellular
ageing used normal non-transformed cells that were able to be maintained in culture for
several months and up to 100 population dublings (Norsgaard et al. 1996; Porter et al.
1997; Huang et al. 2001; Wang et al. 2004b; Vacanti et al. 2005; Phipps et al. 2007).
Thus, for the purpose of this study, a renal epithelial cell line established from the
kidney of an adult female American opossum (Didelphis virginiana) was used. OK cells
are epithelial-like with a stable non-diploid chromosomal modal number (Koyama et al.
1978) and have been widely used as a model of renal cells. All the experiments
performed in this study resulted from multiple serial passaging of an original vial
obtained from the American Type Culture Collection with 36 passages in culture. In
Part A – Chapter I it was observed that serially-passaged OK cells (from 60 to 80
passages in culture) had identical abilities to proliferate and increased levels of H2O2.
The fact that serially-passaged cells did not lose division potential meant that the
alterations observed were not due to the selection of fast-growing populations and also
100
that cells were not senescent. Further passaging would be necessary to evaluate whether
OK gradually cease dividing and become senescent since these cells had not been
transfected with telomerase. The increase in ROS levels is characteristic of ageing and
reinforces the use of cell-passaging as a model of cellular ageing. Furthermore, as
shown in Part A – Chapter III, serially-passaged OK cells exhibited more features
reminiscent of cellular ageing such as increased cell size and granularity, increased
protein content and alterations in cytoskeleton proteins. Therefore, OK cells seem to
constitute a valid model to study Na+,K
+-ATPase regulation in a context of renal
cellular ageing.
The study of Na+,K
+-ATPase regulation was initiated by evaluating Na
+,K
+-
ATPase activity and expression in serially-passaged OK cells. As shown in Part A –
Chapter I, in vitro cellular ageing of OK cells was accompanied by an increase in
basolateral Na+ transport. This could be due to an increase in Na
+-K
+-ATPase affinity
for its substrate or an increase in the number of operational Na+-K
+-ATPase units in the
membrane. The fact that the increase in Na+-K
+-ATPase activity was accompanied by
an increase in the expression of the α1- and β1-subunits and in the Vmax of the enzyme
without changes in the affinity for intracellular Na+, indicates that the observed
differences resulted from an increase in the number of enzyme units inserted in the
basolateral membrane. Age-dependent up-regulation of Na+-K
+-ATPase observed in
serially-passaged OK cells could be due to many factors as previously reviewed in the
introduction. In this work, focus was given to ROS, the cytoskeleton and ouabain,
mainly due to the following: data was emerging concerning ROS as important
mediators in signal transduction and development of age-associated conditions (Wilcox
2005; Iwai et al. 2006; Bedard and Krause 2007; Valko et al. 2007); the cytoskeleton
101
was known to play a role in the regulation of Na+,K
+-ATPase (Devarajan et al. 1994;
Cantiello 1995; Doctor et al. 1998); and high-circulating levels of ouabain were being
related with the development of hypertension (Therien and Blostein 2000; Aperia
2001).
The regulation of Na+,K
+-ATPase by ROS was first addressed in Part A –
Chapter I in which the effect of daily exposure of OK cells to H2O2 (1 and 10 M) was
studied. Such transient increase in ROS failed to induce long-term alterations in Na+-
K+-ATPase protein expression. In fact, the antioxidant system of OK cells rapidly
metabolized external H2O2 whose levels were reduced to basal values within a few
hours (unpublished data). Thus, the use of a high concentration of exogenous H2O2 was
a limited way of studying long-term effects of ROS signalling. In fact, passaged OK
cells have a moderate but continuous increase in H2O2 generation. Moreover, this
approach does not allow the study of O2·--mediated effects on Na
+-K
+-ATPase function.
To evaluate a possible role of ROS in long-term Na+,K
+-ATPase regulation, induced
sustained alteration in intracellular levels of ROS was required. Thus, it was crucial to
gain further information on the mechanisms involved in the observed increase in H2O2
production in OK cells before further addressing the role of ROS in the regulation of
Na+,K
+-ATPase. Hence, in Part A – Chapter II the expression of enzymes involved in
the regulation of redox balance in the cell was evaluated.
One of the possible sources of moderate increase generation of ROS could be
NOX which appears to be especially important for redox signalling (Lassegue et al.
2001; Gill and Wilcox 2006). Given so, the protein expression of NOX-1 and NOX-2
was determined in serially-passaged OK cells. Moreover, as O2·- in cells is dismutated
into H2O2 spontaneously or by superoxide dismutase (SOD), the protein expression of
102
SOD isoforms - SOD-1, SOD-2 and SOD-3, was also evaluated. Serially-passaged OK
cells had increased NOX-1, SOD-1, SOD-2 and SOD-3 protein expression and no
changes in NOX-2 protein expression. NOX and SOD isoforms have been shown to be
altered in animals affected by several conditions such as hypertension, diabetes and
obesity. Treatment with antioxidants such as apocynin, 4-hydroxy-2,2,6,6-
tetramethylpiperidinyloxy (TEMPOL) and both TEMPOL and catalase have been
shown to be helpful in improving ROS-related conditions (Schnackenberg et al. 1998;
Schnackenberg and Wilcox 1999; Zhan et al. 2004; Manning et al. 2005; Asghar and
Lokhandwala 2006; Hu et al. 2006). In light of these findings it was possible to induce a
sustained decrease in intracellular ROS by pharmacological inhibition of NOX with
apocynin or by reducing O2·- with the SOD mimetic TEMPOL. OK cells exposed daily
to apocynin (300 M) had a sustained decrease in ROS, as confirmed by decreased
H2O2 accumulation in extracellular medium and decreased rate of H2O2 production 24
hours after apocynin addition. Under these conditions, Na+,K
+-ATPase activity was
found to be significantly decreased. Moreover, treatment with apocynin also induced a
decrease in the protein expression of the α1-subunit of Na+,K
+-ATPase. Treatment of
OK cells with TEMPOL (300 M) induced a significant decrease in Na+,K
+-ATPase,
activity and did not have an effect on protein expression of the α1-subunit (unpublished
data). Thus, it is believed that in OK cells although both O2·- and H2O2 seem to play an
important role as mediators in the regulation of Na+,K
+-ATPase, the increase in Na
+,K
+-
ATPase activity seems to be mainly regulated by O2·-. In fact, a similar mechanism of
regulation of Na+,K
+-ATPase in vivo was reported by Bełtowski (Beltowski et al. 2006).
They observed that prolonged leptin infusion in rats stimulated Na+,K
+-ATPase activity
and that this response was dependent on ROS generation. Knowing that renal Na+,K
+-
ATPase plays a crucial role in the control of Na+ homeostasis these findings can
103
contribute to a better understanding of the mechanisms through which an up-regulation
of specific NOX and SOD enzyme isoforms may lead to alterations in renal function
that may culminate in the development of renal associated conditions.
Ageing is an extremely complex process known to be driven by a variety of
mutually interacting mechanisms. Thus, despite ROS being able to regulate Na+,K
+-
ATPase function other factors could also be contributing to the observed up-regulation
of Na+,K
+-ATPase in serially-passaged OK cells. The question of whether in vitro aged
OK cells had changes in cytoskeleton proteins and whether these changes could also
have influenced Na+,K
+-ATPase up-regulation was raised and evaluated in Part A –
Chapter III.
Although, serially-passaged OK cells had increased Na+,K
+-ATPase protein
expression and mRNA levels of the α1-subunit of Na+,K
+-ATPase (3 fold and 2 fold,
respectively), this increase was much less pronounced than that observed in basolateral
Na+,K
+-ATPase activity that was increased 7 fold. As mentioned in Part A – Chapter I
the observed increase in basolateral Na+ currents could be due to an increase in the
number of Na+,K
+-ATPase units in the basolateral membrane. The observed 7 fold
increase in enzyme activity may be explained by a simultaneous decrease in
internalization of membrane Na+,K
+-ATPase, an increased half-life of membrane
Na+,K
+-ATPase and/or a depletion of the cytoplasmatic pool. The cytoskeleton is the
cell compartment which participates in cell structure, motility and membrane traffiking
and signalling. Ankyrin, spectrin, and moesin are cytoskeleton proteins known to be
involved in Na+,K
+-ATPase regulation. Moesin and ankyrin bind to the cytoplasmatic
domain of the α1-subunit of Na+,K
+-ATPase and regulate the insertion into the
basolateral membrane, enzyme activity and/or membrane microdomain localization
104
(Hopitzan et al. 2005; Stabach et al. 2008). The main ankyrin expressed in the kidney is
ankyrin-G (Peters et al. 1995). Several ankyrin-G transcripts and proteins have been
identified within different renal cells, though their specialized roles in cell organization,
regulation and function have not been fully elucidated (Peters et al. 1995; Doctor et al.
1998). Results show that serial passaging of OK cells was accompanied by an increase
in the protein expression of moesin, changes in the expression of ankyrin-G isoforms
and down-regulation of spectrin-βII protein expression. Moesin over-expression could,
to a certain extent, be responsible for the observed increase in Na+,K
+-ATPase activity
in serially-passaged OK cells. Studies performed in human bronchial epithelial cell lines
demonstrated that both ankyrin-G and spectrin-βII were necessary for the correct
insertion of Na+,K
+-ATPase in the basolateral membrane (Kizhatil and Bennett 2004;
Kizhatil et al. 2007). The fact that serial passaging of OK cells was accompanied by a
decreased expression of spectrin-βII and increased Na+,K
+-ATPase activity suggests
that an alternative isoform of spectrin may be responsible for Na+,K
+-ATPase anchoring
in the basolateral membrane of OK cells. Regarding ankyrin-G, it was observed that in
OK cells with increasing number of cell passages the expressed ankyrin-G isoform
shifted from a ~220/200 kDa ankyrin-G isoform to a ~190 kDa ankyrin-G isoform.
According to Piepenhagen (Piepenhagen and Nelson 1998) the ~190 kDa ankyrin-G
isoform was found to be responsible for increasing the half-life of the enzyme in the
basolateral membrane (Yeaman et al. 1999). This finding supports the hypothesis that in
OK cells the observed 7 fold increase in Na+,K
+-ATPase activity could not be fully
explained by the observed 2 fold increase in total protein expression but rather by a
parallel increase in the number of active Na+,K
+-ATPase units present in the basolateral
membrane.
105
The question remaining to be answered in Part A was whether ouabain could
also play a role in long-term regulation of Na+,K
+-ATPase in the in vitro model of
cellular ageing. This question was raised in Part A – Chapter I and developed in Part
A – Chapter IV. As shown in Part A – Chapter I, serially-passaged OK cells respond
differently to ouabain. An increase in the protein expression of the α1-subunit of
Na+,K
+-ATPase following prolonged treatment with ouabain (100 nM) occured only in
serially-passaged OK cells. It remained to be elucidated whether the observed increase
in the protein expression of the α1-subunit of Na+,K
+-ATPase was accompanied by an
increase in basolateral Na+ transport and if an ouabain-dependent signal cascade was
being activated only in serially-passaged OK cells. As demonstrated in Part A –
Chapter IV ouabain-induced increase in protein expression of the α1-subunit of
Na+,K
+ATPase was accompanied by an increase in basolateral transepithelial flux of
Na+. Moreover, such an increase was dependent on activation of both PI-3K and MAPK
pathways. However, ouabain was able to activate a signal cascade in both low- and
serially-passaged OK cells as demonstrated by the observed increase in ouabain-
mediated extracellular signal-related kinase 1/2 (ERK-1/2) phosphorylation. Since
activation of ERK-1/2 is considered to be a proximal event in the ouabain-mediated
activation of a signal cascade, downstream events in this cascade must significantly
diverge between low- and serially-passaged OK cells. In fact, recently published data
shows that ouabain failed to activate the PI-3K/Akt pathway in cell lines with low
Na+,K
+ATPase (Tian et al. 2009). It is hypothesized that in low-passaged OK cells
ouabain-treatment did not stimulate this pathway and thus no up-regulation of
Na+,K
+ATPase was observed. One cannot exclude the possibility that membrane based
cytoskeleton alterations in serially-passaged OK cells might also have contributed to the
difference in the ouabain-mediated cell response. Divergent results have been observed
106
concerning both short- and long-term regulation of Na+,K
+-ATPase by ouabain (Liu et
al. 2002; Khundmiri et al. 2006; Khundmiri et al. 2007; Tian et al. 2009). Clearly,
regulation of Na+,K
+-ATPase is dependent not only on the activation of ouabain-
mediated signal cascade but also on additional cell specific factors some of which may
be age-dependent.
One major question in the study of the regulation of Na+,K
+-ATPase using an in
vitro model of cellular ageing is whether the changes observed correlate with what
occurs in an organism. Thus, in Part B – Chapter I, we addressed the question of
whether Na+,K
+-ATPase function was altered in the kidney of aged WKY rats and if a
correlation between redox balance and Na+,K
+-ATPase function exists in vivo. In Part
B – Chapter II, in vivo ouabain-mediated regulation of renal Na+,K
+-ATPase during
ageing was evaluated.
In the experiments performed in Part B – Chapter I WKY rats aged up to 91
weeks were used. Although at the age of 91 weeks WKY rats showed no signs of severe
loss of renal function or renal injury, it was observed that the kidney was becoming
dysfunctional. This was supported by the fact that K+ excretion and fractional excretion
of Na+ were severely affected in WKY rats during ageing. Evaluation of Na
+,K
+-
ATPase activity demonstrated that Na+,K
+-ATPase function was altered in the kidney of
aged WKY rats. In the proximal tubules, where the bulk of Na+ and K
+ reabsorption
takes place, no age-related changes were observed until the age of 52 weeks. However,
at 91-weeks of age, Na+,K
+-ATPase activity was significantly decreased. The fact that
no alterations were observed in protein expression of the α1-subunit of Na+,K
+-ATPase
suggests that the age-related decrease in Na+,K
+-ATPase activity could be due to
increased phosphorylation of basolateral Na+,K
+-ATPase leading to internalization of
107
the enzyme by endocytosis. In fact, this has been also observed in the proximal tubules
of aged Fischer rats (Asghar et al. 2001). In the renal cortex of Fischer rats, age-related
increases in ROS were responsible for higher basal PKC activity and Na+,K
+-ATPase
phosphorylation. This mechanism could also explain what is reported for the renal
cortex of aged WKY rats. Moreover, as presented in Part B- Chapter II, in the
proximal tubules of WKY rats Na+,K
+-ATPase phosphorylation increased with age. In
renal medulla, which is mainly constituted by cortical collecting ducts and where the
final regulation of Na+ and K
+ in the urine takes place, ageing was accompanied by a
significant increase in Na+,K
+-ATPase activity and expression of the α1-subunit.
Furthermore, not only was Na+,K
+-ATPase activity increased in renal medulla of aged
WKY rats but also, in this part of the kidney, H2O2 production was increased with age
and in comparison with renal cortex. Given that Na+,K
+-ATPase regulation differs
between the proximal tubules and distal nephron segments it is possible that, in the renal
medulla increased ROS may activate cell specific signalling pathways that up-regulate
Na+,K
+-ATPase activity. In fact, NOX isoforms are structurally different, localized in
distinct compartments of the cell and are differentially regulated by growth factors and
during development (Dikalov et al. 2008). In the present work this is supported by the
fact that in the proximal tubules of aged WKY rats Na+,K
+-ATPase activity is decreased
and NOX-4 is increased (Part B- Chapter I) whereas in serially-passaged OK cells
both Na+,K
+-ATPase and NOX-1 are increased (Part A – Chapter II). Although the
profile of oxidant and antioxidant enzyme expression was not determined in the renal
medulla, the analogies with the in vitro model of cellular ageing raise the possibility that
increased production of ROS in renal medulla could partially account for the observed
increase in Na+,K
+-ATPase activity.
108
The role of Na+,K
+-ATPase in proximal tubules has been the target of extensive
research due to the fact that, as mentioned before, it plays a crucial role in the bulk of
Na+ and nutrient reabsorption. Moreover, as recently reviewed by Wang (Wang et al.
2009) it is well known that altered renal proximal tubular Na+ reabsorption is implicated
in the development of essential hypertension. Interest in the regulation of Na+ transport
in renal medulla is more recent and mainly due to the existence of a possible role in the
initiation and development of several forms of experimental hypertension (Cowley et al.
1992; Cowley and Roman 1996). In renal medulla ROS appear to directly alter Na+
reabsorption and indirectly alter medullar blood flow, contributing to the development
of hypertension (Taylor et al. 2006a; Taylor et al. 2006b; Cowley 2008). The original
findings showing that aged WKY rats had increased renal medulla H2O2 production and
Na+,K
+-ATPase activity suggest that these alterations may precede the development of
ageing-associated conditions. From this point of view, attention should be paid to ROS
and renal medulla transporter regulation during ageing.
In Part B – Chapter II, it was evaluated whether long-term administration of
ouabain to juvenile (12 week-old) and mature (52 week-old) WKY rats induced
alterations in renal Na+ handling and Na
+,K
+-ATPase function in renal proximal tubules.
After 7 weeks of ouabain treatment both juvenile and mature rats developed moderate
but sustained hypertension. However, Na+,K
+-ATPase activity was only decreased in
the proximal tubules of ouabain-treated juvenile WKY rats. Surprisingly, ouabain-
treated juvenile rats had no changes in renal excretion of Na+. Once more, distal parts of
the nephron seemed to be playing an important role in the fine adjustment of Na+.
The fact that in mature WKY rats no alterations in Na+,K
+-ATPase activity in
the renal proximal tubules were observed suggests that the mechanisms responsible for
109
Na+,K
+-ATPase down-regulation are age-dependent. Taking into account the results
obtained in Part A regarding Na+,K
+-ATPase regulation by ouabain, the results
obtained in vivo may seem contradictory. However, they reflect the integrated
regulation of Na+,K
+-ATPase by several endogenous factors and the complexity of the
ouabain-mediated signal cascade. Activation of other endougenous mechanisms
responsible for Na+,K
+-ATPase regulation in in vivo animal models of ouabain-induced
hypertension are complex and need further research, since they depend not only on the
age and the strain of the rats, but also on the dosage and the duration of the treatment
(Zhang et al. 2010).
In conclusion, a model of in vitro renal cellular ageing, that can be an additional
tool to be used for the study of age-induced alterations in membrane transporters, was
developed and characterized. In this model it was shown that ageing was accompanied
by alterations in cellular redox balance and cytoskeleton composition. Additionally, it
was demonstrated that these two factors played an important role in the regulation of
Na+,K
+-ATPase function. Moreover, findings on ouabain-mediated regulation of
Na+,K
+-ATPase improved the understanding of apparently conflicting data obtained
from both in vitro and in vivo experiments. The role of ROS in the regulation of renal
Na+,K
+-ATPase was additionally evaluated in an in vivo model of ageing. Results
suggest that the mechanisms by which the redox balance regulates Na+,K
+-ATPase in
the in vitro model may also be present in the renal medulla of aged WKY rats. Being so,
during ageing urinary electrolyte homeostasis could be compromised due to alterations
in the medullar fine-tuning of urinary Na+ and K
+. Moreover, results point to the
possibility that the observed changes in Na+,K
+-ATPase function precede the
110
development of ageing-associated renal conditions. Further research will be necessary
to elucidate this hypothesis.
111
112
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113
114
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Summary
125
126
Summary
The kidney is one of the body organs severely affected by ageing. In the kidney Na+,K
+-
ATPase, a basolateral membrane protein expressed throughout the nephron, plays an
essential role in the reabsorption of Na+ and K
+ and in the Na
+-dependent reabsorption
of glucose and amino acids. Changes in renal Na+,K
+-ATPase have been found during
the development of ageing-associated conditions, such as hypertension and diabetes.
However, not much information is available on the age-dependent regulation of renal
Na+,K
+-ATPase. In this study, a model of in vitro renal cellular ageing using opossum
(Didelphis virginiana) kidney cells (OK cells) was developed and characterized. The
use of renal cells aged by serial-passages made it possible to determine that reactive
oxygen species (ROS), the cytoskeleton and ouabain play a role in the regulation of
Na+,K
+-ATPase. In vitro cellular ageing was accompanied by changes in cellular redox
status and the cytoskeleton. Aged cells had increased production of ROS and a
substitution of the ~190 kDa isoform of the cytoskeleton protein ankyrin-G for the
~220/200 kDa isoform. This led to an increase in Na+,K
+-ATPase activity in aged renal
cells. The fact that chronic ouabain-treatment induced a mitogen-activated protein
kinase and phosphoinositide
3-kinase dependent up-regulation of Na+,K
+-ATPase
activity only in aged OK cells shows that age and cell specific factors are responsible
for the regulation of Na+,K
+-ATPase. In addition to in vitro studies, renal Na
+,K
+-
ATPase function was also evaluated in aged Wistar Kyoto (WKY) rats. In aged WKY
rats, different changes in Na+,K
+-ATPase function occurred along the nephron. Na
+,K
+-
ATPase activity decreased in aged renal cortex and increased in renal medulla. The aged
kidney had an increase in H2O2 production. This increase was more pronounced in renal
medulla where it may account for the observed up-regulation of Na+,K
+-ATPase
127
activity. Results obtained with aged ouabain-treated rats indicate that renal regulation of
Na+ mediated by Na
+,K
+-ATPase is dependent on the interaction of several endogenous
factors some of which may be age-dependent. The data presented in this work provides
a useful model for the study of renal cellular ageing and new insights of how multiple
factors can contribute to changes in Na+,K
+-ATPase regulation during in vitro and in
vivo ageing.
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Sumário
O rim é um dos órgãos mais afectados pelo envelhecimento. Por outro lado, sabe-se que
a ATPase do sódio e potássio (Na+,K
+-ATPase) é uma proteína localizada no bordo
basolateral das células do nefrónio que desempenha um papel fundamental na
reabsorção de Na+ e K
+ e na reabsorção de glicose e aminoácidos dependente de Na
+ e
está alterada em patologias associadas ao envelhecimento, como a hipertensão e a
diabetes. No entanto, a informação disponível sobre a regulação da Na+,K
+-ATPase
durante o envelhecimento é escassa. Neste trabalho desenvolveu-se e caracterizou-se
um modelo de envelhecimento celular com recurso a células renais de gambá-da-
Virgínia (Didelphis virginiana) envelhecidas por passagens contínuas. Verificou-se que
no envelhecimento in vitro se alteram o estado redox e o citoesqueleto das células
renais, a actividade da sua Na+,K
+-ATPase e a resposta desta enzima à acção da ubaína.
As células renais envelhecidas produzem mais espécies reactivas de oxigénio (ERO) e
substituem a expressão proteica da isoforma da anquirina-G com 220/200 kDa pela
isoforma com 190 kDa. Estas alterações levam a um aumento da actividade da Na+,K
+-
ATPase nas células envelhecidas. A exposição crónica à ubaína induziu um aumento da
actividade da Na+,K
+-ATPase apenas nas células envelhecidas. Este aumento depende
da estimulação da cínase de proteínas activada por mitogénios e da estimulação da
cínase na posição 3 do fosfatidilinositol. Em ratos Wistar Kyoto (WKY) envelhecidos
verificou-se que a função da Na+,K
+-ATPase no rim também está alterada. Com o
envelhecimento dos ratos WKY a actividade da Na+,K
+-ATPase do nefrónio diminui no
córtex e aumenta na medula, onde ocorre também o aumento maior de produção de
H2O2. Os resultados obtidos com ratos tratados com ubaína indicam que a regulação do
transporte renal de Na+ mediado pela Na
+,K
+-ATPase é dependente da interacção de
vários factores endógenos, alguns dos quais dependentes da idade. Em conclusão, este
129
trabalho apresenta um modelo celular útil para o estudo in vitro do envelhecimento
renal e contribui para o aumento do conhecimento sobre a regulação da Na+,K
+-ATPase
no envelhecimento.
130
