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From the Department of Clinical Science, Intervention and Technology, Division of Renal Medicine, Karolinska Institutet, Stockholm, Sweden
RENAL KLOTHO AND MINERAL METABOLISM
Risul Amin
Stockholm 2014
Cover image: Immunofluorescence staining shows Klotho expression (red) in the distal tubules. The sodium-phosphate co-transporter Npt2a (green) is localized at the brush-border membrane of the proximal tubules. DAPI (blue) stains the cell nuclei.
All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by US-AB. © Risul Amin, 2014 ISBN 978-91-7549-482-1
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I would like to dedicate this thesis to Sweden
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ABSTRACT
Calcium and phosphorous are critical elements in a number of physiological processes,
including maintenance of bone structure, cell signaling and energy metabolism. Their
endocrine regulation is tightly controlled through a number of feedback mechanisms
involving parathyroid hormone (PTH), vitamin D and the recently discovered fibroblast
growth factor 23 (FGF23). The kidney is a key organ in maintaining normal serum
levels of calcium and inorganic phosphorous, and disturbances in mineral metabolism
are commonly observed in patients with chronic kidney disease (CKD). Klotho is a
membrane-bound protein expressed in the renal tubules that acts as a co-receptor for
FGF23. In addition, Klotho can be shedded from the cell surface to extra-cellular
compartments and function as a hormone with effects on mineral metabolism
independent of FGF23. During the progression of CKD the expression of Klotho
rapidly declines, and accumulating evidence point to lack of Klotho as a pathogenic
factor driving clinical complications in CKD. The main focus of this thesis has been to
elucidate the role of renal Klotho in mineral metabolism and on systemic effects.
In Study I we generated distal tubule-specific Klotho knockout mice (Ksp-KL-/-) by
employing cre-lox recombination. Ksp-KL-/- mice were hyperphosphatemic with
elevated serum Fgf23 levels, indicating that distal tubular Klotho affects phosphate
reabsorption in the proximal tubules. The exact mechanism of this proposed distal-to-
proximal tubular signaling remains unknown.
In Study II we generated mice with Klotho deleted throughout the nephron (Six2-KL-/-
). Six2-KL-/- mice were infertile, kyphotic, growth retarded and had a decreased life
span, closely resembling the phenotype seen in systemic Klotho knockout mice. Also
the serum and urine biochemistries, low serum Klotho levels as well as profound
histological abnormalities were indistinguishable from systemic Klotho knockout mice,
unraveling the kidney as the principle contributor to circulating Klotho and mediator of
Klotho anti-ageing traits.
Taken together, the studies presented in this licentiate thesis substantially contribute to
the understanding of renal Klotho function.
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LIST OF PUBLICATIONS
I. Olauson H, Lindberg K, Amin R, Jia T, Wernerson A, Andersson G, Larsson
TE.
Targeted deletion of Klotho in kidney distal tubule disrupts mineral
metabolism.
J Am Soc Nephrol. 2012 Oct; 23 (10): 1641-51.
II. Lindberg K, Amin R, Moe OW, Hu MC, Erben RG, Wernerson A, Lanske B,
Olauson H and Larsson TE.
The kidney is the principal organ mediating Klotho effects.
Accepted for publication in J Am Soc Nephrol (Feb 17, 2014).
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RELATED PUBLICATIONS NOT INCLUDED IN THIS
THESIS
I. Lindberg K, Olauson H, Amin R, Ponnusamy A, Goetz R, Taylor RF,
Mohammadi M, Canfield A, Kublickiene K, Larsson TE.
Arterial Klotho expression and FGF23 effects on vascular calcification and
function.
PLoS One. 2013;8(4):e60658.
II. Olauson H, Lindberg K, Amin R, Sato T, Ting J, Goetz R, Mohammadi M,
Andersson G, Lanske B, Larsson TE.
Parathyroid-specific deletion of the Klotho gene unravels a novel calcineurin
dependent FGF23 signalling pathway that mediates suppression of PTH
secretion.
PLoS Genet. 2013 Dec;9(12):e1003975.
III. Jia T*, Olauson H*, Lindberg K, Amin R, Edvardsson K, Lindholm B,
Andersson G, Wernerson A, Sabbagh Y, Schiavi S, Larsson TE.
A novel model of adenine-induced tubulointerstitial nephropathy in mice.
BMC Nephrol. 2013 May 30;14(1):116.
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CONTENTS
1 Introduction ................................................................................................... 7 1.1 Mineral metabolism ............................................................................ 7
1.1.1 Calcium homeostasis .............................................................. 7 1.1.2 Phosphate homeostasis ........................................................... 9 1.1.3 Fibroblast growth factor-23 ................................................. 11 1.1.4 Klotho ................................................................................... 13
1.2 Mineral disturbances in chronic kidney disease ............................... 15 2 Aims ............................................................................................................. 18 3 Material and Methods .................................................................................. 19
3.1 Ethical approval ................................................................................ 19 3.2 Cre-Lox recombination ..................................................................... 19 3.3 Molecular methods ............................................................................ 20
3.3.1 Enzyme linked immunosorbent assay (ELISA) .................. 20 3.3.2 Immunohistochemistry and immunofluorescence ............... 20 3.3.3 Immunoblot .......................................................................... 21 3.3.4 Real-time qPCR analysis ..................................................... 21
3.4 Statistical analysis ............................................................................. 22 4 Results and discussion ................................................................................. 23
4.1 Distal tubuli-specific Klotho-knockout mice have disrupted mineral metabolism and elevated levels of Fgf23 (Study I) ................................... 23 4.2 The kidney is the main contributor to systemic Klotho and the principal organ mediating Klotho effects (Study II) ................................................. 26
5 Acknowledgements ..................................................................................... 29 6 References ................................................................................................... 31
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LIST OF ABBREVIATIONS
1,25(OH)2D 1,25 dihydroxyvitamin D
ADHR Autosomal dominant hypophosphatemic rickets
CaSR Calcium-sensing receptor
CKD Chronic kidney disease
CVD Cardiovascular disease
CYP24A1 1, 25-dihydroxyvitamin D 24-hydroxylase
CYP27B1 25 dihydroxyvitamin D 1-alpha hydroxylase
ESRD End-stage renal disease
FGF Fibroblast growth factor
FGF23 Fibroblast growth factor-23
FGFR Fibroblast growth factor receptor
GFR Glomerular filtration rate
HFTC Hyperphosphatemic familial tumoral calcinosis
IF Immunofluorescence
IHC Immunohistochemistry
mKL Membrane-bound Klotho
cKL Shedded full-length Klotho
sKL Truncated Klotho
Ksp-KL-/- Distal tubule-specific Klotho knockout mice
Npt The type II sodium-dependent phosphate co-transporter
Pi Inorganic phosphorus
PTH Parathyroid hormone
PTH1R Parathyroid hormone receptor 1
qPCR Quantitative real-time polymerase chain reaction
RAAS Renin-angiotensin-aldosterone system
SHPT Secondary hyperparathyroidism
Six2-KL-/- Renal Klotho knockout mice
TIO Tumor-induced osteomalacia
TRPV5 Transient receptor potential cation channel subfamily V, 5
VDR Vitamin D receptor
XLH X-linked hypophosphatemic rickets
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1 INTRODUCTION
1.1 MINERAL METABOLISM Calcium and phosphorus are vital to life and involved in many biological processes,
including maintenance of bone structure, intracellular signaling and energy metabolism.
Mineral homeostasis are preserved through a balanced influx and efflux from the
intestine, bone and kidney, and orchestrated by several hormones in a complex set of
feedback loops. Abnormalities in mineral metabolism are usually a consequence of
hereditary or acquired dysfunctions in any of these organs. The kidney is a pivotal
organ regulating calcium and phosphorus metabolism, and disrupted homeostasis of
these elements are therefore almost universal in patients with chronic kidney disease
(CKD) [1]. Overwhelming epidemiological evidence link these abnormalities to
adverse clinical outcomes, most prominently cardiovascular morbidity and mortality in
patients with CKD as well as in community-based settings [2, 3].
1.1.1 Calcium homeostasis 1.1.1.1 Calcium
Calcium is the most abundant mineral in the human body and has a central role in bone
metabolism, neuromuscular and secretory functions. The daily dietary intake of
calcium in an adult is around 1000 mg. Approximately 50% of the total serum calcium
is free and biologically active, the rest is bound to albumin and to a less degree other
anions. The concentration of calcium within body fluids is tightly controlled by
homeostatic mechanisms involving influx and effluxes of calcium between
extracellular fluids and the bone, kidney and intestine [4]. Two principal hormones
account for this regulation: parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D
(1,25(OH)2D).
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1.1.1.2 Calcium homeostasis and parathyroid hormone
The parathyroid glands are small endocrine glands located adjacent to or embedded in
the thyroid gland. Their main function is to produce and secrete PTH. Low
concentrations of serum calcium inactivate the parathyroid gland resident calcium-
sensing receptor (CaSR), which rapidly results in increased PTH secretion, while high
serum calcium activates the CaSR and suppresses PTH secretion [5]. PTH is an 84
amino acid peptide hormone with a half-life of approximately 4 minutes. PTH acts on
the G protein-coupled parathyroid hormone receptor (PTH1R) and has three main
effects: 1) directly stimulates bone resorption; 2) increases calcium reabsorption in the
renal distal tubules; and 3) stimulates the renal production of 1,25(OH)2D. The
concerted effect of PTH is to rapidly increase serum calcium, and the relationship
between calcium and PTH is characterized by a sigmoidal curve with the steepest slope
at physiological levels of serum calcium [6].
1.1.1.3 Calcium regulation by vitamin D
Vitamin D can be produced either in the skin or ingested with the diet. It is 25-
hydroxylated in the liver, and subsequently 1-hydroxylated in the kidney to its active
form – 1,25(OH)2D. Active vitamin D stimulates calcium absorption in the gut and
reabsorption in the kidney, and resorption in bone, by binding the nuclear vitamin D
receptor (VDR), which acts as a transcription factor promoting genes involved in
calcium ion transport. Calcium and 1,25(OH)2D are reciprocally regulated through a
negative feedback loop. The enzyme 25-dihydroxyvitamin D 1-alpha hydroxylase
(CYP27B1), which fulfils the final activation step of vitamin D, is induced by PTH,
hypocalcemia, and hypophosphatemia, and in contrast, CYP27B1 is repressed by
hypercalcemia and hyperphosphatemia [7, 8].
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1.1.2 Phosphate homeostasis 1.1.2.1 Phosphorus
Phosphorus (P) is the sixth most abundant element in the body. In nature, phosphorus is
predominantly bound to oxygen as a phosphate (PO43-), and is an essential component
of bone, nucleic acids and the phospholipids in the cell membrane. The total body
content of phosphate is about 500-700 g, of which 85% is in the form of hydroxyapatite
in bones and teeth, 15% distributed in the cellular compartment and less than 1% in
extracellular fluids. The physiological range for serum inorganic phosphorous (Pi) is
0.7-1.5 mmol/l. Phosphate balance is regulated by multiple endocrine factors including
PTH, 1,25(OH)2D3 and the more recently discovered hormones fibroblast growth factor
23 (FGF23) and Klotho [9].
1.1.2.2 Phosphate homeostasis
Phosphate homeostasis is maintained by three major mechanisms: intestinal absorption,
bone resorption, and renal reabsorption. The daily intake of phosphate is approximately
1200 mg, of which 70% is absorbed by the small intestine through both active and
passive transport mechanisms. The active transport is mediated through the sodium-
dependent phosphate transporter type 2b (Npt2b). Npt2b is primarily expressed in the
small intestine and in the lungs, and is regulated by two major stimuli; 1,25(OH)2D and
dietary phosphate. The passive diffusion is primarily load-dependent although the exact
regulation is unknown. The relative contribution of these two mechanisms is not
exactly known and may vary across species, but active transport is estimated to account
for at least 50% of total transport based in interventional studies in Npt2b knockout
mice [10]. The kidney is the most important organ for maintaining phosphate
homeostasis. Under basal condition, approximately 70% of the filtered phosphate is
reabsorbed at the brush border membrane of the proximal tubules. Both animal and
human studies suggest that the sodium-dependent phosphate transporters Npt2a and
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Npt2c are responsible for approximately 80% and 20% of the renal phosphate
reabsorption, respectively. Bone is the largest reservoir for phosphate in the body.
Reciprocally, extracellular phosphate concentration is a determinant of bone formation
and resorption. A number of studies suggest that a physiological level of phosphate
inhibits bone resorption whereas dietary deprivation is associated with higher
osteoclastic activity and increased phosphate release [11].
1.1.2.3 Phosphate regulation
PTH was long considered the best-characterized phosphate-regulating factor.
Hyperphosphatemia stimulates the release of PTH, which acts on the proximal tubule to
decrease the brush-border membrane expression of Npt2a and Npt2c, thus causing
increased urinary phosphate excretion [12]. In contrast, 1,25(OH)2D stimulate intestinal
phosphate absorption through increased expression of Npt2b. Bone also plays an
important role to regulate phosphate homeostasis. A decrease in serum Pi leads to
increased phosphate resorption from bone, both directly and through the actions of PTH
and vitamin D. The discovery of the phosphaturic hormones FGF23 and Klotho has
dramatically changed the concepts of phosphate regulation [13].
The regulation of mineral metabolism is summarized in Figure 1.
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Figure 1. Calcium and phosphorus balance is preserved through a balanced influx and efflux
from the intestine, bone and kidney, and orchestrated by several hormones in a complex set of
feedback loops.
1.1.3 Fibroblast growth factor-23 The Fibroblast growth factors (FGFs) are a highly conserved family of genes that can
be divided into seven phylogenetic subfamilies. The human/mouse FGFs gene family
comprises 22 members, from FGF1 to FGF23. These 22 genes encode molecules that
have the ability to bind to one or several of the fibroblast growth factor receptors
(FGFRs) [14]. The FGFs are also divided based on their function into intracellular,
canonical, and hormone-like FGFs. In contrast to the intracellular and
Parathyroid gland
KidneySmall intestine
Calcium ↓
PTH ↑
1,25(OH)2D ↑
Calcium absorption ↑
Bone
1,25(OH)2D ↑
Calcium reabsorption ↑
Phosphate excretion ↑
Bone resorption ↑
CYP27B1 FGF23 ↑
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autocrine/paracrine actions of the canonical FGFs, hormone-like (e.g. FGF19, FGF21
and FGF23) act systemically as endocrine factors [15].
1.1.3.1 Structure and function of FGF23
The human FGF23 gene is located on the chromosome 12p13 and is composed of three
exons separated by two introns. It encodes a 32 kDa glycoprotein containing 251 amino
acids. In the N-terminus there is a 24 amino acid signal peptide; thus the secreted
FGF23 protein consists of 227 amino acids. FGF23 is produced by osteoblasts and
osteocytes in bone. The biologically active full-length FGF23 is cleaved into inactive
N-terminal (20 kDa) and C-terminal (12 kDa) fragments, both within the cell it is being
produced, and in the circulation [16]. The estimated half-life of FGF23 is 20-60
minutes. FGF23 has three main functions in physiology; 1) lower serum Pi through
decreasing the abundance of Npt2a and Npt2c at the brush border membrane of renal
proximal tubules; 2) reduce serum 1,25(OH)2D levels by decreasing the activation and
increasing its catabolism through regulation of CYP27B1 and the 24-hydroxylase
(CYP24A1), respectively; and 3) decrease the synthesis and secretion of PTH from the
parathyroid glands [17, 18]. Hence, the concerted effects of FGF23 is to directly
decrease serum levels Pi, and indirectly decrease serum calcium through effects on
1,25(OH)2D and PTH.
1.1.3.2 Animal models and model diseases
Intravenous administration of Fgf23 or transgenic overexpression of Fgf23 in mice
results in hypophosphatemia due to increased renal phosphate wasting, and decreased
levels of circulating 1,25(OH)2D (and PTH within the short term) [18-20]. A similar
phenotype is seen in patients with increased FGF23 levels, either through abnormal
processing (for example in autosomal dominant hypophosphatemic rickets, ADHR,
OMIM #193100), regulation (X-linked hypophosphatemic rickets, XLH, OMIM
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#307800) or ectopic production (tumor-induced osteomalacia, TIO) [21-23]. In
addition, reduced bone mineral density and growth retardation are observed in mice
over-expressing Fgf23 [24]. In contrast, Fgf23 knockout mice or humans with
homozygous missense mutations in FGF23 (e.g. hyperphosphatemic familial tumoral
calcinosis, HFTC OMIM #21900) suffer from an opposite phenotype with
hyperphosphatemia, hypercalcemia, high serum 1,25(OH)2D levels and soft-tissue
calcifications [25, 26] .
1.1.3.3 Fibroblast growth factor receptors
FGF23 exerts its actions in the kidneys and parathyroid glands through binding to
FGFRs and activate the MAPK/ERK pathway. The binding to a FGFR requires the
presence of Klotho, a type 1 membrane-bound protein that functions as an FGF23 co-
receptor [27]. Klotho enhances the affinity of FGF23 to multiple FGFRs, most
importantly FGFR1, 3 and 4, by forming Klotho-FGFR binary complexes. The
importance of Klotho as a co-receptor is evidenced by the fact that mice lacking Klotho
(Klotho knockout mice) develop an identical phenotype as the Fgf23 knockout mice,
with hyperphosphatemia, high serum 1,25(OH)2D levels, infertility, growth retardation,
shortened lifespan and ectopic calcifications, despite a 1000-fold increase or more in
circulating Fgf23 levels [28] .
1.1.4 Klotho 1.1.4.1 Discovery of Klotho gene
Klotho is considered an ageing-suppressor gene that was first identified by Kuro-o and
colleagues in 1997 when studying mice with random mutations in a sodium proton
exchanger [28]. It was named after the Greek goddess Clotho, who spins the thread of
life and decides over birth and death. Klotho is highly expressed in the distal
convoluted tubules in the kidney, chief cells in the parathyroid glands and the epithelial
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cells of choroid plexus of the brain. Silencing Klotho resulted in a phenotype
resembling human ageing with shortened life span, muscle and skin atrophy,
pulmonary emphysema, osteopenia, infertility, hyperphosphatemia and vascular and
soft tissue calcification. In contrast, overexpression of Klotho extended life span in
mice, speculatively through suppression of insulin/IGF1signaling [29].
1.1.4.2 Structure and isoforms of Klotho
The human KLOTHO gene contains five exons and encodes a type 1 single-pass
transmembrane polypeptide of 1014 amino acids. The Klotho protein has two internal
repeats named KL1 and KL2 that share homology to family 1 beta-glycosidase, and a
short intracellular C-terminus [30]. Membrane bound Klotho (mKL) can be shedded
from the cell surface (cKL) by membrane-anchored proteases (including ADAM10 and
ADAM17) into blood, urine and cerebrospinal fluid [31]. In addition to mKL and cKL,
a secreted isoform (sKL) is also generated through alternative splicing at exon 3 [32]
(Figure 2). The relative quantity of the various isoforms, at least at the protein level, is
currently unknown.
Figure 2. Klotho has three isoforms; a membrane-bound form (mKL), a shedded form (cKL)
and a truncated form generated through alternative splicing (sKL).
KL1
KL2
KL1
KL2
KL1
Proteolytic cleavage
Alternative splicing
mKL cKL sKL
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1.1.4.3 Mode of action of Klotho isoforms
The three Klotho isoforms have distinct functions. In 2006 mKL was identified as a
permissive co-receptor for FGF23, increasing its affinity to FGFRs by multiple folds. In
contrast to mKLs role as a co-receptor, cKL function as an independent endocrine
factor [33, 34]. Recent studies have revealed that cKL is involved in a variety of
biological processes including modulation of ion transport, suppression of the renin-
angiotensin-aldosterone system (RAAS), anti-senescence and anti-oxidation [33, 35,
36]. Furthermore, injection of cKL in mice suppresses insulin/IGF1 signaling leading to
increased insulin resistance. Finally, it was reported that both sKL and cKL are
antagonists of Wnt signaling, and are able to counteract the development of fibrosis in
various mouse models of renal injury [37, 38].
1.1.4.4 Regulation of Klotho
The regulation of Klotho is still incompletely understood. However, the promoter
region of Klotho contains vitamin D responsive elements, and Klotho expression is
upregulated by administration of vitamin D, both in vivo and in vitro [39, 40].
Similarly, Klotho is increased in mice on a low phosphate diet [41]. Conversely, a
number of factors such as phosphate, calcium, inflammation, oxidative stress, uremic
toxins and FGF23 have been shown to reduce Klotho expression [35]. The upstream
regulators of Klotho shedding and alternative splicing are yet to be resolved.
1.2 MINERAL DISTURBANCES IN CHRONIC KIDNEY DISEASE CKD is defined as the presence of kidney damage, or a progressive loss in renal
function measured as decreased glomerular filtration rate (GFR below 60 ml/min/1.73
m2), for a period of three months or more. CKD is divided into five stages (Table 1)
depending on GFR [42]. In CKD stage 5, also known as End-Stage Renal Disease
(ESRD), the five-year survival rate is approximately 50%. To sustain life in ESRD
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patients, renal replacement therapies such as dialysis or kidney transplantation are
needed. People with CKD have a markedly increased risk of developing cardiovascular
disease (CVD) [43]. Other than traditional risk factors such as diabetes and high blood
pressure, there are also CKD-specific factors known to be associated with disease
progression and mortality including albuminuria, hyperphosphatemia, and activation of
RAAS. Current treatment strategies, aimed at slowing progression of disease, and at
reducing CVD risk, are clearly insufficient to prevent development of ESRD and
alleviating the associated cardiovascular high-risk profile.
Stage Description GFR (mL/min/1.73 m2) Albuminuria
1 Normal or high ≥90
<30 mg/g
30-300 mg/g
>300 mg/g
2 Mildly decreased 60-89
3a Mildly to moderately decreased 45-59
3b Moderately to severely decreased 30-44
4 Severely decreased 15-29
5 Kidney failure <15 (or dialysis)
Table 1. Stages of chronic kidney disease. CKD is defined as either kidney damage (usually
quantified by albuminuria) or glomerular filtration rate (GFR) <60 mL/min/1.73 m2 for more
than three months.
As renal function declines, the kidneys fail to maintain normal calcium and Pi levels in
the blood, leading to a disturbed mineral metabolism with increased risk of CVD and
mortality [3]. Biochemical changes usually become evident at CKD stage 3-4, but the
pathogenesis starts already at earlier stages. The dynamics of these changes have been
re-evaluated after the discovery of FGF23, which increases already at a mild reduction
in renal function and precedes other hormonal changes [44, 45]. Initially, elevated
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FGF23 compensate for the reduced number of nephrons and maintain serum Pi within
the normal range. However, this comes at the expense of reduced 1,25(OH)2D levels
and a subsequent development of secondary hyperparathyroidism (sHPT) [46] . At later
stages these hormonal adaptations becomes insufficient and instead progresses into a
maladaptive response, contributing to the increased risk of disease progression and
mortality. A number of studies have convincingly shown that renal and parathyroid
Klotho expression is decreased in animals and humans with acute renal failure or CKD,
regardless of etiology [47-51]. This suppression may partly be mediated by epigenetic
changes since uremic toxins induce hypermethylation of the Klotho gene [52]. The
progressive decrease in Klotho eventually results in end-organ FGF23 resistance, which
is likely to contribute to the extreme elevations in FGF23 observed in ESRD [53]. In
turn, FGF23 was recently identified as a cardiovascular toxin with direct effects on
myocardial growth [54] .
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2 AIMS The overall aim of this thesis was to refine the understanding of renal Klotho function
and role in regulating mineral metabolism.
Specifically, the objectives were to;
• Elucidate the role of distal tubular Klotho in renal phosphate handling and on
regulation of FGF23 (Study I).
• Investigate the role of renal Klotho on systemic effect and its contribution to
circulating Klotho (Study II).
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3 MATERIAL AND METHODS 3.1 ETHICAL APPROVAL The studies in this thesis were conducted in compliance with the guidelines of animal
experiments of Karolinska Institutet and approved by Stockholm South ethical
committee. Ethical approval numbers are S68-10, S116-12 and S118-12.
3.2 CRE-LOX RECOMBINATION Cre-Lox recombination is a widely used technology to generate conditional knockout
mice. The system consists of two components: the Cre enzyme and the LoxP sites. The
LoxP site is a specific 34 base pair sequence derived from bacteriophage P1. When
LoxP sites are inserted into the mouse genome (floxed mice), recombination occurs
between LoxP sites in cells expressing Cre recombinase, resulting in either activation
or repression of a specific DNA sequence or gene (Figure 3).
Figure 3. The whole or parts of the target gene is flanked by LoxP sequences, allowing for
tissue-specific deletion and disruption of gene function.
There are a few potential problems using Cre-Lox recombination. First, lack of
specificity of certain Cre promoters results in recombination in several tissues, which
can make the interpretation of results more difficult. Using a reporter strain is a
Exon 1 Exon 2 Exon 3
Exon 1 Exon 2 Exon 3 Tissue-specific promoter Cre recombinase
Exon 1 Exon 3
X
Wild-type allele
Floxed allele
Floxed allele after cre excision
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common way to confirm that Cre expression is restricted to the intended target tissue.
Second, Cre-Lox recombination is usually less efficient compared to conventional
knockout techniques.
3.3 MOLECULAR METHODS
3.3.1 Enzyme linked immunosorbent assay (ELISA)
Enzyme linked immunosorbent assay (ELISA) is a standard technique used for
detection and quantification of soluble antigens and antibodies in body fluids. Briefly,
an unknown amount of antigen is affixed to the surface of a well coated with a capture
antibody. A detection antibody is then applied over the surface to bind the antigen. The
detection antibody is covalently linked to an enzyme, and in the final step a substrate is
added which the enzyme converts to a detectable signal measured using a
spectrophotometer. In this thesis ELISAs are used to quantify serum levels of FGF23
and PTH.
3.3.2 Immunohistochemistry and immunofluorescence Immunohistochemistry (IHC) and immunofluorescence (IF) are widely used methods
to detect the presence and location of a target protein on a tissue section or in cultured
cells. The technique utilizes antibodies for detection of specific target antigens. Either
monoclonal or polyclonal antibodies may be used. Polyclonal antibodies recognize
different epitopes on the same antigen, whereas monoclonal antibodies bind to a
specific epitope. Usually IHC and IF relies on one primary and one secondary antibody.
The primary antibody is raised against the epitope of interest, and the secondary
antibody is raised against the immunoglobulins of the primary antibody. The secondary
antibody is conjugated with a linker molecule or directly bound to a reporter molecule.
The antibody-antigen interaction is visualized using chromogenic detection (IHC),
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which can be seen in a regular microscope, or fluorescent detection (IF), in which a
fluorophore is conjugated to the antibody, which requires a fluorescence microscope for
detection. Potential drawbacks of IHC and IF are unspecific staining and
autofluorescence. Furthermore, IHC and IF provides only semi-quantitative assessment
of protein expression and are less sensitive than other assays such as immunoblotting or
ELISA. In this thesis, the specificity of all antibodies used for IHC and IF were verified
by immunoblots or other techniques.
3.3.3 Immunoblot Immunoblotting is the golden-standard for specific protein detection and quantification
in cell or tissue lysate. This process is accomplished through three steps: 1) size
separation by gel electrophoresis, 2) transfer to solid support by electrical current
induction, 3) staining with primary and secondary antibodies for visualization. A
reporter enzyme or fluorophore is linked to the secondary antibody that produces a
color that allows detection of the protein of interest. Antibody specificity in
immunoblotting can be confirmed through validation of the protein size, and by using
positive and negative controls.
3.3.4 Real-time qPCR analysis Real-time quantitative PCR (qPCR) analysis is a common and sensitive method to
amplify and quantify specific gene products. The method can use either DNA binding
dyes such as SYBR Green, or sequence specific fluorescent reporter probes. In this
thesis, total RNA was extracted and reverse-transcribed into cDNA, and real-time
qPCR subsequently performed using gene-specific primers in a SYBR Green based
assay. The relative gene expression was calculated with the 2-ΔΔ Cq or Ct method
normalizing the gene of interest to a reference gene in the same sample. GAPDH was
used as a reference gene in study I and β-actin in study II.
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3.4 STATISTICAL ANALYSIS Gaussian distribution was tested using D’Agostino and Pearson omnibus normality test.
Variables fulfilling the criteria for normal distribution were tested with two-tailed t-test.
Non-normally distributed variables were compared using Mann-Whitney test.
Differences were considered statistically significant when p<0.05. Regression analysis
was performed using linear regression. A minimum of five mice of each genotype was
analyzed in all experiments. In study I, Ksp-KL-/- mice with relative Klotho levels >
70% were excluded during comparisons between groups. All mice were included in
correlation tests.
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4 RESULTS AND DISCUSSION
4.1 DISTAL TUBULI-SPECIFIC KLOTHO-KNOCKOUT MICE HAVE DISRUPTED MINERAL METABOLISM AND ELEVATED LEVELS OF FGF23 (STUDY I)
Renal Klotho has a key role in maintaining a normal mineral metabolism, both through
direct regulation of calcium- and phosphate co-transporters, and by facilitating FGF23
signaling. However, there are still a number of unanswered questions regarding Klotho
function and mechanism(s) of action. For example, the exact site by which Klotho
mediates FGF23 effects on urinary phosphate wasting and vitamin D metabolism is
unknown. Klotho is nearly exclusively expressed in the distal tubules whereas
phosphate reabsorption is confined to the proximal tubules. Some studies report that
there is sufficient Klotho expression in the proximal tubules to allow FGF23 signaling,
while other studies have shown activation of downstream signaling events upon FGF23
administration exclusively in the distal tubules [55, 56]. The systemic Klotho knockout
mouse develops a phenotype with severely dysregulated mineral metabolism, and is
therefore unsuitable to dissect Klotho’s role in physiology. To better understand renal
FGF23-Klotho function and regulation, we generated distal tubule specific Klotho
knockout mice (Ksp-KL-/-) employing Cre-Lox combination.
Ksp-KL-/- mice were viable, fertile, had normal longevity and a normal gross
phenotype. The knockout efficiency varied between 0 and 74% as measured with qPCR
and immunofluorescence staining (Figure 4). To determine the impact of distal tubular
Klotho deletion we examined serum and urine biochemistries, renal histology as well as
protein and transcript expression.
24
Figure 4. Partial deletion of distal tubular Klotho in Ksp-KL-/- mice was confirmed with
immunofluorescence staining. Klotho co-localized fully with TRPV5 in wild-type mice, and
partially in Ksp-KL-/- mice.
Ksp-KL-/- mice were hyperphosphatemic with elevated Fgf23 levels, corresponding to
the residual Klotho expression. PTH levels were decreased in Ksp-KL-/- mice whereas
calcium and 1,25(OH)2D remained normal. Urinary calcium excretion was increased,
which could speculatively be due to decreased renal levels of TRPV5, as confirmed
with immunoblot. Unexpectedly, urinary phosphate excretion was unaltered in Ksp-KL-
/- mice. In contrast, brush-border membrane expression of Npt2a was markedly
increased, providing a plausible explanation to the observed hyperphosphatemia.
Further, immunoblotting showed increased renal levels of VDR. Transcript levels of
renal Cyp27B1 were increased in Ksp-KL-/- mice and Klotho transcript levels correlated
to those of Cyp27B1, VDR, Npt2a, and FGFR1.
In a subgroup of Ksp-KL-/- mice with normal serum Pi, Fgf23 levels were elevated,
supporting that additional factors than serum Pi contribute to Fgf23 regulation.
20 µm
WT Ksp-KL-/-Kl
otho
TRPV
5M
erge
d
25
Speculatively, end-organ resistance or soluble Klotho could be involved. Further
studies are needed to explore this potential relationship. Histological analysis revealed a
normal renal morphology. The lack of renal fibrosis and vascular calcification argues
against direct paracrine effects of Klotho, at least during low cellular stress. These
characteristics might be tested in subsequent studies by induction of renal failure or
with ageing mice.
This is the first study to present genetic and functional evidence that partial deletion of
Klotho in the distal tubules has a major impact on renal phosphate handling in the
proximal tubules. The factor(s) responsible for this proposed distal-to-proximal tubule
signaling are currently unknown, but could speculatively be soluble Klotho (Figure 5).
Although distal tubular Klotho appears essential to renal phosphate handling, our
results indicate a limited effect on vitamin D metabolism. Importantly, our data does
not exclude a role for Klotho in the proximal tubule, and this should be further
examined in future studies.
Figure 5. Deletion of Klotho in the distal tubules has a fundamental impact on renal phosphate
handling in the proximal tubules, suggesting the presence of a distal-to-proximal mechanism.
3 Na+
HPO42-
Proximal tubular cell Distal tubular cell
Luminal side
2 Na+
Npt2a
Npt2c
Klotho
FGFRs
FGF23
Distal-to-proximal mechanism?
Glomerulus
Proximal tubule Distal tubule
Loop of Henle
26
4.2 THE KIDNEY IS THE MAIN CONTRIBUTOR TO SYSTEMIC KLOTHO AND THE PRINCIPAL ORGAN MEDIATING KLOTHO EFFECTS (STUDY II)
Klotho has been reported to be expressed in multiple organs including the kidneys,
parathyroid glands, choroid plexus, pancreases, breast, testis, ovaries and urinary
bladder, yet its tissue-specific role outside the kidney remain uncertain. Systemic
Klotho knockout mice develop a phenotype resembling accelerated ageing with organ
abnormalities such as soft tissue calcifications, skin atrophy, pulmonary emphysema
and osteomalacia, and biochemical abnormalities including extremely elevated Fgf23
and 1,25(OH)2D, hypercalcemia and hyperphosphatemia. However, the organ-specific
contribution to the ageing phenotype in Klotho knockout mice, and relevant tissue
source of soluble Klotho are yet to be identified. In order to elucidate these issues we
generated mice with Klotho deleted throughout the nephron (Six2-KL-/-) using Cre-Lox
combination. Successful deletion of renal Klotho was confirmed with
immunofluorescence (Figure 6) and western blotting. Renal Klotho transcript levels
were reduced by approximately 80% compared to wild-type mice.
Figure 6. Immunofluorescence staining reveals efficient deletion of renal Klotho (red) in Six2-
KL-/- mice. Lotus tetragonolobus lectin (LTL, green) is a proximal tubule-specific marker and
DAPI (blue) stains cell nuclei.
Klot
ho/L
TL/D
API
WT Six2-KL-/-
27
Six2-KL-/- mice were infertile, kyphotic, growth retarded and had a decreased life span,
closely resembling the phenotype seen in systemic Klotho knockout mice. Their serum
biochemistries were characterized by hyperphosphatemia and remarkably elevated
Fgf23 and 1,25(OH)2D levels. PTH levels were decreased in the face of hypercalcemia.
Circulating levels of Klotho was measured using a combined technique of
immunoprecipitation and immunoblotting, and revealed an 80% reduction in Six2-KL-/-
mice. This strongly supports that the kidney is the principal contributor to circulating
Klotho. To confirm this, renal shedding was examined ex vivo using kidney explants. In
stark contrast to wild type mice, Klotho protein was undetectable in conditioned media
from Six2-KL-/- kidneys. Further, immunostaining showed preserved expression of
parathyroid Klotho in extra-renal organs such as the parathyroid glands and choroid
plexus in Six2-KL-/- mice. Thus, it can be concluded that the kidney is the principle
source of soluble Klotho and that extra renal tissues are not capable of correcting a
systemic deficiency of Klotho.
Next we examined the impact of renal Klotho ablation on kidney histology and
detected a higher proliferation rate with increased cellular density, loss of
differentiation between proximal and distal convoluted tubules as well as loss of
cuboidal Bowman’s epithelium of glomeruli in male Six2-KL-/- mice. Additional
histomorphological changes included mild interstitial fibrosis and widespread
nephrocalcinosis. To evaluate the impact of local Klotho expression on the ageing-like
phenotype we performed an extensive histological comparison between Six2-KL-/- mice
and systemic Klotho knockout mice. Notable histological findings in Six2-KL-/- mice
included pulmonary emphysema, reduced subcutaneous fat and skeletal
28
hypomineralization. Importantly, we found no differences in renal or extra renal
abnormalities between Six2-KL-/- and systemic Klotho knockout mice.
In conclusion, our data unequivocally unravels the kidney as the primary source of
circulating Klotho and mediator of Klotho function. This study have several
implications in the field of kidney research and provides support that therapeutic
Klotho delivery in CKD patients, a state of Klotho deficiency, may improve the uremic
complication and help to slow disease progression.
29
5 ACKNOWLEDGEMENTS
During the time of these studies, my mother got diagnosed with CKD and my aunt died
from kidney failure, bringing this research to a very personal level for me. Hopefully,
the combined efforts of the scientific community will eventually lead to a greater
understanding of CKD and allow for better treatment(s) for my mother and everyone
else in this world that suffer from this disease.
The work in this licentiate thesis was performed at the Division of Renal Medicine,
Department of Clinical Science, Intervention and Technology at Karolinska Institutet,
Sweden. I would like to express my great appreciation to everyone who has contributed
during the past years. I would especially like to thank the following people:
My supervisor Tobias Larsson Agervald for his excellent guidance that made me go
into research in the first place. Your outstanding knowledge in FGF23 and Klotho
biology and everything surrounding it is very inspiring and indeed I am truly grateful
for your generosity and support during the past years.
Annika Wernerson, my co-supervisor, for your scientific support, and for always
helping out when needed.
Hannes Olauson, my co-supervisor, for guiding me through this journey. You have
always been there, available for scientific discussions and support. You are an
enthusiastic, creative scientist that I learned a lot from. Your encouragement and
friendship never fails to motivate me.
30
My former and present colleagues, Karolina Lindberg, and Karin Edvardsson, for
making the endless hours in the lab so much more enjoyable, for all discussions about
work, life and everything else. Christina Hammarstedt, for excellent technical
assistance, always being so helpful and taking care of our last minutes orders. Your
kind attitude towards me made me feel like you were my Swedish mother. Ting Jia, for
your kind friendship and motivation.
Thanks to all the nice people at CLINTEC and Kliniskt Forskningscentrum (KFC).
Peter Stenvinkel, Bengt Lindholm, Tony Qureshi, Karolina Kublickiene, Björn
Anderstam, Monica Eriksson, Ann-Christin Bragfors and Samsul Arefin.
Special thanks to the members of Edvard Smith’s team for their kind help, Leonardo
Vargas, Manuela Gustafsson, Hossain Nawaz, Dara Mohammad, Sylvain Geny,
Burcu Bestas. Also thanks to Göran Andersson, for his kindness and help to allow
me to do imunohistochemistry in his laboratory.
A big thanks you to my all KI friends, including, Philipp, Mei, Brigitte, Sara,
Ibrahim, Abdul, Ferdous, Miraz, Asif Aziz.
Hearty thanks to my Bangladeshi friends, Samsul Boss, Nishu Apu, Jowel Viya and
Vave, for cooking and taking good care of me the last couple of years when I was
spending long hours in the lab.
And finally, my parents Ruhul Amin and Rekha Amin, and my sisters Dr. Ireen
Amin and Safrin Amin, who gave me a happy childhood with love and all mental
support and motivation during these studies.
31
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