ABSTRACT
La famiglia dei trasportatori dei monocarbossilati (MCT) comprende 14 membri
caratterizzati da diverse proprietà nel meccanismo di trasporto e con una specifica
distribuzione tissutale. Nel rene sono presenti diversi membri della famiglia MCT,
ma poche sono le conoscenze sulla loro esatta localizzazione e funzione. Per
questo siamo andati ad approfondire lo studio nel rene di topo. MCT1, MCT2,
MCT7 e MCT8 sono localizzati sulla membrana basolaterale delle cellule epiteliali
presenti lungo il nefrone. MCT1 e MCT8 sono localizzati nelle cellule del tubulo
prossimale, mentre MCT7 e MCT2 nel tratto spesso ascendente dell’ansa di
Henle. MCT7 è presente anche nelle cellule del tubulo distale. CD147, una β-
subunità delle proteine MCT1 e MCT4, ha mostrato, con l’indagine della
localizzazione, una parziale sovrapposizione con MCT1 e MCT2, e un
espressione a livello delle cellule intercalate. Inoltre, SMCT1 e SMCT2, due
cotrasportatori di monocarbossilati Na+dipendenti, sono espressi sulla membrana
apicale delle cellule intercalate di tipo A. Analisi sul trascritto del rene di topo, in
una condizione di acidosi, hanno mostrato delle alterazioni sulle quantità di mRNA
di diversi componenti della famiglia Slc16. Di conseguenza, i topi sono stati trattati
con un carico acido per un periodo compreso tra 2 e 7 giorni. Al termine del
trattamento gli animali hanno mostrato un marcato ma transitorio incremento dei
livelli di lattato nelle urine. Durante l’acidosi è stata riscontrata una down-
regolazione dei livelli di mRNA di MCT1, MCT8 e SMCT2, mentre i livelli di mRNA
di MCT7 e SMCT1 sono aumentati. Per quanto riguarda i livelli di espressione
proteica solo MCT7 e MCT2 risultano essere down-regolati, mentre tutti gli altri non
presentano variazioni. Riassumendo, in questo lavoro è stata studiata la
localizzazione di vari membri della famiglia dei trasportatori MCT nel rene di
mammifero ed è stato dimostrato che l’acidosi metabolica induce un transitorio
incremento dei livelli di lattato nelle urine parallelamente ad una diminuzione del
livello di espressione proteica di MCT2 e MCT7.
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INTRODUCTION
Lactate and pyruvate are the major monocarboxylates circulating in the blood (11).
The liver can utilize lactate arising from anaerobic glucose metabolism in muscle
tissue to perform gluconeogenesis. This process is known as Cori-cycle and can
also take place in the kidney (1). Gluconeogenic enzymes are highly abundant in
the cells of the proximal tubule (27, 40) and uptake and metabolic experiments
have identified glutamine and lactate as major substrates for renal
gluconeogenesis (8,15). Conversely, lactate can serve as a substrate for further
oxidation and thus, energy production in cells that are not in an anaerobic state.
Lactate utilization supports renal transport activity and appears to be stimulated
during alkalosis and reduced during acidosis (3). Depending on the energy and
oxidative capacity of a cell, lactate can either be extruded from the cytosol or
inversely, taken up by the cell. Lactate and other monocarboxylates are
transported by transport proteins belonging to two families of the Solute Carrier
(SLC) family, SLC5 (24, 44) and SLC16 (28). Two members of the SLC5 family,
namely SLC5A8 (SMCT1) and SLC5A12 (SMCT2) were recently reported to be
expressed in apical membranes of proximal tubule cells, suggesting to facilitate the
uptake of lactate from the urinary ultrafiltrate (26). Deletion of both transport genes
in a double knockout mouse model resulted in a 29-fold increase in urinary lactate
levels, indicating that the transporters are responsible for the major reabsorption of
lactate (45). Similar results were obtained with Slc5a8 knockout mice, where
lactate levels in urine and saliva were significantly increased due to lactate
reabsorption deficiency (19). The SLC16 family of monocarboxylate transporters
comprises of at least 14 members (28). However, the function, transport mode, and
exact tissue distribution of several members have not been elucidated to date.
MCT1 (SLC16A1), MCT2 (SLC16A7), MCT3 (SLC16A8), and MCT4 (SLC16A3)
have been demonstrated to be genuine proton-driven monocarboxylate
transporters. MCT1 has been shown to be present in basolateral membranes of
proximal tubule cells (18), and its transport function has been investigated
intensively (13, 14, 48). In addition, MCT1 and MCT2 have been shown to be
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present in the same tissues but in different cell types (22, 30, 37). In brain tissue, a
potential lactate and pyruvate shuttling mechanism between MCT1 in neurons and
MCT2 in astrocytes was demonstrated (12, 13, 16, 42, 46). The MCT4 isoform is
predominantly expressed in skeletal muscle. Both, MCT1 and MCT4 interact with
CD147 (also known as basigin or OX-47) which may act as chaperone enhancing
membrane expression and transport activity (28). Two other members of the
SLC16 family are atypical transporters. MCT8 (SLC16A2) does not transport
monocarboxylates, instead it is an efficient transporter for thyroid hormone
isoforms T3 and T4 (20). Mutations of SLC16A2 cause X-linked mental retardation
(17, 21). TAT1 (SLC16A10) has been identified as a transporter for aromatic amino
acids, independent from proton transport and not accepting monocarboxylates
(31). The transport substrate, mode, and tissue expression of the other SLC16
family members have not been elucidated to date. However, in a previous study
investigating changes in mouse kidney transcriptome we detected mRNA for
several members of the SLC16 family under basal conditions: Slc16a2 (MCT8),
Slc16a4 (MCT5), Slc16a9 (MCT9), and CD147 with high mRNA abundance,
Slc16a6 (MCT7), Slc16a13 (MCT13), and Slc16a14 (MCT14), with intermediate
mRNA levels, and lower mRNA levels for Slc16a1 (MCT1), Slc16a5 (MCT6),
Slc16a7 (MCT2), Slc16a10 (TAT1), and Slc16a11 (MCT11) (36). Metabolic acidosis
involves many adaptative and compensatory mechanisms. Previously, we had
performed a transcriptome analysis of mouse kidney and detected massive
changes in mRNA expression levels of genes involved in ammoniagenesis,
gluconeogenesis, Krebs cycle, oxidative phosphorylation an many transport
proteins (36). Among the mRNAs highly altered were several members of the
SLC5 and SLC16 families including MCT1, MCT7, MCT8, MCT9, SMCT1 and
SMCT2 (36). Thus, the aim of the present study was to elucidate the localization of
several members of monocarboxylate transporters in mouse and rat kidney and to
investigate their regulation during metabolic acidosis.
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CHAPTER 1
THE KIDNEY’S MICROSCOPY
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THE KIDNEY’S MICROSCOPY
Organization of the Renal Vasculature
The kidneys are paired organs, which have the production of urine as their primary
function. Kidneys are seen in many types of animals, including vertebrates and
some invertebrates. They are part of the urinary system, but have several
secondary functions concerned with homeostatic functions. These include the
regulation of electrolytes, acid-base balance, and blood pressure. In producing
urine, the kidneys excrete wastes such as urea and ammonium; they are also
responsible for the reabsorption of glucose and amino acids. Finally, they are
important in the production of hormones including vitamin D, renin and
erythropoietin. Each kidney has concave and convex surfaces. The concave
surface, the renal hilum, is the point at which the renal artery enters the organ, and
the renal vein and ureter leaves. The kidney is surrounded by tough fibrous tissue,
the renal capsule, which is itself surrounded by perinephric fat, renal fascia (of
Gerota), and paranephric fat. The anterior (front) border of these tissues is the
peritoneum, while the posterior (rear) border is the transversalis fascia. The
substance, or parenchyma, of the kidney is divided into two major structures:
superficial is the renal cortex and deep is the renal medulla. Grossly, these
structures take the shape of 8 to 18 cone-shaped renal lobes, each containing
renal cortex surrounding a portion of medulla called a renal pyramid (of Malphigi).
Between renal pyramids, which are composed of medulla, are projections of cortex
called renal columns (of Bertin). Nephrons, the urine-producing functional
structures of the kidney, span the cortex and medulla (fig1). The initial filtering
portions of the nephron, the renal corpuscles, are located in the cortex and each
sends a renal tubule that passes from the cortex deep into the medullary pyramids.
As part of the renal cortex, and medullary ray there is a collection of renal tubules
that drain into a single collecting duct. The tip, or papilla, of each pyramid empties
urine into a minor calyx, minor calyces empty into major calyces, and major
calyces empty into the renal pelvis. Along the length of the nephron in each of
these, there are six morphologically distinguishable segments, each occurring at a
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particular level in the cortex or medulla. The epithelium lining of each segment has
a characteristic microscopic structure related to its specialization for a specific
function in the formation of urine. Traditionally, only four segments of the renal
tubule were recognized, namely, the proximal convolution, the loop of Henle, the
distal convolution, and the collecting duct. Each of these is further subdivided into
two or more segments. At the proximal end of each nephron, there is a closed,
thinwalled expansion of the tubule that is deeply invaginated to form a cup-shaped
hollow structure called Bowman’s capsule. The concavity of this blind end of the
nephron is occupied by a globular tuft of highly convoluted capillaries, the
glomerulus. Two segments of the proximal convoluted tubule (PCT pars convoluta),
situated in the cortex, and the proximal straight tubule (PST pars recta), extending
from the cortex into the outer stripe of the medulla. This is followed by the
intermediate tubule which is subdivided into the descending thin limb (DTL pars
descendens), traversing the inner stripe of the outer medulla and extending deep
into the inner medulla, and a recurrent portion, the ascending thin limb (ATL pars
ascendens). At the junction of the inner and outer medulla, the ascending limb is
continuous with the distal straight tubule (DST thick ascending limb) which
traverses the outer medulla and continues into the cortex, where it becomes the
distal convoluted tubule (DCT). In the cortex, the distal convoluted tubule is joined
by a connecting tubule (CT), to a collecting duct (CD), which passes downward
through the cortex and medulla to the area cribrosa of the renal papilla, where it
opens into a minor calyx. The portion of the nephron, traditionally called the loop of
Henle, includes the segments now called the thick descending limb of the proximal
tubule, the thin descending and ascending limbs of the intermediate tubule, and the
thick ascending limb of the distal tubule.
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