Download Lipid-binding proteins in rat and human kidney

Kidney International, Vol. 56, Suppl. 71 (1999), pp. S-159–S-162
Lipid-binding proteins in rat and human kidney
HIDEKI KIMURA, HIROSHI FUJII, SATORU SUZUKI, TERUO ONO, MASAAKI ARAKAWA,
and FUMITAKE GEJYO
Department of Clinical and Laboratory Medicine, Faculty of Medicine, Fukui Medical University, Fukui, Japan,
Department of Biochemistry, Niigata University School of Medicine, Niigata, Japan, and Departments of Medicine (II),
Niigata University School of Medicine, Niigata, Japan
Lipid-binding proteins are expressed in the glomeruli and cultured mesangial cells of rat and human kidneys.
Background. The kidney metabolizes actively lipophilic molecules. Several species of lipid-binding proteins (LBPs) have
been well characterized, including fatty acid-binding proteins
(FABPs), acyl-CoA binding protein (ACBP), sterol carrier
protein 2 (SCP2), cellular retinol binding protein (CRBP), and
phosphatidylinositol transfer protein (PITP).
Methods. To clarify which LBPs are expressed in isolated
rat glomeruli (RG), cultured rat mesangial cells (RMC) and
human kidney, RT-PCR, immunoblot analysis and immunohistochemistry were performed.
Results. Protein and mRNA expression of heart type (H-)
FABP was found in RMC, but not in RG. Immunohistochemistry using antihuman H-FABP antibody revealed that an
H-FABP like protein was present in the capillary wall and
distal tubules of human glomeruli. Immunoblot analysis using
the antibody showed that a 110-kDa protein related to
H-FABP was present in human isolated glomeruli but not in
any other tissues tested including blood, liver, and heart, and
that the 14-kDa protein, H-FABP itself was localized in the
distal tubules of human kidney. mRNA for SCP2, ACBP and
PITP was detected in RG and RMC. CRBP mRNA was detected in RG but not RMC.
Conclusions. A variety of lipid-binding proteins are present
in rat glomeruli. In human glomeruli, a novel 110-kDa H-FABPrelated protein is localized specifically in the capillary wall.
The kidney is one of the organs that most actively
metabolize hydrophobic ligands. Partial segments of
nephrons rapidly uptake long-chain fatty acids from
blood stream, synthesize prostanoids from arachidonic
acid in response to humorous factors, and modulate renal
functions [1]. Diet-induced or endogenous hyperlipidemia in animal models of glomerular injury results in
accelerating the progression of glomerulosclerosis [2]. A
recent report of O’Donnell and coworkers [3] showed
that mesangial cell proliferation is mediated by isoprenoid products of the mevalonate pathway where cholesKey words: fatty acid-binding protein, sterol carrier protein, acyl-CoA
binding protein, cellular retinol binding protein, phosphatidylinositol
transfer protein, glomeruli.
 1999 by the International Society of Nephrology
terol is endogenously synthesized. Lipid-binding proteins (LBPs), cytosolic proteins with low molecular
masses (10–20 kDa), are capable of binding lipophilic
molecules and are thought to play important roles in cell
signaling pathways involving lipophilic molecules as well
as intracellular trafficking and metabolism of lipids [4,5].
The family of LBP mainly contains fatty acid binding
protein (FABP), acyl-CoA binding protein (ACBP), sterol carrier protein 2 (SCP2), cellular retinol binding protein (CRBP) and phosphatidylinositol transfer protein
(PITP). Recently, we reported that heart type (H-)
FABP was present in distal tubules of rat kidney [6] and
that H-FABP like protein was distributed in glomerular
capillary walls as well as distal tubules of human kidney
[7]. However, whether other LBPs are present in glomeruli remains unknown.
METHODS
Normal glomeruli and tubules of rat and human kidneys were isolated by the mesh sieving technique. Rat
mesangial cell (RMC) strains were isolated from male
WKY rats. Cell passages 3 through 6 were used. Polyclonal antibodies against human H-FABP (HFPoA) and
rat H-FABP were prepared by subcutaneous immunization of rabbit as described previously [6]. After Tricine
glycine SDS-PAGE on polyacrylamide (10%) slab gels,
immunoblot analysis was performed with a semidry
transfer system (Bio-Rad, USA) and an ECL detection
kit, (Amersham, Buckinghamshire, UK).
Analysis of LBP gene expression
Expression of rat H-FABP mRNA was examined by
Northern blot analysis using rat H-FABP cDNA as a
probe. Gene expression of other LBPs were examined
with reverse transcriptase-polymerase chain reaction using specific primers.
Immunohistochemistry
Sections of frozen tissues (1 mm thickness) were stained
by indirect immunofluorescence. Sections of paraffinembedded specimens (2 mm thickness) were examined
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Kimura et al: Lipid-binding proteins in the kidney
Fig. 1. Immunolight micrographs of human kidney with polyclonal antibody against human heart-type fatty acid-binding protein. (A) In glomeruli,
the immunostaining is found in the capillary walls but not in the mesangial area. Frozen sections were not counterstained with hematoxylin and
eosin. (B) In interstitium, the staining is observed in the distal tubules. Sections from the paraffin-embedded specimens were counterstained with
hematoxylin and eosin. Magnification: (A) 3400; (B) 3200.
Kimura et al: Lipid-binding proteins in the kidney
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Fig. 2. Immunoblot analysis of different tissue homogenates probed with polyclonal antibody against human heart-type fatty acid-binding protein.
Several species of human tissues were obtained from autopsy samples from a single patient. Homogenates (30 mg) of the tissues and a blood
sample (30 mg) were subjected to immunoblot analysis. Lane 1, isolated glomeruli; lane 2, heart; lane 3, isolated renal tubules; lane 4, blood; lane
5, adrenal tissues; lane 6, small intestine; lane 7, lung; lane 8, spleen.
by the streptavidin-biotin-complex/horseradish peroxidase method.
of CRBP mRNA was detected in rat liver and RG, but
not RMC (data not shown).
RESULTS
H-FABP and its related protein
Expression of H-FABP protein and mRNA was found
in RMC, but not in rat glomeruli (data not shown). Immunolight micrography of normal human kidney showed
that the specific staining for H-FABP was observed exclusively in the capillary walls of glomeruli (Fig. 1A)
and in the distal tubules of the interstitium (Fig. 1B).
Immunoblot analysis using HFPoA showed that the immunoreactive proteins described above displayed molecular masses of 110 kDa in the glomerular homogenate
and 14 kDa in the tubular homogenate (Fig. 2). Preabsorption of HFPoA by purified H-FABP at the molar
ratio of antigen to antibody [50/1] diminished equally
the immunoreactivities of both the 110- kDa protein in
glomeruli and purified human H-FABP (data not shown),
demonstrating that the two proteins shared a common
antigen. This unique 110-kDa protein was not able to be
immunochemically detected in homogenates from any
other tissues examined, including liver, heart, skeletal
muscle, adrenal gland, adipose tissue and small intestine
(Fig. 2), although the 14-kDa protein was detected in
heart and skeletal muscle tissue.
DISCUSSION
Other lipid-binding proteins
Expression of mRNA for ACBP, SCP2 and PITP was
detected in rat liver, RG, and RMC, while expression
In the present study, we showed for the first time that
mRNA for H-FABP, SCP2, ACBP, CRBP, and PITP is
expressed variably in rat glomeruli or RCM. Because
lipophilic molecules, such as retinoic acid, and phosphatidylinositol are known to be elements of several cell-tocell signaling pathways, these LBPs may modulate lipidmediated signal transduction in mesangial cells and
glomeruli [5]. PITP is a requisite component for epidermal growth factor signaling [8], while CRBP and retinoids probably participate in the process of smooth muscle cell activation [9]. FABP may modulate the activity
of peroxisome proliferator-activated receptors owing to
its affinity for long-chain fatty acids, putative ligands
for the receptors, and thereby may influence the gene
expression of other LBPs. However, whether or not
some pathogenetic relationships between FABP and
other LBPs exist in progressive renal disease remains to
be clarified.
We also obtained the interesting finding that a 110kDa protein, immunochemically related to human
H-FABP, is confined to the capillary wall in human glomeruli, while H-FABP itself, (14 kDa) was localized
exclusively to the distal tubules, from the thick ascending
loop of Henle to collecting ducts. The distribution of
H-FABP (14 kDa) in the human kidney is consistent
with our previous report that H-FABP is present in the
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Kimura et al: Lipid-binding proteins in the kidney
distal tubules of the rat kidney [6]. In the distal tubules,
mitochondrial b-oxidation of long-chain fatty acids is
predominant and the high rate in prostaglandin synthesis
is observed. It is intriguing that a 100-kDa ion channel
in neuron called the NMDA1 receptor had about 30%
homology with H-FABP in a putative fatty acid-binding
domain, residue 263–393 [10], although the detailed molecular nature of the 110-kDa H-FABP-related protein
remains unknown. Therefore, The H-FABP in the distal
tubules may be involved in lipid metabolism such as
intracellular trafficking and b-oxidation of long-chain
fatty acids and prostaglandin synthesis, whereas the 110
kDa H-FABP related protein in the glomerular capillary
wall may serve as a receptor that is activated by fatty
acids, such as the NMDA-1 receptor. Additional studies
will be required to characterize the 110 kDa protein and
to clarify pathogenetic roles of LBPs in renal disease.
In conclusion, many species of LBPs are present in
rat glomeruli and cultured mesangial cells. In the human
kidney, a novel 110-kDa H-FABP-related protein is
present only in the glomerular capillary wall, while
H-FABP is distributed in the distal tubules.
ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid for Scientific
Research 09671159 from the Ministry of Education, Science, and Culture of Japan. The skillful technical assistance of Drs. Naofumi Imai
and Hirokazu Sato is highly appreciated.
Reprint requests to Hideki Kimura, M.D., Department of Clinical and
Laboratory Medicine, Faculty of Medicine, Fukui Medical University, 23
Shimoaitsuki, Matsuoka, Yoshida, Fukui 910-1193, Japan.
E-mail: [email protected]
REFERENCES
1. Breyer MD, Badr KF: Arachidonic acid metabolites and the kidney. In: The Kidney, 5th, ed., edited by Brenner BM, Philadelphia,
WB Saunders, 1996, pp. 754–788.
2. Schmitz PG, Kasiske B, O’Donnell MP, Keane WF: Lipid and
progressive renal injury. Seminars Nephrol 9:354–369, 1989.
3. O’Donnell MP, Kasiske BL, Atluru D, Kim Y, Keane WF:
Mesangial cell proliferation is dependent on isoprenoid products
of mevalonate metabolism. J Clin Invest 91:83–87, 1993.
4. Peeters RA, Veerkamp JH, Demel RA. Biochim Biophys Acta
1002:8–13, 1989.
5. Glatz JFC, Borcher T, Spener F, Van der vusse GJ: Fatty acids in
cell signaling: modulation by lipid binding proteins. Prostaglandins
Leukot Essent Fatty Acids 52:121–127, 1995.
6. Kimura H, Odani S, Nishi S, Sato H, Arakawa M, Ono T: Primary
structure and cellular distribution of two fatty acid binding proteins
in adult rat kidneys. J Biol Chem 266:5963–5972, 1991.
7. Kimura H, Fujii H, Nishi S, Nakagawa Y, Ono T, Arakawa M:
Purification and localization of two fatty acid-binding proteins in
the human kidney [Abstract]. J Am Soc Nephrol 2:763, 1991.
8. Kauffmann-zeh A, Thomas GMH, Ball A, Prosser S, Cunningham E, Cockcroft S, Hsuan JJ: Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling. Sci
268:1188–1190, 1995.
9. Neuville P, Geinoz A, Benzonana G, Redard M, Gabbiani
F, Ropraz P, Gabbiani G: Cellular retinol-binding protein 1 is
expressed by distinct subsets of rat arterial smooth muscle cells in
vitro and in vivo. Am J Pathol 150:509–521, 1997.
10. Petrou S, Ordway RW, Singer JJ, Walsh JV Jr: A putative fatty
acid-binding domain of the NMDA receptor. Trends Biosci:41–42,
1993