It C

Identification of a renal-specific oxido-reductase in

newborn diabetic mice

Qiwei Yang*, Bharat Dixit, Jun Wada*, Yufeng Tian*, Elisabeth I. Wallner*, Satish K. Srivastva,

and Yashpal S. Kanwar*

*Department of Pathology, Northwestern University Medical School, Chicago, IL 60611; and Department of Biochemistry, University of Texas Medical

Branch, Galveston, TX 77555

Communicated by Emanuel Margoliash, University of Illinois, Chicago, IL, June 8, 2000 (received for review February 23, 2000)

generated from sorbitol because the synthesis of these ECM

Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase

proteins can be normalized by the use of an ALR2 inhibitor,

(AKR), is widely distributed in mammalian tissues and has been

sorbinil (8, 9). The linkage of these two pathways also is

implicated in complications of diabetes, including diabetic ne-

supported by studies that showed ALR2 to have a high substrate

phropathy. To identify a renal-speci c reductase belonging to the

affinity for glycated methylglyoxal (10). Because of the original

AKR family, representational difference analyses of cDNA from

description of ALR2, a number of aldo-keto reductase (AKR)

diabetic mouse kidney were performed. A full-length cDNA with an

family members have been discovered in various mammalian

ORF of 855 nt and yielding a 1.5-kb mRNA transcript was isolated

tissues (11). So far, among this superfamily, there is no known

from a mouse kidney library. Human and rat homologues also were

renal-specific reductase that has been described and can be

isolated, and they had 91% and 97% amino acid identity with

linked to hyperglycemia in diabetes mellitus. In view of the above

mouse protein. In vitro translation of the cDNA yielded a protein

considerations, we initiated studies to search for such a reduc-

product of 33 kDa. Northern and Western blot analyses, using the

tase, using representational difference analysis (RDA) of cDNA

cDNA and antirecombinant protein antibody, revealed its expres-

(12), in the kidneys of streptozotocin-induced diabetes in new-

sion exclusively con ned to the kidney. Like ALR2, the expression

born mice. A number of cDNA fragments were isolated that had

was up-regulated in diabetic kidneys. Its mRNA and protein ex-

up-regulated mRNA expression. In this communication, we

pression was restricted to renal proximal tubules. The gene neither

describe the identification of a kidney-specific oxido-reductase

codistributed with Tamm Horsfall protein nor aquaporin-2. The

that is up-regulated in the hyperglycemic state.

deduced protein sequence revealed an AKR-3 motif located near

the N terminus, unlike the other AKR family members where it is

Materials and Methods

con ned to the C terminus. Fluorescence quenching and reactive

Induction of Diabetes in Newborn Mice. Hyperglycemic state was

blue agarose chromatography studies revealed that it binds to

induced in ICR newborn mice (Harlan) by an i.p. injection of

NADPH with high af nity (KdNADPH 66.9 2.3 nM). This binding

streptozotocin (200 mg kg of weight) in citrate buffer. Control

domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the

mice received buffer only. After 3 weeks, kidneys of mice with

AKR-3 motif that is similar to the other AKR members. The iden-

blood glucose levels 250 mg dl were harvested and snap-

ti ed protein is designated as RSOR because it is renal-speci c with

frozen in liquid nitrogen, and total RNA was extracted.

properties of an oxido-reductase, and like ALR2 it may be relevant

in the renal complications of diabetes mellitus.

RDA. The method of cDNA-RDA was used to isolate glucose-

induced genes in the kidneys of diabetic mice (DM) (12). Briefly,

diabetes mellitus diabetic nephropathy

total RNA from normal mouse (NM) and DM kidneys was

isolated, poly(A) RNA was selected, and first- and second-

R enal complications are a common manifestation of diabetes

strand cDNAs were synthesized. The double-stranded cDNAs

mellitus. Characteristics of these complications are an in-

were subjected to subtractive hybridization, where the NM

crease of extracellular matrix (ECM) proteins, i.e., type I and

kidney cDNA was used as the driver and that of the DM kidney

type IV collagens and decorin and fibronectin, synthesized by

as the tester. After three rounds of subtractive hybridization, the

glomerular, tubular, and interstitial cells (1). The increase in

difference products (DPs) were isolated and analyzed by 2%

ECM may be multifactorial, but recent studies have narrowed it

agarose gel electrophoresis. After removal of the linkers by

down to two or three pathogenetic mechanisms that are affected

digestion with DpnII, they were ligated into BamHI-digested

by hyperglycemia. The hyperglycemia may increase the mRNA

pBluescript KS (Stratagene). After transformation, bacterial

expression and bioactivity of certain cytokines that modulate the

colonies were picked to prepare plasmid DNAs for nucleotide

synthesis of various ECM proteins, e.g., transforming growth

sequencing. The sequence of various DPs was subjected to

factor (2, 3). Nonenzymatic glycation is another mechanism by

homology search by the BLAST program via the National Center

which various Amadori intermediaries lead to the generation of

advanced glycation products (AGEs). The AGEs further cross-

link the glycated proteins with one another and render them

Abbreviations: AGE, advanced glycation product; AKR, aldo-keto reductase; ALR1, alde-

extremely resistant to proteolytic degradation, resulting in an hyde reductase; ALR2, aldose reductase; DM, diabetic mouse; DP, difference product; ECM,

accumulation of ECM in the kidney (4). Such an AGE-mediated extracellular matrix; NC, nitrocellulose; NM, normal mouse; RBG, reactive blue agarose;

cross-linking process is not restricted to the kidney tissue pro- RDA, representational difference analysis; RSOR, renal-speci c oxido-reductase.

teins alone, but it affects other tissue proteins as well, e.g., ocular Data deposition: The sequences reported in this paper have been deposited in the GenBank

lens crystallins (5, 6). Another mechanism that is also relevant database (accession nos. AF197127, AF197128, and AF197129).

to diabetic nephropathy is the polyol pathway, which consists of To whom reprint requests should be addressed at: Department of Pathology, Northwest-

two major reactions. First, glucose is reduced by aldose reduc- ern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail:

*-******@***.***.

tase (ALR2) to sorbitol by using NADPH as the hydrogen donor.

The publication costs of this article were defrayed in part by page charge payment. This

The sorbitol then is oxidized by sorbitol dehydrogenase to article must therefore be hereby marked advertisement in accordance with 18 U.S.C.

fructose by using NAD as the hydrogen acceptor (7). Conceiv- 1734 solely to indicate this fact.

ably, there is a link between the polyol pathway and the glycation Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073 pnas.160266197.

of collagens and fibronectin by excessive amounts of fructose Article and publication date are at www.pnas.org cgi doi 10.1073 pnas.160266197

9896 9901 PNAS August 29, 2000 vol. 97 no. 18

for Biotechnology Information on-line service. The DNA frag- Expression of fusion proteins was induced by the addition of 1

ments of the DPs that were novel and differentially regulated and mM isopropyl-1-thio- -D-galactopyranoside in the medium, and

had definitive mRNA transcripts by Northern blot analysis were the culture was extended for another 3 h. The cells were

further investigated. harvested by centrifugation at 5,000 g, and the pellet was

resuspended in an ice-cold binding buffer (5 mM imidazole 0.5

M NaCl 20 mM Tris HCl, pH 7.9) and sonicated, and then the

cDNA Library Screening, Isolation of Full-Length cDNA, and Nucleotide

Sequencing. A DP with 130 bp that yielded mRNA transcript of lysate was centrifuged at 39,000 g. The supernatant was loaded

1.5 kb was further characterized and was used for screening a onto a nickel-charged column. The column was successively

mouse kidney library (13). Nitrocellulose (NC) filter lifts were washed with the binding buffer and a washing buffer (60 mM

made, prehybridized, and hybridized with the radiolabeled imidazole 0.5 M NaCl 20 mM Tris Cl, pH 7.9). The bound

screening probe. The cDNA clones with overlapping sequences proteins were eluted with an elution buffer (1 M imidazole 0.5

were isolated and subcloned into pBluescript II KS (Strat- M NaCl 20 mM Tris Cl, pH 7.9). The fractions with high

agene), and nucleotide sequencing, homology, and protein content of proteins were pooled and dialyzed against 0.1 M PBS

structural analyses were performed. Rat and human kidney and deionized water. The purity of the isolated fusion protein

cDNA libraries (Stratagene) were screened to isolate mouse was assessed by SDS PAGE analysis.

homologues.

Generation and Characterization of the Antibody. The fusion protein

Northern and Southern Blot Analyses. For Northern analyses, 30 g was used for raising polyclonal antibody in rabbits (14). For

of total RNAs extracted from NM and DM kidneys was glyoxy- Western blot analyses, protein lysates were prepared by homog-

lated, subjected to 1% agarose gel electrophoresis, and capillary- enizing various mouse tissues in ice-cold extraction buffer (10

transferred to nylon membranes. The membrane blots were mM Hepes 1.5 mM MgCl2 10 mM KCl 0.5 mM DTT 1 mM

hybridized with -32P-dCTP-labeled full-length human or mouse PMSF, pH 7.4). The nuclei were pelleted by centrifuging the

cDNA as isolated above. The blots were washed and hybridized homogenate at 3,000 g at 4 C. The concentration of the protein

under high stringency conditions, and autoradiograms were in the supernatant was adjusted to 20 mg ml, followed by 10%

prepared (13). In addition, RNAs isolated from various mouse SDS PAGE under reducing conditions. The gel proteins were

tissues and kidneys of rat and human were processed for electroblotted onto a NC membrane (14). The membrane blot

Northern blot analyses. For Southern blot analyses, kidney was immersed in a blocking solution containing 5% nonfat milk

tissues from adult mouse, rat, and human were obtained, and and Triton X-100 TBS (0.5% Triton X-100 0.1 M Tris 0.15 M

genomic DNA was isolated (14). The DNA (10 g) was subjected NaCl, pH 7.5). This was followed by successive incubations, 60

to a 0.8% agarose gel electrophoresis after digestion with various min each, with the polyclonal antifusion protein antibody (1:100

restriction endonucleases, which included EcoRI, XbaI, PstI, dilution) and anti-rabbit IgG conjugated with horseradish per-

BamHI, HindIII, and SalI. The gels were treated with 0.2 M HCl, oxidase (1:1,000 dilution) at 37 C. The blot was washed three

BIOCHEMISTRY

followed by successive treatments with denaturing and neutral- times with Triton X-100 TBS and immersed in a chemilumines-

izing solutions (14). Duplicate blots were prepared by transfer- cent reagent solution (Bio-Rad), and autoradiograms were

ring the digested DNAs onto nylon membranes. The blots developed. The analyses also were carried out on blots with the

were hybridized either with the -32P-dCTP-radiolabeled 5 intact fusion protein and another aliquot that had been absorbed

end-specific or 3 end-specific probes under high stringency with the antibody.

conditions.

Tissue Expression Studies. For in situ hybridization studies, the

In Vitro Translation Studies. The studies were performed to con- full-length cDNA clone was selected as a template to generate

-33P-UTP-labeled sense and antisense riboprobes by using the

firm the ORF of the mouse cDNA clones and to verify the size

of the putative protein product. Two full-length cDNAs were Riboprobe in vitro Transcription System (Promega). The ribo-

selected as the template. They were subcloned into pCR2.1 probes then were used for in situ hybridization with the mouse

vector (Invitrogen) by using PCR and sense (5 -CATGTCTT- kidney sections (13). For protein expression, 4- m-thick cryostat

TCATTCTTTATTGATACCCAGC-3 ) and antisense (5 - kidney sections were prepared, incubated with the polyclonal

TTGCTCCCTCAGGATGAAGG-3 ) primers. A TNT-coupled antifusion protein antibody, and reincubated with goat anti-

reticulocyte lysate in vitro translation system was used (Pro- rabbit IgG antibody conjugated with FISH, and then examined

mega), and the translation was carried out in the presence of with an UV light microscope. To delineate a definitive spatial

[35S]methionine and T7 RNA polymerase. The reaction products distribution of the fusion protein in the kidney, its expression

were subjected to 10% SDS PAGE, and autoradiograms were relative to other tubular proteins, i.e., Tamm Horsfall protein

prepared. A positive control included luciferase-encoding plas- and aquaporin-2, was investigated. Tamm Horsfall and aqua-

mid that yields a translated product of 61 kDa. porin-2 are markers of distal tubular and collecting duct epithe-

lia, respectively (15, 16). Serial cryostat tissue sections were

Isolation of the Fusion Protein. Two expression constructs were prepared and stained either with anti-Tamm Horsfall (ICN) and

generated by PCR using full-length cDNA isolated from the antifusion protein or antiaquaporin-2 (a gift from Mark Knep-

mouse kidney library. A XhoI site (CTCGAG) was introduced by per, National Institutes of Health, Bethesda, MD) and antifusion

using sense primer 5 -GGGGGGCTCGAGATGAAGGTC- protein. The sections then were stained with secondary antibod-

GATGTGG-3 and antisense primer 5 -GGGGGGCTCGAGT- ies that were conjugated with fluorescein or rhodamine and

CACCAGCTCAGGGTGCCG-3 . Flanking GC clamps examined.

(GGGGGG) also were included into the primer sequences. The

PCR-amplified products were digested with XhoI, agarose gel- Characterization of the Fusion Protein. Initially, immunologic cross-

purified, and ligated into the XhoI-digested pET-15b vector reactivity of the fusion protein with other enzyme proteins

carrying an N-terminal His Tag sequence (Novagen). The con- [aldehyde reductase (ALR1) and ALR2] that may be involved in

structs were sequenced to ensure proper in-frame ligation, Taq hyperglycemic state was investigated by dot blot method. One

polymerase fidelity, and their 5 and 3 end orientation. Trans- microgram of recombinant ALR1 or ALR2 (17, 18) was applied

formation was performed by using bacterial host BL21(DE3) onto a NC paper and air-dried. After blocking with 5% fat-free

(Novagen). A single colony was picked, and the bacteria were milk solution, the NC paper was incubated with antifusion

allowed to grow in LB medium until an A600 of 0.6 was achieved. protein antibody. The NC paper was washed with PBS contain-

Yang et al. PNAS August 29, 2000 vol. 97 no. 18 9897

ing 0.5% Tween 20 and incubated with goat anti-rabbit IgG

conjugated with alkaline phosphatase. The NC paper was re-

washed, and color development was performed by using AP kit

(Bio-Rad). BSA was used as a negative control. A mild reactivity

of the antibody with the AKR suggested that the isolated fusion

protein might belong to the family of AKRs that contain

NADPH-binding domain(s). This led us to investigate its affinity

characteristics for NADPH by fluorescence spectrophotometry.

All fluorescence titrations were performed at 25 C in 50 mM

potassium phosphate, pH 7.0 by using an excitation wavelength

of 280 nm and emission wavelength of 340 nm. Various amounts

of protein were titrated in 1.8 ml of assay buffer with 2- to 10- l

additions of NADPH of known concentration. A control titra-

tion without the enzyme in the same volume of buffer was carried

out to correct for nonenzymatic changes in the fluorescence of

NADPH. Assuming that the fluorescence quench is directly

proportional to the protein coenzyme complex, the molar frac-

tion of the protein bound at each concentration of NADPH is

given by: [E.NADPH] [E]t F Fmax, where Fmax is the

maximal fluorescence change with complete saturation, F is the

observed fluorescence change and [E]t is the total protein

concentration. Fmax can be calculated from a linear double-

reciprocal plot (1 f luorescence change versus1 [NADPH])

and the dissociation constant (Kd) is calculated from the replot

of 1 (1 ) versus [NADPH]total according to Ward s

method (19).

To confirm the NADPH binding characteristics of the fusion Fig. 1. (A C) Northern blot analyses of RSOR in kidneys of control (NM) and

protein and its immunologic cross-reactivity with other reduc- DM. RSOR is up-regulated in DM kidneys (A). The -actin expression is unaf-

tases, i.e., ALR1 and ALR2, Western blot analyses were per- fected (C). B is the same blot stained with methylene blue, and it shows

formed. In addition, NADPH binding characteristics of proteins intactness of the RNAs and their loading of equal amounts in various lanes. (D)

present in the kidney cortex were investigated. Mouse kidney Alignment of the amino acid sequences of mouse, rat, and human RSOR. Dots

cortices were homogenized in 0.1 M potassium phosphate buffer indicate the identity of the amino acids. Mouse RSOR has 97% and 91%

sequence homology with rat and human forms, respectively. Arrows indicate

containing 1 mM EDTA, pH 7.0. The extract was centrifuged at

N and C terminals of the RSOR. * indicates glycosylation site. MAKSKDSFR-

13,000 g, and the supernatant was passed through a reactive

NYTSGPL, AKR motif.

blue agarose (RBG) column (Sigma) equilibrated with the

phosphate buffer. The column was washed with the same buffer

and eluted with 1 mM NADPH in the buffer solution. The the nonmammalian proteins isolated from Arabidopsis thaliana

fractions were pooled and concentrated. Aliquots of the super- and Pinus radiata. Among the homologues that were isolated

natant of the extract, eluant, fusion protein, ALR1, ALR2, and from the cDNA libraries (Fig. 1D), it had 97% and 91%

BSA were subjected to SDS PAGE after adjusting the protein amino acid sequence homology with rat and human, respectively.

concentration. The proteins were electroblotted onto a NC In the mouse cDNA, an eukaryotic consensus polyadenylation

membrane, and Western blotting was performed as described site (AATAAA) was present at positions 142 147 downstream

above by using antifusion protein antibody. from the termination codon, suggesting that the 3 untranslated

region of the cDNA is virtually complete.

Results

Isolation and Characterization of Differentially Expressed Genes in

Northern Blot and Southern Blot Analyses. Using the mouse RSOR

Streptozotocin-Induced DM Kidney. Several DNAs that were dif-

cDNA as a probe, a single mRNA transcript of 1.5 kb was

ferentially expressed by cDNA-RDA were identified, and one of

observed in kidneys of various species (Fig. 2A). The blot stained

them (130 bp) yielded a mRNA transcript of 1.5 kb (Fig. 1A)

with methylene blue (Fig. 2C) and hybridized with -actin probe

Compared with control, its expression was up-regulated in

(Fig. 2D) indicated equal amounts of RNA loading in various

several different DM kidneys (Fig. 1 A). The amount of total

lanes; however, the hybridization signal was weak in human

RNA loaded (30 g) in each lane was comparable, and that is

kidney RNA. This weak signal may be caused by the use of the

reflected in the same blot stained with methylene blue (Fig. 1B).

mouse cDNA probe and suggested that there may be differences

The mRNA expression of -actin was similar in DM and NM

among the various species in the genomic organization of the

kidneys (Fig. 1C). Using the 130-bp DNA difference product as

RSOR. This is conceivable because the blot hybridized with

a screening probe, several clones were isolated. Two clones had

human RSOR cDNA as a probe revealed a relatively weak signal

initiation and termination codons, and they contained an ORF

in mouse kidney RNA (Fig. 2 B). Southern blot analyses yielding

of 285 aa with a predicted putative protein product of 33 kDa.

different banding pattern of the DNA digested with restriction

The structural analyses of the protein revealed multiple phos-

endonucleases confirmed that there are significant differences in

phorylation sites and an N-terminal AKR motif, MAKSKDS-

the genomic organization of the RSOR among various species

FRNYTSGPL, encompassing 21 36 amino acid residues (Fig.

(Fig. 2 E J).

1D). A single potential N-glycosylation site was present, and

interestingly, it was located within this motif. The protein had a

high aspartic acid content with a predicted pI of 4.88. In view Verification of the RSOR cDNA and Characterization of the Fusion

Protein and the Antibody. The in vitro translation revealed a

of these characteristics, the protein was tentatively designated as

translated product of 33 kDa that corresponded to the ORF of

renal-specific oxido-reductase (RSOR), although it does not

the putative protein product. Because the translated product

have any homology with other members of the AKR family.

Intriguingly, it had homologous sequences in certain segments of from two different cDNA clones had identical molecular

9898 www.pnas.org Yang et al.

Fig. 4. (A) Northern blot of RNAs from various mouse tissues hybridized with

RSOR cDNA. A 1.5-kb mRNA transcript is observed only in the kidney. The

-actin mRNA expression is similar in all of the tissues (B). (C) Western blot of

protein extracts of various tissues hybridized with antifusion protein anti-

body. A 33-kDa band is seen exclusively in the kidney.

Fig. 2. (A D) Northern blot of kidney mRNA of normal mouse, rat, and

the various tissues examined (Fig. 4A). Similarly, Western blot

human, hybridized with mouse (A) and human (B) cDNA probes. A single

analyses also indicated a single band of 33 kDa confined only

transcript of 1.5 kb is observed in all of the species (A and B). Loading of equal

to the kidney (Fig. 4C). The in situ tissue autoradiograms

amount of RNAs of various species is re ected by the similar intensity of 28S

and 18S bands in the blot stained with methylene blue (C) and by comparable revealed that it is exclusively expressed in the tubular epithelium

-actin expression (D). (E J) Southern blots of genomic DNA of mouse, rat, and

of the renal cortex whereas it is absent in the glomeruli and

human, digested with restriction endonucleases and hybridized with 5 end-

medulla (Fig. 5 A C). To further delineate its expression in

speci c (E, G, and I) and 3 end-speci c (F, H, and J) RSOR cDNA probes. The

various renal tubular segments, immunofluorescence studies

banding pro le is variable, suggesting that the genomic organization of the

BIOCHEMISTRY

were performed on serial tissue sections stained with anti-

RSOR is different among the three species.

RSOR, anti-Tamm Horsfall, and antiaquaporin-2 antibodies. A

strong intracellular immunoreactivity was seen in the tubular

weights, it suggested that the isolated cDNA has a corresponding epithelia throughout the renal cortex of the kidney (Fig. 5 D F

authentic protein (Fig. 3A). Similarly, the presence of a 35-kDa and J L). The immunoreactivity of RSOR was absent in tubular

band in SDS PAGE of the fusion proteins from two different segments exhibiting immunoreactivity for Tamm Horsfall (Fig.

clones established the authenticity of the ROSR protein (Fig. 5 G I) and aquaporin-2 (Fig. 5 M O), suggesting that the RSOR

3B). The excess 2.5-kDa mass is caused by the addition of is expressed exclusively in the renal proximal tubular epithelium.

c-myc-(His)6-tag. The detection of a single 35-kDa band by

Western blot analyses confirmed identity of the protein and Functional Characterization of the Fusion Protein. An increasing

specificity of the polyclonal antibody (Fig. 3C). The specificity of degree of fluorescence quenching was observed with the addi-

the latter is further reflected by the reduction in the intensity of tion of increasing amounts of NADPH, and it is graphically

the band when the antibody-absorbed fusion protein was sub- depicted in the titration curve (Fig. 6A). These results suggest

jected to SDS PAGE (Fig. 3C, lane 2). that the fusion protein has a high affinity NADPH binding site

or domain with KdNADPH 66.9 2.3 nM.

Tissue Expression Analyses of RSOR. Northern blot analyses re-

The Western blot analysis, using antifusion protein antibody,

vealed a single 1.5-kb mRNA transcript in the kidney among

also indicated the presence of such an NADPH binding protein

in the kidney cortex because the eluant from the RBG column

yielded a band of 33 kDa (Fig. 6B, lane 4). The immunoreac-

tivity of the band was less compared with the whole homogenate

of the kidney cortex (Fig. 6B, lane 3), although comparable

amounts of protein were loaded for SDS PAGE analyses. It is

conceivable that exact protein concentration may be difficult to

assess by the Bio-Rad protein assay in the eluant from the RBG

column because protein is still bound to the NADPH. The fusion

protein band was 35 kDa, where 2.5 kDa is caused by the

presence of c-myc-(HIS)6-tag (Fig. 6B, lane 5). No immunore-

activity with the BSA (Fig. 6B, lane 6) and negligible reactivity

Fig. 3. (A) Pro les of in vitro-translated products. Two different cDNA clones

with the recombinant ALR2 (Fig. 6B, lane 2) were observed.

yield identical 33-kDa products (lanes 2 and 3). The 61-kDa product in lane

Interestingly, a mild immunoreactivity was detected with the

1 is generated from the control plasmid. (B) SDS PAGE of fusion proteins

generated from two different constructs. Identical 35-kDa bands are seen in recombinant ALR1 (Fig. 6B, lane 1), suggesting that polyclonal

gel stained with Coomassie blue. Additional 2.5 kDa is caused by the c-myc-

antifusion antibody has some cross-reactivity with other mem-

(His)6-tag in the fusion protein. (C) Western blot analyses of the fusion protein

bers of the AKR family. By densitometric analysis, the intensity

before (lane 1) and after (lane 2) absorption with the anti-RSOR antibody. The

of the recombinant ALR1 band was 25-fold less compared with

intensity of the 35-kDa band is notably reduced after absorption, suggesting

the novel fusion protein (Fig. 6B, lane 1 vs. 5).

that the antibody is speci cally directed against the 35-kDa RSOR.

Yang et al. PNAS August 29, 2000 vol. 97 no. 18 9899

Fig. 5. Low (A), medium (B), and high (C) magni cation photomicrographs of in situ autoradiograms of kidney tissue sections hybridized with RSOR riboprobe.

The RSOR mRNA is exclusively expressed in tubules of the renal cortex and is absent in the medulla and glomeruli (arrowheads). (D F and J L) Photomicrographs

with different magni cations of the kidney sections stained with anti-RSOR antibody. The spatial protein expression of RSOR is similar to the mRNA message,

and it is absent in the medulla and glomeruli (arrowheads). (G I) Immuno uorescence photographs of kidney sections stained with anti-Tamm Horsfall protein

antibody, a marker of distal tubular epithelium (arrows). The photographs depicted in G I are the serial tissue sections of micrographs shown in D F. (M O)

Immuno uorescence photographs of kidney sections stained with anti-aquaporin-2 antibody, a marker of collecting duct epithelium (arrows). The photographs

depicted in M O are the serial tissue sections of micrographs shown in J L. Absence of RSOR immunoreactivity in the distal and collecting tubules suggests that

it is exclusively expressed in the proximal tubules. Magni cations: A, D, G, J, and M, 10; B, E, H, K, and N, 20; C, F, I, L, and O, 40.

where identical molecular weight bands were observed by using

Discussion

two different cDNA clones. Similarly, generation of identically

AKRs are a family of monomeric oxido-reductases that catalyze

sized recombinant proteins from different cDNA clones further

the NADPH-dependent reduction of a wide variety of aliphatic

supports the translatability of the isolated cDNA.

and aromatic aldehydes and ketones (11). The functions of most

Unlike the ALR1 and ALR2 that are widely distributed in

of the family members are not very well defined, nevertheless,

various tissues, the mRNA transcript of the isolated clone was

ALR1 and ALR2 have been under intense investigation (7, 10,

exclusively expressed in the kidney. Both ALR1 and ALR2 and

17, 20 25), and the latter has received the most attention

AKR-related family members have been isolated from the

because of its pathophysiologic relevance to diabetic complica-

kidney, but their sequences are nonhomologous with this renal-

tions (26). Besides, the increased amounts of glycated proteins

specific cDNA (29, 30). The Western blot, using antifusion

and sorbitol, the ALR2 activity and expression of its 1.5-kb

protein antibody, confirmed that it is exclusively expressed in the

mRNA transcript have been found to be up-regulated in kidneys

kidney. Next, intrarenal spatial distribution of this renal-specific

of diabetic rats (27, 28). Because a similar-sized mRNA tran-

gene was studied in view of the fact that ALRs also are expressed

script was found to be up-regulated in several DM kidneys, our

in the kidney (30). The ALR2 is expressed in the mesangial cells

initial considerations were that the 130-bp clone isolated by

where it is involved in the pathophysiology of the glomerulus in

cDNA-RDA is a fragment of the known mammalian ALR2.

diabetes (31), and it is also present in the medulla where it is

However, the fact that no sequence homology was observed led

believed to serve as an osmolyte regulator (32). Unlike the

us to isolate the full-length cDNA. The latter, with a putative

ALRs, both mRNA and protein expression of this novel gene

protein product of 33 kDa, also did not reveal any sequence

were confined to the cortical tubules and not in the glomeruli.

homology with AKR family members or other known mamma-

Further delineation of its expression revealed that it neither

lian sequences in the GenBank database. However, the isolated

codistributes with Tamm Horsfall protein nor with aquaporin-2,

cDNA had partial homology with proteins derived from A.

indicating that it is restricted to the proximal tubular epithelia,

thaliana and P. radiata. The fact that rat and human cDNA

and thus can be regarded as one of their specific markers. In

sequences were quite homologous and yielded similar ORFs and

addition, immunofluorescence studies suggest that it is an in-

mRNA transcripts indicated that it is likely a translatable gene.

Such a notion was supported by in vitro translational studies tracytoplasmic protein, and that is in line with its deduced

Fig. 6. (A) Titration curve depicting a high af nity of the RSOR fusion protein for NADPH. (B) SDS PAGE of various proteins analyzed by Western blot hybridized

with antifusion protein antibody. Lane 1, ALR1; lane 2, ALR2; lane 3, kidney cortex extract; lane 4, RBG column eluant of the kidney extract, lane 5, RSOR fusion

protein; lane 6, BSA. Reactivity with the eluant from RBG column (lane 4) indicates recognition of NADPH binding protein by the anti-RSOR antibody. Mild

cross-reactivity with the ALR1 also is observed (lane 1). (C) Comparison of amino acid sequence of AKR motifs. Motif sequences are boxed. The NADPH binding

sites are bolded and underscored. M-RSOR*, mouse RSOR; M-17 HSD, mouse 17 -hydroxysteroid dehydrogenase; B-PGFS, bovine lung prostaglandin F synthase;

H-BABPDD, bile acid binding protein dihydrodiol dehydrogenase; Frog-Rho, -crystallin; R-ALR1, rat ALR1; M-ALR2, mouse ALR2.

9900 www.pnas.org Yang et al.

protein sequence where no extended transmembrane hydropho- mutation of amino acids downstream of the AKR-3 motif, i.e.,

Arg311 in ALR1, and Cys298 and Cys303 in ALR2, resulted in their

bic stretches were observed.

Interestingly, a stretch of amino acids identified as AKR-3 altered catalytic properties (17, 24, 34). Such studies are deemed

signature was observed in this renal proximal tubular epithelial- necessary to determine the kinetic properties and substrate

specific protein. The fact that its molecular weight was compa- specificity of the RSOR. Initial attempts to study the kinetic

rable to most of the AKR members, i.e., 30 40 kDa, and being properties of RSOR, using traditional substrates, e.g., D-glucose

acidic, i.e., pI 4.9, suggested that it may have properties that are and DL-glyceraldehyde, were not successful. It may be that the

characteristic of this superfamily. Fluorescence quenching stud- AKR-3 motif is located near the N terminus and contains an

ies indeed indicated that it has a high affinity binding site for N-linked glycosylation site. The latter may be needed for the

NADPH with KdNADPH 67 nM. The Western blotting of the catalytic activity of RSOR, although it seems not to be essential

eluant proteins from the RBG column also attests that it is a for the activity of ALR1 or ALR2, in which the AKR-3 motif is

NADPH binding protein and can be designated as RSOR. The not glycosylated. Certainly, studies are anticipated in which

NADPH binding site is located within the AKR-3 motif, and a RSOR would be expressed in insect cells to isolate its glycosy-

comparison of this domain with other AKR members is shown lated form for the delineation of its substrate specificity, catalytic

in Fig. 6C. It is conceivable that because of the sequence domains, and kinetic properties.

similarity in AKR motif and NADPH domain a weak RSOR- In summary, a RSOR with high affinity for NADPH that is

antibody cross-reactivity with the ALR2 was observed. The up-regulated in experimental streptozotocin-induced diabetes

domain is comprised of a tetrapeptide, Ile-Pro-Lys-Ser (IPKS), mellitus is described, and the mechanism(s) by which its expres-

where the first two amino acids may be variable. The lysine sion is modulated by hyperglycemia, whether related to the

residue seems to be the critical amino acid residue because its oxidant or osmotic stress, should be the subject of further studies.

modification by pyridoxal 5 phosphate affects the catalytic Finally, because the RSOR s expression is tubular-specific, the

efficiency of ALR1 and ALR2 (21, 23). Intriguingly, the AKR-3 studies also would yield important clues as to the pathogenesis

motif in the RSOR is located near the N terminus, whereas it is of tubular lesions, which incidentally also play a major role in the

confined to the C terminus in most of the AKR members, pathogenesis of renal complications in diabetes mellitus (35).

including some of the structural proteins, e.g., -crystallin (33).

The significance of the C terminus, in terms of substrate binding This work was supported by National Institutes of Health Grants

sites of the ALRs, is emphasized by studies where substitution or DK28492 and DK36118.

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