(Received for publication, January 17, 1996; and in revised form, February 20, 1996)
From the
We report the isolation and characterization of RK-1, a novel
peptide found in the kidney. RK-1 is related to the
corticostatin/defensins and has the sequence
MPCSCKKYCDPWEVIDGSCGLFNSKYICCREK but differs from the very cationic
corticostatins/defensins in having only one arginine and a calculated
charge at pH 7 of +1. Like some myeloid corticostatin/defensins
RK-1 inhibits the growth of Escherichia coli. Since
corticostatin/defensins effect ion flux in responsive epithelia we used
volume changes in villus enterocytes as a model system to study the
effects of RK-1 on ion channels in epithelial cells. At concentrations
10
M RK-1 decreased enterocyte volume
in a dose-dependent manner through a pathway that requires
extracellular calcium and is inhibited by niguldipine, a
dihydropyridine-sensitive ``L''-type
Ca
-channel blocker. In other assay systems for
corticostatin/defensins, such as the inhibition of
adrenocorticotropin-stimulated steroidogenesis, or cell lysis, RK-1 was
inactive or only weakly active. These results demonstrate the existence
of a novel system of biologically active peptides in the kidney
represented by RK-1 which is antimicrobial and can activate epithelial
ion channels in vitro.
Corticostatin/defensins (CS/defs) ()are low molecular
weight arginine-rich peptides with between 29 and 34 amino
acids(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) and three
disulfide bridges(12) . Many CS/defs are antimicrobial at
micromolar concentrations (1) probably as a result of their
ability to create voltage-dependent pores by insertion into the lipid
phase of biological membranes(13) . Some CS/defs, the
corticostatins, are specific antagonists of the hormone
adrenocorticotropin(3, 5, 6, 9, 11, 14, 15) ,
some are chemotactic for monocytes at 10
M(16) , some are cytostatic for HL-60
promyelocyte-like cells at 10
and 10
M(17) , and some inhibit natural killer-cell
activity in vitro at 10
M(18) .
Many CS/defs stimulate a
calcium-dependent volume reduction in villus enterocytes at
concentrations of 10 to 10
M(19) in response to changes in the ion flux across the
cell membrane. The maintenance of cell volume in villus epithelial
cells in isotonic medium reflects a balance between the rates of salt
influx and efflux(20) . The addition of calcium ionophore to
these cells under isotonic conditions activates charybdotoxin-sensitive
K
channels (21) and calcium-activated
Cl
channels(21) . The consequent loss of
these osmolytes together with water results in a reduction in cellular
volume. The volume response to CS/defs requires extracellular calcium
and is dihydropyridine-sensitive implying a pivotal role for L-type
calcium channels in this process.
The expression of genes for
CS/def-like polypeptides is not confined to myeloid cells. The
intestine contains antimicrobial CS/def-like peptides called cryptdins
found in Paneth's cells of the crypt but not in cells of the
inflammatory
system(22, 23, 24, 25) . The ability
of some CS/defs to elicit water movement in enterocytes (19) may contribute to the onset of diarrhea, as has been
suggested for guanylin, another peptide found in Paneth's
cells(26) . Interestingly a further system of cationic
cystine-rich peptides, the -defensins, that are structurally
distinct from CS/defs occurs in bovine tracheal epithelia (27) and in bovine neutrophils(28) , suggesting that
specialized peptidergic defense systems may occur in several tissues.
The existence of two specialized families of CS/def-like peptides, the defensins and the cryptdins, raises the possibility of other tissue-specific CS/def-related systems. Urinary tract infections may lead to kidney failure and are a significant source of Gram-negative septicemia(29) . Neither myeloid nor enteric CS/defs occur in the kidney, but other kidney-specific CS/def-like peptides might contribute to the defense mechanisms of the kidney.
We used a
homology screening system based on the unusual physicochemical
properties of the CS/defs to detect renal-specific peptides. Chemical
assays have been successful in the isolation of biologically active
gastroendocrine peptides (30) but have rarely been attempted in
other fields. Here we used three selection criteria: the elution
position on reverse phase HPLC, an apparent size of approximately 30
residues assessed by size-exclusion HPLC and amino acid analysis, and a
cystine content of about 20%. Using these criteria we isolated a
peptide, RK-1, that is abundant in the kidney and is a distant relative
of the myeloid/enteric CS/defs. Having proven the existence of a renal
CS/def-like peptide, RK-1 was then subjected to a series of assays
characteristic of the myeloid CS/defs to compare its activities to
those of the classic CS/def system. These experiments show that RK-1
possesses the Ca channel activating effects
associated with the myeloid CS/defs.
Figure 3: A comparison of the effects of RK-1 (open circles, n = 14) and CS-4/NP-2 (closed circles, n = 5), a rabbit myeloid defensin on the growth of E. coli (strain Y1090), expressed as percent of control incubations (no peptide). Error bars represent S.E.
Figure 1:
Stages in the purification of RK-1. A, the material eluting from C Sep-Pak at between
15% acetonitrile in 0.1% trifluoroacetic acid and 40% acetonitrile in
0.1% trifluoroacetic acid was fractionated on RP-HPLC using a linear
gradient of 0-80% acetonitrile in 0.1% trifluoroacetic acid as
described under ``Materials and Methods'' and monitored for
absorbtion at 215 nM. UV absorbing fractions were analyzed for
cystine content (y axis and histogram). The arrow indicates the elution position of RK-1. The elution positions of
myeloid defensins CS-1/NP3a and HP-4 are also indicated. CS-1/NP3a is
the earliest eluting defensin of those we have tested and HP-4 the
latest. B, the final purification of RK-1 by RP-HPLC using a
12-24% gradient of acetonitrile in 0.1% trifluoroacetic acid over
60 min as described under ``Materials and
Methods.''
This sequence is compared with representative members of the myeloid and enteric CS/defs in Table 2and as a dendrogram in Fig. 2. The distance between peptides along the branches represents their relatedness expressed in arbitary units. For example, HP-1 and HP-3 which differ by one amino acid are close, whereas HP-1 and HP-4 which have only 11 amino acids in common are placed far apart on the dendrogram. In this analysis there is no more difference between defensins and cryptdins than between defensins of different species. In contrast RK-1 maps on a separate limb of the dendrogram even though it possesses the cystine motif of the CS/defs.
Figure 2: A dendrogram of the degree of sequence relatedness among the CS/defs and RK-1 was obtained using the program CLUSTAL in PC/GENE. CS-1 to CS-5 are rabbit myeloid CS/defs, HP-1 to HP-4 are human defensins, R-1 to R-5 are rat CS/defs, Cry 1 to Cry 5 are murine cryptdins(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 21, 22) . For simplicity only some of the enteric cryptdins are shown(44) .
Figure 4:
The effect of 10M RK-1 on cell volume of villus enterocytes in a sodium containing (A) and sodium free (B) medium. In A, open circles represent the effect of RK-1 on volume (n = 4), closed circles show the volume of untreated
cells in isotonic medium over the same time course (n =
4), and closed squares show the effect of RK-1 on villus cell
volume in the presence of 0.2 µM niguldipine (n = 4). In B, open circles show the effect on cell
volume of RK-1 alone (n = 7), open diamonds show the effect of RK-1 in a calcium-free medium (n = 7), closed diamonds show the effect of RK-1 in
the presence of 1 nM GVIA
-conotoxin (n = 7), and closed squares the effect of RK-1 in the
presence of 0.2 µM niguldipine (n = 7). Vertical bars represent the S.E.
Neither 0.2 µM niguldipine
nor 1 nM GVIA -conotoxin alone had any effect on
enterocyte volume. Niguldipine added together with RK-1 prevented
volume reduction (final volume 0.91 ± 0.01 versus 0.83
± 0.01, p < 0.001, Fig. 4B). GVIA
-conotoxin (10 nM) added with RK-1, partially attenuated
the volume reduction (final relative volume, 0.88 ± 0.1 versus 0.84 ± 0.01, p < 0.05). Together
these results suggest that the action of RK-1 is mediated through
dihydropyridine-sensitive Ca
channels in villus
epithelial cells.
Figure 5:
The effect of HP-1 and RK-1 on the growth
of mammalian cell lines. The vertical axis represents the
percentage incorporation of [H]thymidine as a
percentage of incorporation in control wells. Experiments are the mean
of four independent determinations and are expressed as ± S.E.
, A549/RK-1;
, A549/HP-1;
, CHO/RK-1;
,
CHO/HP-1;
, SK-MES-RK1;
,
SK-MES-HP1.
Here we demonstrate the existence of a novel peptide found in the kidney which we call RK-1 and which has antimicrobial activity against E. coli, and activates ion channel activity. RK-1 possesses the cystine motif of the CS/defs and has potent biological activities. It is, however, structurally distinct from both the myeloid and enteric peptides indicating the evolution of at least three families of CS/def-like peptides from a common ancestor with each family restricted in its distribution to either neutrophils and alveolar macrophages, the intestinal crypts, or the kidney. RK-1 is the least cationic of the CS/def-like peptides and unlike the myeloid and enteric CS/defs the positive charge is carried mainly on lysine residues. To distinguish RK-1 and related peptides, peptides from the myeloid or enteric CS/defs, and to avoid the potential problems associated with naming a peptide from a tissue source or a bioassay, we propose the generic term lysyl-CS/def with the individual peptides given initials according to their species.
Having proven the existence of a CS/def-related peptide in the kidney we performed several assays to compare its biological characteristics with those of myeloid and enteric CS/defs. Among these we examined the antimicrobial activity of RK-1 against E. coli, and its ability to kill mammalian epithelial cells. E. coli was selected because it is a common bacterial pathogen in urinary tract infections (29) and is a standard test organism for CS/defs and cryptdins. RK-1 is antimicrobial in this assay, although less so than the bone marrow-derived peptide CS-4/NP-2. Although the role of CS/defs in host defense is well established (1) some CS/defs are only weakly antimicrobial(32) . The conditions used here were based on those described by Lehrer et al.(32) and may not reflect the conditions in the kidney. Further experiments using other conditions and organisms will be required to show the optimal conditions for RK-1 antimicrobial activity.
The villus enterocyte volume assay is a useful model to study the effects of biological mediators on ion channels in epithelial cells (19, 20, 21) . RK-1, like the corticostatins(19) , stimulates the shrinkage of villus cells in isotonic media both in the presence and absence of sodium. The decrease in volume requires extracellular calcium and is inhibited by niguldipine, an L-type calcium channel blocker(34) . Thus the effects of RK-1 on calcium dependent volume changes in villus enterocytes and, by implication on dihydropyridine-sensitive L-type calcium channels in these cells, are similar to those previously reported for the CS/defs(19) .
There is considerable
evidence that dihydropyridine-sensitive voltage-dependent
Ca channels are important in the kidney. The
dihydropyridine-sensitive voltage-dependent Ca
channel is composed of five subunits of which the
-subunit forms the ion pore. At least five forms of
the
-subunit exist with various electrophysiological
properties and sensitivities to pharmacological probes and each encoded
by separate genes(35) . RNA transcripts of four of the
-subunit genes have been demonstrated in the kidney
and are differentially distributed between the cortex and the
medulla(36) . Possible roles for these channels include
Ca
-reabsorption(37) , tubular epithelial cell
volume regulation(38, 39, 40, 41) ,
in mesangial cell function(42) , and in glomerular
hemodynamics(43) . This does not prove that the properties of
RK-1 as a functional agonist of Ca
channels in
epithelial cells are physiologically relevant but indicates that the
molecular and cellular machinery exists for RK-1-like peptides to act
through Ca
channels in the kidney.
In summary, the major finding of this paper is the existence of a novel and potently biologically active peptide in the kidney. The similarity of this molecule with the CS/defs reveals a network of CS/def-like peptides with each branch of the family restricted to a very narrow range of tissues. The evidence presented suggests two hypotheses for the function of RK-1. First, based on its homology with the defensins and cryptdins and its antimicrobial activity, RK-1 may be involved in disease resistance in the kidney. Second, the biological effects of RK-1 on calcium dependent volume regulation may indicate a role for RK-1 as a local regulator of calcium channels. Our demonstration of a novel peptide in the kidney exemplified by RK-1 should open important new avenues of investigation into renal physiology and pathology.