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Medizinische Klinik IV, Universität Erlangen-Nürnberg, Germany.
Correspondence to Dr. Roland Veelken, Medizinische Klinik IV, Nephrologisches Labor, Unversität Erlangen-Nürnberg, Loschgestrasse 8, D-91054 Erlangen, Germany. Phone: +49 9131 85 6314; Fax: +49 9131 85 9202; E-mail: mfm431{at}rzmail.uni-erlangen.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study in rats was designed to test the hypothesis that induction of iNOS in glomeruli or preglomerular vessels contributes to glomerular hyperfiltration in diabetes mellitus caused by streptozotocin. We investigated the renal functional effects of L-iminoethyl-lysine (L-NIL), which is a 30-fold more potent inhibitor of iNOS than of ecNOS (9,10). For comparison, we also used different doses of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective blocker of all NOS isoforms. In addition, we investigated the glomerular expression of iNOS and ecNOS in kidney sections and isolated glomeruli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal Hemodynamics
Two weeks after successful streptozotocin injection, measurements of GFR
and renal plasma flow (RPF) were performed as described previously
(11). Briefly, femoral
arterial and venous catheters, as well as a bladder fistula, were implanted
under intraperitoneal methohexital (50 mg/kg) anesthesia. The animals were
then placed in a restrainer and allowed to recover for at least 4 h. In
conscious rats, continuous infusions of inulin and para-aminohippurate in
saline were started, and MAP was monitored by pressure transducers connected
to a polygraph (Grass Medical Instruments, Quincy, MA). The infusion rate was
set at 62.5 µl/min in control and normoglycemic diabetic animals. To
account for polyuria, extra saline was administered to diabetic animals to
match the urinary losses during the procedure. After 2 h, urine samples were
collected for determination of flow rate. Urine volume was measured
gravimetrically. Steady-state conditions were assumed when diuresis matched
infusion rate during three subsequent 10-min intervals. After reaching
steady-state conditions, arterial blood was drawn for measurements of
hematocrit, sodium, potassium, chloride, as well as inulin and
para-aminohippurate to determine GFR and RPF.
Subsequently, diabetic rats received 0.1, 1, or 10 mg (six rats per dose) of L-NIL, a selective inhibitor of iNOS (Alexis, Grünberg, Germany) (9,10,12). Six nondiabetic control rats received 10 mg of L-NIL. Measurements of GFR and RPF were performed before and after intravenous bolus injection of the putative iNOS inhibitor at the following time points: -20 min, -10 min, +10 min, +20 min, +40 min, +60 min, and +120 min.
In addition, 0.1, 1, and 10 mg of the nonselective NOS inhibitor L-NAME (Sigma) were administered to six diabetic and six nondiabetic control rats, as described for L-NIL-treated animals. GFR and RPF were measured before the injection of each dose as well as 15, 45, and 120 min after administration.
Measurements of para-aminohippurate and inulin, as well as calculation of GFR and RPF, were performed as described previously (11).
Extraction of RNA and Protein from Isolated Glomeruli
Nine diabetic and six nondiabetic control animals that did not undergo
renal hemodynamic studies were sacrificed for the assessment of glomerular NOS
RNA and protein. Rats were killed in deep thiobarbital anesthesia (100 mg/kg
body wt, intraperitoneally), and the left kidney was snap-frozen in liquid
nitrogen for immunohistochemistry. The right kidney was used for isolation of
glomeruli as described previously
(13,14).
Glomerular RNA and protein were extracted using TRI-Reagent (Molecular
Research Center, Inc., Cincinnati, OH)
(15). Two different positive
controls were used for iNOS detection. First, three rats were sacrificed 4 h
after an intraperitoneal injection of lipopolysaccharides (LPS) (3 mg/kg body
wt; Sigma), which induces iNOS expression in the kidney
(16). Second, we used six rats
that were treated orally with a 3.7 nmol/L (10 mg%) solution of L-NAME
(17) and had undergone surgery
(arterial and venous catheters) 1 d before sacrifice. Others had previously
reported induction of renal iNOS by surgery
(18), and increased expression
of iNOS in gut mucosa after L-NAME treatment
(19). We have confirmed renal
glomerular induction of iNOS RNA and protein after surgery in L-NAME-treated
rats (see Results).
Northern Blot Analysis
Total RNA was size-fractionated on a 1 % agarose formaldehyde gel and
transferred onto Hybond nylon membranes (Amersham, Braunschweig, Germany). The
blot was baked at 80°C for 2 h, prehybridized with 5x Denhardt's
solution, 5x SSC, 50% formamide, 50 mM Na3PO4,
0.1% SDS, and 0.25 mg/ml salmon sperm DNA at 40°C for 2 h. DNA
hybridization probes were labeled with 
-[32P]-dCTP, using a
random-primed labeling kit (Boehringer, Mannheim, Germany). Blots were
hybridized in prehybridization solution containing 2 x 106
cpm/ml of probe at 40°C overnight. Blots were washed twice for 15 min with
2x SSC containing 0.1% SDS and then 30 min with 0.1x SSC
containing 0.2% SDS. Blots were exposed to Kodak XAR-2 films with intensifying
screens at -80°C. For hybridization with iNOS cDNA, a 611-bp
EcoRI/PstI cut insert of the murine iNOS cDNA clone piNOS B2
was used, as described previously
(20). As a probe for GAPDH, a
470-bp fragment corresponding to nucleotides 510 to 980 of the rat GAPDH
sequence (GenBank accession no. X022331) was amplified using the primers and
PCR conditions specified below.
Reverse Transcription-PCR
For reverse transcription (RT), 1 µg of total glomerular RNA was
reverse-transcribed by Moloney murine leukemia virus reverse transcriptase
using oligo-dT. Five microliters of a 20-µl RT reaction were used for
subsequent PCR. PCR for iNOS was performed by 35 PCR cycles at 94°C for 1
min, 55°C for 1 min, and 72°C for 1 min in a Touchdown thermocycler
(Hybaid, London, United Kingdom). The primers 5'-CCG GAT CCT CTT TGC TAC
TGA GAC AGG-3' and 5'-CCG AAT TCG GGA TCT GAA TGC AAT
GTT-3', corresponding to nucleotides 1613-1634 and 2057-2078 of the
full-length rat iNOS cDNA, yielded a 465-bp product
(21). To exclude amplification
of genomic DNA, primers were selected to be localized in different exons.
Because the genomic organization of the rat iNOS gene was not available,
primer selection was based on the assumption of structural homology between
rat and human iNOS genes. If this assumption holds true, the sense primer
would be localized in exon 14, and the antisense primer in exon 18
(22), approximately 7 kb
distant from each other. For GAPDH, the primers 5'-AAT GCA TCC TGC ACC
ACC AA-3' and 5'-GTC ATT GAG AGC AAT GCC AGC-3' were used in
22 PCR cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 2
min, as described previously
(20).
Western Blot Analysis
Protein samples containing 20 µg of total protein were denatured by
boiling for 5 min and were separated on an 8% denaturing SDS-PAGE gel. After
electrophoresis, the gels were electroblotted onto polyvinylidene difluoride
membranes (Pall Filtron, Karlstein, Germany), blocked with 3% bovine serum
albumin (BSA)/Tris-buffered saline (TBS)/0.2% NaN3/0.1% Tween 20
overnight, and incubated with the primary antibodies for 2 h. Antibodies N
32030 and N 30020 from Transduction Laboratories (Lexington, KY) were used to
detect iNOS and ecNOS, respectively. NOS protein was visualized with a
secondary horseradish peroxidase-conjugated anti-rabbit or antimouse IgG
antibody, respectively, using the enhanced chemiluminescence system
(Amersham).
Immunolocalization
Immunohistochemical analysis for iNOS and ecNOS in renal tissue sections
was performed using the APAAP technique as described
(23). Five-micrometer cryostat
kidney sections were air-dried for 10 min, fixed in cold acetone for 10 min,
washed twice in TBS/1% BSA, and incubated with 100% fetal calf serum for 30
min at 37°C. Sections were then layered with the primary antibodies and
incubated at 4°C overnight. As a negative control, equimolar
concentrations of preimmune rabbit IgG was used. After rinsing in TBS/1% BSA,
the sections were incubated with secondary antibodies mouse anti-rabbit and
subsequently rabbit anti-mouse or only rabbit anti-mouse (M 737 and Z 456;
both from Dako, Hamburg, Germany) for 1 h at 37°C each. Sections were then
layered with the APAAP complex (D 651; Dako) for 30 min at room temperature. A
Neufuchsin kit (K 698; Dako) was used as a chromogen. Finally, sections were
counterstained with hematoxylin and embedded in Aquatex (Merck, Darmstadt,
Germany). For immunofluorescence detection of ecNOS, an analogous protocol was
used, except that sections were incubated with CY2-labeled goat anti-mouse IgG
secondary antibody from Dianova (Hamburg, Germany) for 2 h. Washed sections
were then covered with Trisbuffered mowiol, pH 8.6 (Hoechst, Frankfurt am
Main, Germany). Microscopy was performed using a Leitz Aristoplan microscope
with a x40 Fluotar objective (Leica Instruments, Nussloch, Germany). For
CY2 fluorescence detection, 520-nm emission filters were used.
Statistical Analyses
Statistical analyses were performed using the SAS software package (SAS
Institute, Cary, NC). For normally distributed data, t test was used;
otherwise nonparametric methods were applied. Data are given as mean ±
SEM. A P value < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Administration of the iNOS inhibitor L-NIL in doses of 0.1 and 1 mg caused no changes of systemic or glomerular hemodynamics in diabetic rats (Figure 1). Only use of the highest dose of L-NIL (10 mg) induced a mild yet significant increase of MAP and significantly reduced RPF from 10360 to 7260 µl/min (Figure 1). This maximal dose of L-NIL had no effect on glomerular hemodynamics in control rats.
L-NAME treatment caused significant changes of GFR, RPF, and MAP (Figure 2). In diabetic rats, even the lowest dose of L-NAME (0.1 mg) decreased GFR and RPF significantly, reducing renal hemodynamics to control values. This low dose of L-NAME had no effect in control rats. After the two higher doses of L-NAME, GFR and RPF (Figure 2) declined in diabetic and control rats, showing no further differences between the groups. One milligram of L-NAME increased MAP significantly by 15 ± 6 mmHg in diabetic animals and by 13 ± 5 mmHg in controls, whereas 0.1 mg of L-NAME had no effect on MAP in either group. Ten milligrams of L-NAME increased MAP significantly by 25 ± 4 mmHg in diabetic animals and by 30 ± 4 mmHg in controls.
In RNA extracted from isolated glomeruli of diabetic and control rats, iNOS mRNA could not be detected either by Northern blot (data not shown) or by RT-PCR (Figure 3). Control hybridization or RT-PCR amplification for the housekeeping gene GAPDH was positive in all glomerular RNA samples used (data not shown), excluding RNA degradation as a reason for the negative results on iNOS. In contrast, iNOS mRNA was detectable by RT-PCR in glomeruli from control rats treated with LPS 24 h before sacrifice. Positive results were also obtained from chronically L-NAME-treated animals that had undergone surgery the day before (Figure 3). A second, slightly larger amplicon (approximately 550 bp) was also detected in positive control samples. Contamination with genomic DNA cannot explain this finding, as evidenced by the lack of an amplicon in reactions without RT (Figure 3, lane 6) and the fact that genomic DNA should yield a 7-kb product (22). We can only speculate that alternative splicing might be responsible for a larger iNOS transcript (24).
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Similarly, by Western blot, iNOS protein could not be detected in diabetic and control rats but was detected in positive controls (Figure 4). Furthermore, immunohistochemistry revealed no iNOS staining in diabetic and control rat glomeruli, whereas marked glomerular iNOS staining was present in both types of positive control, i.e., in rats pretreated with LPS or L-NAME (Figure 5). Here, glomerular immunostaining for iNOS was uniformly diffuse in a predominantly mesangial pattern.
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In contrast to iNOS, ecNOS protein was detectable in glomeruli from diabetic as well as control animals (Figure 6). Compared with controls, ecNOS protein was clearly elevated in diabetic rat glomeruli (Figure 6). Western blotting was performed in three groups of diabetic rats and controls, and the increase of ecNOS levels in diabetic rat glomeruli was averaged 174% over control, determined by densitometry. By immunohistochemistry and immunofluorescence microscopy, ecNOS protein in glomeruli appeared to be localized to the endothelium of preglomerular vessels and glomerular tufts. Positive ecNOS staining was more abundant in diabetic than in control rat glomeruli (Figure 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data confirm the important role of NO in the pathogenesis of glomerular hyperfiltration in early diabetes mellitus: Actue inhibition of NOS with a low dose of the nonselective NOS inhibitor (L-NAME) decreased GFR and RPF significantly in diabetic animals back to normal control values, whereas this dose did not affect renal hemodynamics in control rats. This finding indicates that glomerular hyperfiltration is markedly dependent on increased NO generation and action at this early stage of experimental diabetic nephropathy. These results agree with previous reports, which postulated a significant role of NO in diabetic hyperfiltration (4,5,27,28), even though the involvement of specific NOS isoforms in this process remains unclear.
To test the potential contribution of iNOS to NO-mediated glomerular hyperfiltration, we performed experiments with a relatively selective iNOS blocker, L-NIL. We found that in awake rats, selective iNOS inhibition with doses up to 1 mg (approximately 4 mg/kg body wt) had no effect on renal hemodynamics and BP (Figure 1). Studies of others had shown that this dose was sufficient to completely block iNOS activity (9). However, the highest dose of L-NIL (10 mg) significantly decreased RPF (with no statistically significant change of GFR) and increased BP. This effect of the highest dose of L-NIL is most likely explained by nonselective inhibition of all NOS isoforms, because L-NIL is only an approximately 30 times more specific inhibitor of iNOS than of ecNOS (10). A 100-fold lower dose of the nonselective inhibitor L-NAME (0.1 mg L-NAME versus 10 mg L-NIL), however, exerted similar or greater effects on renal hemodynamics than 10 mg of L-NIL. Recently, Schwartz et al. reported that in conscious LPS-treated rats, the glomerular effects of increased NO production by iNOS could be prevented when the rats had been pretreated with 3 mg/kg L-NIL intraperitoneally (16). BP and heart rate were not affected. These observations indicated that doses of L-NIL, which correspond to the middle dose of our dose—response curve established after intravenous application of the inhibitor, are able to block the effects of iNOS in the rat kidney.
Aminoguanidine, an inhibitor of nitric oxide synthase (NOS) and of advanced glycation end products, has been described to attenuate the progression of renal disease in experimental diabetes (29). The results of this study showed that the protective effects of aminoguanidine were not due to inhibition of iNOS but rather to inhibition of advanced glycation end product formation. These observations and our results would argue against a significant involvement of iNOS in the functional changes of the kidney in early and late diabetes mellitus.
We did not detect iNOS mRNA or protein in glomeruli of diabetic rats. Although we cannot completely exclude the possibility that the methods we used were not sufficiently sensitive to detect iNOS in diabetic animals, we consider this quite unlikely for the following reasons: (1) We used several independent methods (Northern blot, RT-PCR, Western blot, immunohistochemistry) on tissue of diabetic animals and positive control tissue of LPS-treated rats and after iNOS induction by L-NAME; (2) We were able to detect iNOS mRNA and protein with each method in positive control tissue of the same organ (kidney) that had undergone the same extraction procedures (isolation of glomeruli, RNA and protein, or snap-freezing for immunohistochemistry). This finding is in agreement with previous reports showing the inducing effects of LPS treatment (16) or surgery (18) on iNOS expression and the effects of L-NAME on gut mucosa iNOS expression (19). The induction of iNOS under these experimental circumstances has substantial relevance, because both nonsterile surgery and L-NAME treatment are commonly performed by researchers concerned with NOS, and the results obtained may be confounded by artifactual iNOS induction. In a recent study, iNOS and ecNOS protein expression was reported to be increased in kidneys of rats with streptozotocin-induced diabetes mellitus (30). These investigations, however, were limited to Western blot analyses of total renal cortex and no data were provided on isolated glomeruli. Furthermore, it is unclear whether the observed increase of iNOS protein expression was effected by nonsterile surgery in anesthetized rats.
In contrast to iNOS, we found increased amounts of ecNOS in glomeruli of diabetic rats. Besides its well known regulation by calcium, ecNOS can be regulated by phosphorylation (26) or translocation (31). In addition, expressional control of ecNOS has been recently outlined in various tissues (25). For example, vascular endothelial growth factor, which is increased in the hyperglycemic state (32), can stimulate the expression of ecNOS (33). By immunohistochemistry, we could localize ecNOS to the endothelium of preglomerular arteries and arterioles as well as to the glomerular tuft. In light of the hymodynamic effects of L-NAME observed by us (Figure 2) and others (27), our findings suggest a key role for ecNOS in glomerular hyperfiltration observed in early streptozotocin-induced diabetes mellitus. During the preparation of this manuscript, it was reported that increased ecNOS production was detected in the afferent arterioles of glomeruli in rats with experimental diabetes mellitus in the first 4 wk of disease development (34). It should be noted, however, that the importance of NO for glomerular hemodynamics may decrease over time in experimental diabetes mellitus (35). It has been suggested that this phenomenon could be due to progressive decline of NO-dependent cGMP generation (36).
In conclusion, the present results confirm that NO plays an important role for the development of glomerular hyperfiltration in early diabetic nephropathy of rats. Our findings did not reveal evidence for the induction of iNOS in glomeruli of diabetic rats. Instead, we observed that greater glomerular abundance of ecNOS causing increased generation of NO contributed to glomerular hyperfiltration. The mechanisms leading to increased levels of ecNOS in glomeruli of diabetic rats and their potential relevance for the initiation and/or progression of diabetic nephropathy require further study.
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