Tubular and Glomerular L-Arginine Transport (Uptake and Transporters) and the Nitric Oxide Synthases in Ischemic Acute Renal Failure (iARF) in Streptozotocin-Induced Diabetic Rats (STZ-DM)

Abstract

Background. L-arginine or its metabolites may be important pathogenetic factors in ischemic acute renal failure (iARF) in rats. It was found that the L-arginine-nitric oxide synthase-nitric oxide system plays an important role in the renal hemodynamic alterations in the early stages of diabetes. The iARF in diabetic rats is much more severe than the normal rats exposed to a same ischemia time. The purpose of the present study was to evaluated L-arginine uptake and its transporters and nitric oxide synthase isoform expression in tubuli and glomeruli of STZ-induced diabetic rats with iARF. Methods. iARF was induced by right nephrectomy and left renal artery clamping for 60 min followed by a 60 min reflow period. iARF was induced in STZ diabetes rats two weeks after intraperitoneal streptozotocin (60 mg/kg body weight) and in normal control rats. L-arginine uptake, L-arginine transporters (CAT1 and CAT2) and nitric oxide synthases (iNOS, eNOS, and bNOS) were determined by RT-PCR) in both glomeruli and tubuli preparations. Results. The STZ diabetic rats compared with the non diabetic normal rats have a higher glomerular L-arginine uptake, higher iNOS mRNA, lower eNOS mRNA, and lower tubular CAT1 mRNA, eNOS mRNA, and bNOS mRNA. The diabetic iARF after one hour of reperfusion had lower glomerular L-arginine uptake, lower CAT1 mRNA, lower eNOS mRNA, lower bNOS, and higher tubular iNOS mRNA compared with iARF in normal rats.

Conclusions. Our findings suggest a prolonged and more severe post-glomerular vasoconstriction very early after the reflow in the iARF of STZ diabetic rats compared with the iARF in the normal control rats. That may be a plausible explanation to the very significant decline in GFR and tubular necrosis that characterize the iARF in diabetic rats.

Keywords:

• STZ diabetes
• iARF
• L-arginine transport

INTRODUCTION

Different mechanisms have been purposed to induce the renal injury in iARF, including anoxic damage, release of free oxygen radicals during the reperfusion, neutrophils accumulation, and release of lytic enzymes.[1],[2]

Several studies examined the role of the L-arginine-nitric oxide synthases (iNOS, eNOS, bNOS)-nitric oxide (NO) system, in the pathogenesis of iARF in rats.[3–15] Renal NO causes increased pre-glomerular arteriolar vasodilatation, improving renal blood supply and renal oxygenation.[7],[8] Conger et al.[9] have found that in iARF, eNOS activity is not suppressed as expected but is maximally active, and therefore could not be further stimulated by endothelial-dependent vasodilators.

We found increased NO₂+NO₃ (the stable NO metabolites) levels in the urine and plasma of STZ-induced diabetic rats with iARF. L-arginine administration caused modest improvement in creatinine clearance. It has been also found that renal ischemic injury is significantly worse in diabetic rats.[10] In the same study, it has been proven that NO system regulation is likely responsible for these differences between diabetic and non-diabetic rats.

L-arginine, the only NO precursor, enters the cells via cationic amino-acids transporters. iNOS activity is regulated at the transcriptional level, but directly depends on L-arginine presence extra-cellular. L-arginine availability determines intra-cellular NO production rate.[16],[17]

Renal L-arginine transport is mediated by the y⁺-carriers system.[18] The y⁺-system has high affinity to cationic amino acids. This system is activated by substrate presence in the counter membrane side (trans) and is inhibited by substrate presence in the same membrane side (cis).[19–21]

Several L-arginine transporters have been identified and cloned, termed amino acid transporters 1, 2, and 3 (CAT1, CAT2, and CAT3).[19–21] CAT1 is expressed and acts more than CAT2A by trans-stimulation. CAT2B has been identified in activated macrophages and lymphocytes. CAT2A is a low-affinity transporter and is expressed exclusively in adult rodent's liver.[22] CAT3 is expressed in the adult rat's brain and recently has been identified in the inner medullary collecting ducts in rat's kidneys.[23–25] L-arginine uptake mechanisms have been studied in the recent years, in different clinical settings, including iARF.[26]

Cumulative evidence in the literature suggests either L-arginine or its metabolite, NO, plays a role in the renal hemodynamic alterations during the early stages of diabetes.[27–29]

We have also examined L-arginine uptake and L-arginine transporters expression in the diabetic rat's glomeruli. L-arginine uptake was significantly augmented in diabetic rats. The increased L-arginine uptake was through increased glomerular CAT1 expression in the diabetic rats. This increased L-arginine uptake caused an elevated GFR that characterizes the early stages of diabetic nephropathy.[29]

The purpose of the present study was to evaluate L-arginine uptake, its transporters, and NOS isoform expression in tubuli and glomeruli of STZ-induced diabetic rats with iARF after one hour of reperfusion, and attempts to explain the mechanisms leading to the severe renal injury in iARF in diabetic rats.

MATERIAL AND METHODS

All animal experiments described in this study were conducted in accordance with the protocol approved by the institutional committee on ethics in animal experiments. Studies were performed using male Wistar rats weighting 250–300 g.

DIABETES INDUCTION

Diabetes has been induced by intra-peritoneal administration of streptozotocin (STZ) (fresh preparation with 5% dextrose, suspended in Na-citrate buffer, pH 4.5, 65 mg/Kg). Blood glucose levels were determined using tail blood samples. Animals that exhibited blood glucose levels above 250 mg/dL were included in the studies.

Animals were allowed free access to regular rat chow and tap water. Experiments were performed two weeks following STZ administration. Age-matched normal rats served as controls.

I/R Acute Renal Failure Model

iARF was induced under general anesthesia with ketalar (8 mg/100 g BW) and xylazine (0.25 mg/100 g BW) intra-peritoneally. Immediately after right nephrectomy, the left renal artery was clamped for 60 minutes; after 60 minutes, the left renal artery was declamped for another 60 minutes. The right kidney harvested before the I/R served as control before I/R in the different experimental groups.

Experimental Groups

• Group 1: Control non-diabetic rats before I/R

• Group 2: Diabetic rats before I/R

• Group 3: Control non-diabetic rats with iARF and reperfusion of 60 minutes

• Group 4: Diabetic rats with iARF and reperfusion of 60 minutes

Each experimental group included 8–10 rats. After completion of the surgical procedure, the left kidney was harvested for further examinations.

Isolation of Glomeruli and Tubuli

Kidneys from all experimental groups before and after I/R were harvested and decapsulated. The cortex was carefully dissected free. Glomeruli and tubuli were isolated using a sieving technique. Cortices were minced to a fine paste with a razor blade and gently pressed through a 106 μm stainless steel sieve.

The resulting material was suspended in HEPES buffer (KCl 5 mmol/L CaCl₂ 0.9 mmol/L, MgCl₂ 1 mmol/L, D-glucose 5.6 mmol/L, HEPES 25 mmol/L, NaCl 140 mmol/L), at 4°C, pH 7.4. The suspension was forced through a 20-gauge needle to decapsulate the glomeruli and then passed through a 75 μm sieve. Glomeruli trapped on the sieve were washed and pelleted by centrifugation at 1000 rpm for one minute three times. This fraction consisted of more than 95% glomeruli, the majority of which were decapsulated, while the fraction that was washed through the sieve consisted primarily the tubuli. The freshly isolated glomeruli and tubuli were further used for RNA extraction and for arginine uptake measurements.

L-Arginine Uptake

L-arginine uptake was determined as described by Gazoll et al.[30] Glomerular and tubular suspensions from the various experimental groups were incubated and shaken for 10 minutes in HEPES buffer at 37°C, pH 7.4. [³H]L-arginine and L-arginine, in a final concentration of 1 mmol/L, were added to a total volume of 1 mL for an additional four minutes. The duration of four minutes was chosen because previous studies demonstrated that transport of 1mmol/L of [³H]L-arginine by both tubuli and glomeruli increased over time, and was linear at approximately four minutes. Transport activity was determined by a rapid aspiration of medium and washing the cells with ice-cold phosphate-buffered saline (PBS, four times, 2 mL/tube). The glomeruli and tubuli were then dried and solubilized by 1 mL of 0.5% sodium dodecyl sulphate (SDS) in 0.5N NaOH. Seven hundred micro liters of the extracts were used to monitor radioactivity, using liquid scintillation spectrometry (Betamatic; Germany), and the remaining 300 microliters for protein content determination by the Lowry method.

To correct for non-specific uptake or cell membrane binding, glomeruli were incubated with 10 mmol/L unlabeled arginine in HEPES buffer, and the associated radioactivity was subtracted from each data point. The results are expressed as mean ± SD of at least five different experiments.

Analysis of mRNA Levels of CAT1, CAT2, iNOS, eNOS, and bNOS by RT-PCR

Total cellular RNA was extracted from glomeruli and tubuli, following the method described by Chomczynski and Sacchi.[31] Reverse transcription (RT) was carried out for 1.5 hours at 42°C, followed by polymerase chain reaction (PCR) in 1X PCR buffer, for a total of 35 cycles, each at 94°C for one minute, 60°C for one minute, 72°C for two minutes and seven minutes (final cycle). The first pair of primers was designed to bind to a portion of the rat CAT1 gene:

• forward: 21-mer, 5′-CGG ATC GTC ATC TCC TTC CTG-3′,

• reverse: 21-mer, 5′-CCC TCC CTC ACC GTA TTT CAC-3′.[32]

The second pair of primers, which hybridize to a sequence common to both CAT 2 and CAT2A, were:

• forward: 21-mer, 5′-AAC GTG CTT TTA TGC CTT TGT-3′,

• reverse: 21-mer, 5′-GGT GAC CTG GGA CTC GCT CTT-3′.[33]

The primers used for iNOS were:

• forward: 21-mer, 5′-GTG TTC CAC CAG GAG ATG TTG-3′,

• reverse: 21-mer, 5′-CTC CTG CCC ACT GAG TTC GTC-3′.

The primers used for eNOS were:

• forward: 21-mer, 5′-CCG GAA TTC GAA TAC CAG CCT GAT CCA TGG AA-3′,

• reverse: 21-mer, 5′-GCC GGA TCC TCC AGG AGG GTG TCC ACC GCA TG-3′.

The primers used for bNOS were:

• forward: 21-mer, 5′-GAA TAC CAG CCT GAT CCA TGG AAC-3′,

• reverse: 21-mer, 5′-TCC TCC AGG AGG GTG TCC ACC GCA-3′.[34]

To exclude the possibility of contamination by genomic DNA amplification, and to assess the adequacy of cDNA, experiments were carried out in the absence of reverse transcriptase, and amplification of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was performed, respectively. PCR products were electrophoresed on a 1.6% agarose gel and visualized by ultraviolet (UV) induced fluorescence. All PCR reactions resulted in the amplification of a single product of the predicted size for CAT1, CAT2, iNOS, eNOS, bNOS, and GAPDH.

Statistical Analysis

Data are presented as the mean ± SD. Student's t test was used to assess significant differences between groups; p < 0.05 was considered to be statistically significant.

RESULTS

Effects of I/R on L-Arginine Transport

Glomeruli

Glomerular L-arginine transport before I/R in diabetic rats was significantly higher compared to non-diabetic rats before I/R (26.7 ± 0.94, 13.4 ± 4.3, respectively; p < 0.001; see Figure 1A and Table 1). I/R caused a significant decline in glomerular L-arginine transport (10.5 ± 3.7, p < 0.001) in diabetic rats. I/R did not cause any change in glomerular L-arginine transport in non-diabetic rats (12.3 ± 2.6).

Figure 1. Glomerular and tubular L-arginine transport before and after I/R: A, glomerular L-arginine transport before and after I/R in normal and diabetic rats; B, tubular L-arginine transport before and after I/R in normal and diabetic rats.

Table 1 Glomerular and tubular L-arginine transport and CAT1 and CAT2 mRNA expression before and after I/R

	Glomeruli		Tubuli
	Normal	Diabetes	Normal	Diabetes
L-arginine transport
Before I/R	13.4 ± 4.3	26.7 ± 0.94	14.1 ± 10	11.9 ± 8.2
After I/R	12.3 ± 2.6	10.5 ± 3.7	27.0 ± 11.6^†	11.7 ± 4.7
CAT1 mRNA expression
Before I/R	1.25 ± 0.16	1.86 ± 0.22^‡	0.92 ± 0.15	0.64 ± 0.76^§
After I/R	1.19 ± 0.25	1.14 ± 0.30	0.88 ± 0.24	0.76 ± 0.26
CAT2 mRNA expression
Before I/R	0.72 ± 0.22	0.77 ± 0.6	0.56 ± 0.23	0.46 ± 0.10
After I/R	0.63 ± 0.49	0.79 ± 0.59	0.76 ± 0.11^‡	0.43 ± 0.23

*p < 0.001 vs. normal and vs. diabetic I/R.

^†p < 0.01 vs. normal and vs. diabetic I/R.

^‡p < 0.05 vs. normal and vs. diabetic I/R.

^§p < 0.05 vs. normal.

Tubuli

Tubular L-arginine transport before I/R was similar in both diabetic and non-diabetic rats (11.9 ± 8.2, 14.1 ± 10, respectively; see Figure 1B and Table 1). I/R did not cause a significant change in tubular L-arginine transport in diabetic rats (11.7 ± 4.7).However, in non-diabetic rats, tubular L-arginine transport was significantly increased following I/R (27 ± 11.6, p < 0.01).

Effects of I/R on CAT1 mRNA (CAT1/GAPDH Optic Density)

Glomeruli

Glomerular CAT1 mRNA expression before I/R was significantly higher in non-diabetic rats (1.86 ± 0.22, 1.25 ± 0.16, respectively; p < 0.05; see Figure 2A and Table 1). I/R caused a significant decline in glomerular CAT1 mRNA expression in diabetic rats (1.14 ± 0.3, p < 0.05), as opposed to non-diabetic rats, which showed no significant change in glomerular CAT1 mRNA expression following I/R (1.19 ± 0.25).

Figure 2. Glomerular and tubular CAT1 and CAT2 mRNA expression before and after I/R in normal and diabetic rats: A, glomerular CAT1; B, tubular CAT1; C, glomerular CAT2; D, tubular CAT2.

Tubuli

Tubular CAT1 mRNA expression before I/R was significantly lower in diabetic rats compared to non-diabetic rats (0.64 ± 0.11, 0.92 ± 0.15, respectively; p < 0.05; see Figure 2B and Table 1). I/R did not changed significantly tubular CAT1 mRNA expression in diabetic or non-diabetic rats (0.76 ± 0.26, 0.88 ± 0.24, respectively).

Effects of I/R on CAT2 mRNA (CAT2/GAPDH Optic Density)

Glomeruli

No significant differences have been found in glomerular CAT2 mRNA expression between diabetic and non- diabetic rats before I/R (0.77 ± 0.6, 0.72 ± 0.22, respectively; see Figure 2C and Table 1). I/R did not cause significant changes in glomerular CAT2 mRNA expression in diabetic or non-diabetic rats (0.79 ± 0.59, 0.63 ± 0.45, respectively).

Tubuli

No significant differences have been found in tubular CAT2 mRNA expression between diabetic and non-diabetic rats before I/R (0.46 ± 0.1, 0.56 ± 0.23, respectively; see Figure 2D and Table 1). I/R did not cause significant changes in tubular CAT2 mRNA expression in diabetic rats (0.43 ± 0.23). On the contrary, in non-diabetic rats, I/R caused a significant increase in tubular CAT2 mRNA expression (0.76 ± 0.11, p < 0.05).

Effects of I/R on iNOS mRNA (iNOS/GAPDH Optic Density)

Glomeruli

Glomerular iNOS expression in diabetic rats before I/R was significantly higher compared to non-diabetic rats (1.16 ± 0.33, 0.76 ± 0.35, respectively; p < 0.05; see Figure 3A and Table 2). I/R caused a significant increase in glomerular iNOS mRNA expression in diabetic rats (1.54 ± 0.41, p < 0.01). I/R also caused a significant increase in glomerular iNOS expression in non-diabetic rats (1.88 ± 0.28, p < 0.05). However, the increased glomerular iNOS mRNA expression was modest in the diabetic rats.

Figure 3. Glomerular and tubular iNOS, eNOS, and bNOS mRNA expression before and after I/R in normal and diabetic rats: A, glomerular iNOS; B, tubular iNOS; C, glomerular eNOS; D, tubular eNOS; E, glomerular bNOS; F, tubular bNOS.

Table 2 Glomerular and tubular NOS isoforms mRNA expression before and after I/R

	Glomeruli		Tubuli
	Normal	Diabetes	Normal	Diabetes
iNOS mRNA expression
Before I/R	0.76 ± 0.35	1.16 ± 0.33^†^‡	0^§	0^§
After I/R	1.88 ± 0.28	1.54 ± 0.41	0.67 ± 0.11^¶	0.16 ± 0.1
eNOS mRNA expression
Before I/R	0.74 ± 0.15	0.47 ± 0.12^†	0.85 ± 0.18^│	0.53 ± 0.08^†
After I/R	0.57 ± 0.14	0.58 ± 0.27	0.68 ± 0.12	0.57 ± 0.28
bNOS mRNA expression
Before I/R	1.15 ± 0.10	1.14 ± 0.24	0.75 ± 0.08	0.41 ± 0.21^†
After I/R	0.94 ± 0.12	1.19 ± 0.30	0.71 ± 0.09	0.59 ± 0.30

*p < 0.05 vs. normal I/R.

^†p < 0.05 vs. normal.

^‡p < 0.01 vs. diabetic I/R.

^§p < 0.001 vs. normal I/R and diabetic I/R.

^¶p < 0.001 vs. diabetic I/R.

^│p < 0.01 vs. normal I/R.

Tubuli

Tubular iNOS mRNA expression was undetected before I/R in both diabetic and non-diabetic rats. I/R caused a significant iNOS mRNA expression increase in diabetic rats (0.16 ± 0.1, p < 0.01; see Figure 3B and Table 2). I/R also caused a significant increase in tubular iNOS mRNA expression in non-diabetic rats (0.67 ± 0.11, p < 0.001). However, the increased tubular iNOS mRNA expression was significantly lower in the diabetic rats (p < 0.001).

Effects of I/R on eNOS mRNA (eNOS/GAPDH Optic Density)

Glomeruli

Glomerular eNOS mRNA expression was significantly lower in diabetic rats before I/R, compared to non-diabetic rats (0.47 ± 0.12, 0.74 ± 0.15, respectively; p < 0.05; see Figure 3C and Table 2). I/R did not cause significant changes in glomerular eNOS mRNA expression in diabetic or non-diabetic rats (0.58 ± 0.27, 0.57 ± 0.14, respectively). There was a tendency to a decline in glomerular eNOS mRNA expression in non-diabetic rats after I/R, which did not reached a statistical significance.

Tubuli

Tubular eNOS mRNA expression was significantly lower before I/R in diabetic rats, compared to non-diabetic rats (0.53 ± 0.08, 0.85 ± 0.12, respectively; p < 0.05; see Figure 3D and Table 2). I/R did not cause a significant change in tubular eNOS mRNA expression in diabetic rats (0.57 ± 0.28). However, I/R in non-diabetic rats caused a significant decrease in tubular eNOS mRNA expression (0.68 ± 0.12, p < 0.01).

Effects of I/R on bNOS mRNA (bNOS/GAPDH Optic Density)

Glomeruli

No significant differences have been found in glomerular bNOS mRNA expression between diabetic and non-diabetic rats before I/R (1.14 ± 0.24, 1.15 ± 0.1, respectively; see Figure 3E and Table 2). I/R did not cause significant changes in glomerular bNOS mRNA expression in diabetic or non-diabetic rats (1.19 ± 0.3, 0.94 ± 0.12, respectively).

Tubuli

Tubular bNOS mRNA expression before I/R was significantly lower in diabetic rats compared to non-diabetic rats (0.41 ± 0.21, 0.75 ± 0.08, respectively; p < 0.05; see Figure 3F and Table 2). I/R did not cause significant changes in tubular bNOS mRNA expression in diabetic or non-diabetic rats (0.59 ± 0.3, 0.71 ± 0.09, respectively).

DISCUSSION

In a previous publication, Schwartz et al.[26] evaluated L-arginine uptake and L-arginine mRNA transporters expression in tubuli of rats with iARF. The iARF in rats is characterized by a 5–10-fold increase on tubular L-arginine uptake, 60 minutes after reperfusion. The increased tubular L-arginine uptake is through an augmented tubular expression of CAT2 mRNA lasting as long as 24 hours after reperfusion. It was also accompanied with increased tubular iNOS mRNA expression. It was assumed that in the stage immediately after reperfusion, there is a significant renal L-arginine uptake, leading to augmented iNOS expression and activity, and increased renal NO generation. NO immediately is oxidized to peroxy-nitrite, due to the presence of free oxygen radicals after the ischemia and reperfusion. This toxic radicals cause severe damage to tubular cell membranes.

The present work is the first study that evaluated the tubular NO system in diabetic rats. We have found that there were no differences in tubular L-arginine transport in diabetic rats. However, these rats have a decreased expression of the constitutive NOS isoforms (eNOS, bNOS) and decreased tubular CAT1 mRNA expression.

The main findings in the present study were a different response of the NO system to 60 minutes of I/R in diabetic rats, compared to non-diabetic rats. At the glomerular level, I/R caused a significant decline in glomerular L-arginine transport, compared to no change in non-diabetic rats. This decreased glomerular transport after I/R was accompanied by a decreased glomerular CAT1 mRNA expression in the diabetic rats.

I/R induced activation of iNOS both in diabetic and non-diabetic rats, but was modest in the diabetic rats. I/R did not affect the glomerular expression of the constitutive NOS isoforms in diabetic or non-diabetic rats.

At the tubular level, I/R did not cause the expected increase in tubular L-arginine uptake in diabetic rats, as in non-diabetic rats. The elevation in tubular CAT2 mRNA expression was not detected in the diabetic rats. Furthermore, I/R caused only a slight elevation in tubular iNOS mRNA expression in the diabetic rats.

In the diabetic rats, there is an activation of the NO system even before the I/R. The glomerular findings support an elevated intra-glomerular NO production state, probably due to CAT1 activation and augmented glomerular L-arginine transport. The elevated NO production by glomerular iNOS, causes pre-glomerular vasodilatation (afferent). In parallel to these changes, there is a decreased constitutive NOS isoforms expression, leading to post-glomerular vasoconstriction.

In diabetic rats, some degree of tubular ischemia is already present before the I/R, either from the glomerular changes described, or due to the decreased tubular constitutive NOS activity. Furthermore, L-arginine availability to tubular cells is decreased due to decreased L-arginine transport and uptake.

I/R causes only a slightly elevation in tubular iNOS expression, along with blunted NO production because lack of substrate. This leads to worsened tubular vasoconstriction and tubular ischemia.

The combination of the glomerular and tubular changes in the L-arginine transport and NOS isoforms could explain the severe renal injury in diabetic rats after I/R, already in the earliest stages of iARF. We assume that in the diabetic rats, the renal injury after I/R is combined glomerular and tubular, in contrast to non-diabetic rats, where the renal injury after I/R is milder and mainly tubular.