Abstract
Objectives:
Colistin is a vital antibiotic used in multidrug-resistant infections. Its most important side effect is nephrotoxicity. Colistin is a weak acid. This study aims to evaluate whether urine alkalinization is protective in the nephrotoxicity of colistin.
Methods:
Twenty-eight male Sprague-Dawley rats were divided into groups. Group I (n = 4) was injected with intramuscular distilled water twice a day for 7 days. Group II (n = 8) was injected with 750,000 IU/kg/day colistin for 7 days. Group III (n = 8) was injected with the same dose of colistin after their urinary pH was ≥7 through the addition of bicarbonate in their drinking water. Group IV (n = 8) was injected with the same dose of colistin after their urine density fell below 1010 through the addition of NaCl molds in their food and 12.6 mg/L NaCl in their drinking water.
Results:
According to tubular degenerations (scored 0–5), group I scored 0, group II scored 4.25, group III scored 2, and group IV scored 1.5. In groups III and IV, protection was achieved (p = 0.001). The bicarbonate group was not superior to the NaCl group (p = 0.789). In transmission electron microscopy, group III had more microvilli integrity and autophagic vacuoles compared to group IV. Group IV had mitochondrial swelling and cristae lysis. A lower urine density was related to lower tubular scores (p = 0.001).
Conclusions:
Colistin was highly nephrotoxic without protection. Light microscopy findings revealed that urinary alkalinization and NaCl hydration were similarly protective. Urine alkalinization further prevents ultrastructural changes as revealed by electron microscopy.
Introduction
Colistin resurrected as a potent alternative for infections due to extensively resistant gram-negative bacteria. 1 Colistin is eliminated mainly by renal excretion: up to 80% of colistin undergoes renal tubular reabsorption. The primary side effect that limits its use is nephrotoxicity. In many studies, the incidence of nephrotoxicity in therapeutic doses was reported to be 33% and 61%. 2,3
Recent studies have suggested that the nephrotoxicity of colistin is related to its chemical structure. The colistin molecule has a
Colistin is a cyclic cationic decapeptide, bound to a fatty acid with an α-amide bond. It is a weak acid (with a pKa value of 10) and ionizes partially in the water. The mammalian cell membrane is more permeable to liposoluble substances and ionized forms than nonionized forms. Diffusion from the renal lumen to renal tubular cells is minimal when a substance is maximally ionized. As a weak acid’s ionization can be increased in an alkaline pH, its renal excretion can consequently be increased by urine pH manipulation.
Urine alkalinization is a safe and easy way which could be implemented in clinical practice easily, and nephroprotection with sodium bicarbonate is widely and safely used for many indications, such as intravenous contrast exposure and high dose methotrexate treatment. 9 – 13 Also, urine alkalinization is the preferred treatment for toxicities with weak acid substances. 13 –18
In this study, we evaluated the use of urine alkalinization as protection on colistin nephrotoxicity. Sodium bicarbonate indigestion causes thirst and high water intake. To distinguish whether the protective effects are due to alkaline urine pH or high water intake caused by sodium bicarbonate, a sodium chloride (NaCl) group was used for comparison. NaCl was used to enhance water intake in the comparison group.
Materials and methods
Ethical statements
Ethical board permission was acquired from Hacettepe University Animal Research Ethical Board at March 10, 2015 meeting with number 2015/23-02.
Antimicrobial agent and dosage
The antimicrobial agent used in this study is Colimycin® 150 mg IM/IV flacon (Kocak Farma Ilac ve Kimya Sanayi A.S., Turkey). One vial of Colimycin contains 150 mg colistimethate sodium, which is the equivalent of 4,500,000 IU colistin; it spontaneously converts to its active form (colistin) in serum.
In humans with normal kidney function, the therapeutic dose of colistin is 150,000 IU/kg/day, which is highly nephrotoxic even in normal kidney functions. 19 Plasma protein binding of 55% was reported for colistin in rats and 15% in humans. Therefore, in rats, similar levels for nephrotoxicity require higher doses. 20 In recent studies, doses of 300,000, 450,000, and 750,000 IU/kg/day were used to generate nephrotoxicity. 20 –22 In this study, 750,000 IU/kg/day of colistin was used to achieve nephrotoxicity within 7 days of drug administration without acute neurotoxicity.
Each vial was diluted with 6 mL of distilled water; 0.05 mL of solution was injected for every 100 g of the weight of the animal twice a day.
Animals and laboratory conditions
Twelve-week-old male Sprague-Dawley rats, weighing 350 g (±20 g), were obtained from Hacettepe University Laboratory Animal Research Center. Rats were kept in a controlled environment (temperature 22 ± 2°C and 12-h light/dark cycles). Animals were randomly divided into four groups, and each group kept in standardized cages. Rats were given ad libitum access to food and water.
Urinary alkalinization and daily urine sampling
For urinary alkalinization, sodium bicarbonate was added to the rats’ drinking water. The amount of sodium bicarbonate was calculated according to the doses recommended for urinary uric acid stones in rodents. The recommended dose is 0.15 mol/L (12.6 mg/L). Considering that rats drink 30–50 mL of water daily, the maximum starting dose was approximately 630 mg/day for each animal. Every day, urine samples were collected from each animal via bladder stroking. Urine samples were checked for density and pH with Roche Combur 10 Test® dipsticks. Sodium bicarbonate was increased while taking into consideration the acute oral lethal dose for rats (LD50: 4000 mg/kg) until reaching alkaline urine (pH ≥ 7).
Study design
Animals were randomly divided into four groups (Table 1). Group I (control) had 0.1 mL of intramuscular sterile distilled water injection twice a day for 7 days. Group II (colistin group) had intramuscular colistin injection of 750,000 IU/kg/day divided into two doses for 7 days. Group III (colistin and bicarbonate) had sodium bicarbonate in their drinking water. Their urine was collected every day and checked by dipstick for density and pH. The amount of sodium bicarbonate was increased until reaching alkaline urine (pH ≥ 7). After reaching alkaline urinary pH, the same doses of colistin were injected for 7 days. Their urinary density remained <1010 for 7 days due to excess sodium and water intake. Group IV (colistin and NaCl) had NaCl in their drinking water (initial dose of 12.6 mg/L) and NaCl molds in their food. Their urine was collected every day and checked by dipstick for density and pH. The amount of NaCl in the drinking water was increased until the urine density reached and remained at <1010. After that, the same doses of colistin were injected for 7 days.
Study groups and study design.
Material collecting procedure
For each animal, during the study, daily fresh urine samples were collected via bladder stroking and rapidly checked with a dipstick. After the study, the animals were anesthetized with inhaler sevoflurane, and blood samples were collected via cardiac puncture. Rats were euthanized by immediate decapitation, and their kidneys were removed. Serum specimens were allowed to clot by leaving undisturbed at room temperature for 30 min. Clot removed by centrifuging samples at 3500 r/min for 10 min. Serum immediately transferred into a polypropylene tube using a Pasteur pipette and stored at −80°C.
Biochemical examination
Serum urea and creatinine values were examined in properly preserved serum samples in 24 h. Erba Mannheim® instruments and Erba Mannheim® Crea 275 and Urea 275 reagents were used in this study.
Kidney sample preparation and histopathological examination
Light microscopy
Kidney tissues were fixed in 10% neutral buffered formalin for 48 h. Tissue samples were taken into Leica TP 1020 tissue processor for preparation for light microscopy. Dehydration was achieved by immersing tissues in a series of alcohol solutions (70%, 80%, 90%, 96%, and 100%). Samples were incubated in xylol twice for 1 h for clearing and treated with paraffin for 2.5 h. They were embedded in paraffin using a Leica Eg1150H embedding station, and they were cut into 5-µm sections. Sections were kept in the incubator overnight at 60°C and deparaffinized with xylol for 45 min. They were rehydrated with a series of alcohol solutions (100%, 96%, and 80%). They were stained with hematoxylin–eosin and then dehydrated with alcohol. After clearing with xylol, specimens were covered with mounting media (Entellan, Merck).
For periodic acid–Schiff (PAS) staining, sections were kept in the incubator overnight at 60°C and deparaffinized with xylol for 45 min. They were rehydrated with a series of alcohol solutions (100%, 96%, and 80%), each for 10 min, and rinsed in tap water. The sections were exposed to the periodic acid solution for 5 min and rinsed in tap water. Then the sections were exposed to Schiff’s reagent for 5 min and rinsed in tap water. They were stained with hematoxylin for 2 min and then dehydrated with alcohol. After clearing with xylol, specimens were covered with mounting media (Entellan, Merck). At least three microscopic areas of both glomeruli and tubuli were examined by two investigators (IU and FFK), and tubules were scored blindly using light microscopy (Leica DM6000 B trinocular upright research microscope, Wetzlar, Germany). Tubular degeneration was scored from 0 to 5, as described in Table 2. 23
Renal tubular degeneration scoring system. 23
Transmission electron microscopy
Kidney tissues were quickly separated into 1 mm3 pieces. They were fixed with 2.5% glutaraldehyde solution in phosphate buffer at 4°C for 24 h and postfixed with 1% osmium tetroxide in phosphate buffer for 1 h in the dark. After fixation, tissue samples were placed in the Leica EM TP tissue processor for preparation. Tissues were washed with a buffer solution for 45 min, and then they were dehydrated in a graded series of solutions: ethanol to absolute ethanol. Tissue samples were treated two times with propylene oxide for 15 min. They were infiltrated with a 3:1 mixture of propylene oxide: epoxy resin for 2 h, a 1:1 mixture of propylene oxide: epoxy resin for 2 h, a 1:3 mixture of propylene oxide: epoxy resin for 8 h, and a 10-h infiltration in 100% epoxy resin. Tissue samples were embedded in fresh epoxy resin and polymerized for 48 h at 60°C. One micrometer semi-thin sections were obtained using a Leica RM 2265 microtome and stained with 1% methylene blue-azure II in 1% borax solution. Representative areas were selected by light microscopy (Leica DM 6000B), and 70-nm ultrathin sections were cut using a Leica Ultracut R microtome. Ultrathin sections were double stained with uranyl acetate and lead citrate (Leica EM AC 20, Wetzlar, Germany). These sections were examined in a JEOL-JEM 1400 electron microscope and photographed by a charge-coupled device camera (Gatan Inc., Pleasanton, California, USA). Three random rats’ kidney tissues were selected from each group and were prepared; a minimum of 10 slides was examined by two blinded investigators (IU and FFK).
Power and statistical analysis
In this study, it is aimed that the groups’ variation coefficients were 10%, the differences between two groups were at least 20% (e.g. for creatinine, a difference of 0.002 mg/dL) to reach statistical significance; with 90% power and 5% type-1 error; it is calculated that each group should contain at least six rats. Study groups were designed with eight rats to compensate losses; the control group was formed with four rats.
Numeric values of the study were shown with mean and standard deviation, categorical data, frequency, and percentage. Statistical comparisons between groups were evaluated with Kruskal–Wallis for more than two groups and with Mann–Whitney U for two groups for biochemical measurements, urinary parameters, and tubular degeneration scores. Multiple groups were analyzed using a one-way analysis of variance with a Bonferroni test for post hoc analysis. A p value lower than 0.05 was considered a statistically significant result in all comparisons. SPSS 21 (IBM Inc., Armonk, New York, USA) was used for the analyses of the study.
Results
After starting the study protocol, a total of four rats (two rats from group III and two rats from group IV) immediately died after the first injection of colistin. Necropsy revealed no gross pathology. These subjects were presumed to die due to anaphylaxis. All other rats completed the study without further problems.
Serum urea and creatinine
Mean serum creatinine levels in groups I–IV were similar (cre: 0.68 ± 0.8; 0.68 ± 0.33; 0.56 ± 0.08; 1.06 ± 0.65 mg/dL; respectively; p = 0.131). Urea levels of the groups were 40.55 ± 6.24; 62.11 ± 42.69; and 62.3 ± 16.28 mg/dL for groups I, II, and III respectively, and similar between these three groups (p = 0.002). Group IV had significantly high mean urea of 109.51 ± 55.95 mg/dL (p = 0.001). Groups’ mean serum urea levels were not clinically correlated with histopathologic scores. Mean serum urea level of group II that had advanced tubular degeneration was similar to group III and lower than IV that had mild tubular degeneration (Table 3).
Urinary parameters, serum biochemistry, and tubular degeneration scores of groups.
I: group I (control group); II: group II (colistin group); III: group III (colistin and bicarbonate group); IV: group IV (colistin and NaCl group); SD: standard deviation; TDS: tubular degeneration score.
Histopathological examination
Light microscopy
Light microscopic findings of glomeruli were as follows: The control group (group I) had normal glomeruli; the unprotected colistin group (group II) had slightly increased mesangial matrix; and the alkaline urine group (group III) and saline protection group (group IV) had a minimal decrease in urinary space.
In group I, tubules and glomeruli had no evidence of injury (grade 0). The kidney samples showed normal tubule epithelium (Figure 1(a)). PAS staining showed the prominent brush borders of the proximal tubular epithelium (Figure 1(e)). There was no vacuolization, cylinders, necrosis, interstitial edema, or infiltration.
Light microscopic findings of renal tubuli. (a) Normal structure of renal tubular epithelial cells, no marked injury is visible, group I; (b) spilled epithelial cells in tubule lumen (arrows), vacuolization, and intense necrosis (asterisks), group II; (c) basal membrane separation (arrow) and epithelial thinning (asterisk), group III; (d) dilatation in fewer tubuli, separation between epithelial cells and interstitial edema (arrows), group IV; (e) normal structure of brush border (arrows), proximal tubular epithelium, group I; (f) disorganization of brush border in the proximal tubule epithelium (asterisks), thinning of the epithelium, separation of the lateral connection of epithelial cells (arrows), group II; (g) cast formations (asterisk), vacuolization (arrows), necrosis, and tubular epithelial thinning, group III; and (h) brush border thinning (arrows), proximal tubular epithelium, group IV ((a–d) hematoxylin–eosin, (e–h) periodic acid–Schiff, scale bar: 50 µm).
The kidney specimens of unprotected group II showed varying proportions of tubular degeneration from mild to severe (grade 3–5). Tubular necrosis was evident with degeneration, necrosis, and cast formation. Spilled tubule epithelial cells, thinning of the epithelium, and dilated tubules were observed (Figure 1(b)). PAS staining revealed disorganization of the brush border in the proximal tubule epithelium. In some areas, the tubule epithelial cells separated from lateral connections and also separated from the basement membrane (Figure 1(f)). Vacuolization, the presence of casts, congestion in the veins, necrosis, pyknotic nuclei, and interstitial edema were common findings.
In group III, less tubuli were affected, and mild-to-moderate tubular degeneration was observed (grade 1–4). In some areas, basal membrane separation and epithelial thinning were present. The tubule epithelial cells had eosinophilic cytoplasm and pale basophilic stained accumulations in the tubule lumen (Figure 1(c)). The cast formations, vacuolization, and necrosis were present in some regions, but these findings were less prominent than in group II (Figure 1(g)).
In group IV, dilatation in fewer tubuli, separation between epithelial cells, and slightly eosinophilic tubule epithelial cells were observed. In some areas, interstitial edema was prominent, and exudative material was present in tubules (grade 1–2) (Figure 1(d)). PAS staining showed brush border thinning of the proximal tubular epithelium (Figure 1(h)).
Tubular degeneration scores based on the light microscopic findings of the groups are calculated and shown in Table 3. According to tubular degeneration, average scores were as follows (scored from 0–5, where 0 is normal and 5 is histological necrosis): group I scored 0, group II scored 4.25 ± 0.89, group III scored 2 ± 1.26, and group IV scored 1.5 ± 0.55.
Post hoc analysis showed that group II had a significantly different tubular degeneration score from groups III and IV (p = 0.016 and p = 0.001, respectively). There was no significant difference between groups III and IV (p = 0.789) (Table 3).
Transmission electron microscopy
The kidney samples of group I showed normal tubule epithelium. There was no vacuolization, cylinders, necrosis, interstitial edema, or infiltration. In glomeruli, the urinary spaces were open (Figure 2(a)), and normal structure of capillaries with podocytes was observed. The renal tubular epithelial cells had prominent microvilli. They showed characteristic, ovoid mitochondria with densely packed cristae (Figure 3(a)).
Ultrastructural changes in glomeruli. (a) Normal structure of glomeruli with open urinary space, group I (m: mesangial matrix); (b) increase in mesangial matrix, group II; (c) podocyte hypertrophy and hyperplasia, decrease in urinary space, congestion in capillaries (arrows), group III; and (d) decreased in urinary space, congestion in capillaries (arrows), group IV; ((a–d) renal cortex, uranyl acetate, and lead citrate (scale bar: 2 µm) (m: mesangial matrix, p: podocyte)).
Ultrastructural changes in renal tubular epithelial cells. (a) Normal ovoid mitochondrial structure with densely packed cristae in tubular epithelium (arrows), group I (N: nucleus); (b) mitochondrial swelling, electron-dense accumulations in basement membrane (thick arrows), vacuolization and pseudocysts in parietal epithelial cells of Bowman capsule (asterisk), group II; (c) prominent microvilli (thin arrows), autophagolysosomes (thick arrows) in tubular epithelial cells, mitochondrial swelling, group IV; and (d) increased number of mitochondria, irregular mitochondrial shapes, and fragmented cristae – mitochondrial cristae lysis (arrows) ((a–d) renal cortex, uranyl acetate, and lead citrate; scale bar: 1 µm for (a) and (b), scale bar: 2 µm for (c) and (d) (N: nucleus)).
In the colistin only group (group II), the mesangial matrix was increased in glomeruli, and urinary spaces were narrowed. The glomerular capillaries were occluded (Figure 2(b)). Cytoplasmic vacuoles and pseudocysts in the parietal epithelial cells of the Bowman capsule were observed. Severe tubular degeneration, including mitochondrial swelling, cristae lysis, and vacuolization, were observed. There were electron-dense accumulations in the tubular basal membranes (Figure 3(b)). Interstitial edema and epithelial casts in the collecting tubule lumen were seen. Perinuclear cisternae were dilated and, secondary lysosomes were observed in the cytoplasm. Vacuolization in endothelial cells and congestion in the veins and peritubular capillaries were examined. Tubule epithelial cells were detached from the basement membrane.
In the alkaline urine group (group III), less tubuli were affected than in the unprotected group. Vacuolizations were fewer in tubular epithelial cells. Podocyte hyperplasia and hypertrophy in glomeruli, decreased urinary space, and congestion in the glomerular capillaries were common findings (Figure 2(c)). The detachment of the tubular epithelial cells from the basal membrane and interstitial edema was rarely observed in comparison to the group II. In some tubular epithelial cells, the microvilli structure was more protected. Ovoid mitochondria were seen in tubule epithelia. In some epithelial cells, mitochondrial swelling and autophagic vacuoles were of varied sizes (Figure 3(c)).
In the saline hydration group (group IV), podocyte proliferation and hypertrophy in glomeruli were observed. In some tubuli, an increased number of mitochondria was seen. Mitochondrial swelling and cristae lysis were present frequently. Brush border loss was prominent. In some areas, swelling of interstitial cells and fatty changes in the tubular epithelium were observed. Congestion in capillaries and a decrease in urinary space were observed in glomeruli (Figure 2(d)). The renal tubular epithelia of the saline hydration group showed an increased number of mitochondria, with irregular mitochondrial shape and fragmented cristae (Figure 3(d)). Fewer autophagolysosomes and more severe mitochondrial pathological changes were observed in this group.
The relationship between urinary density and tubular degeneration scores
Table 4 shows the relationship between daily measured urinary density scores and tubular degeneration scores of all rats, independently of the groups. Tubular degeneration scores of the rats those with urinary density under 1010 were mainly first and second degree. Adversely, rats with higher urinary density were scored third to fifth degree. This finding is statistically significant (p = 0.001).
Comparisons of tubular degeneration scores and mean urinary density independent of groups.
aRenal tubular degeneration scoring system. 23
Daily urinary findings and statistical analysis of the study
Differences between the groups that were analyzed included daily urine pH, daily urinary densities, serum creatinine and urea, and tubular degeneration scores. Daily urinary pH values were different and statistically significant. Groups I, II, and IV had an acidic urinary pH (<7), and group III had an alkaline urinary pH (≥7). Daily urinary densities between groups were significantly different. Groups I and II had high a urinary density of >1010 and groups III and IV had a low urinary density of <1010 (p < 0.001). In post hoc analysis, tubular degeneration scores were significantly higher in unprotected group II than in both groups III and IV (p = 0.008 and p = 0.002, respectively). Groups III and IV were similar in terms of light microscopic tubular degeneration scoring (p = 0.601) (Table 3).
Discussion
Since multidrug-resistant gram-negative bacteria became prevalent in after the 1990s, colistin began to be used widely, despite its nephrotoxicity. Prevention of colistin nephrotoxicity has become a focus of interest. Colistin is eliminated mainly by renal excretion. Up to 80% of colistin undergoes renal tubular reabsorption.
24,25
The nephrotoxicity is related to colistin’s antimicrobial mechanism. As a consequence of
Ghlissi et al. studied astaxanthin and vitamin E as antioxidants against colistin nephrotoxicity in rats’ kidneys and found that they were protective. 20 Yousef et al. demonstrated the protective effects of melatonin in rat kidneys with colistin exposure. 28 The well-known antioxidant N-acetylcysteine failed to improve nephrotoxicity in rats. 21 In another study, the administration of grape seed proanthocyanidin extract, a natural antioxidant, with colistin significantly diminished blood urea nitrogen and creatinine levels, renal histopathological score, caspase 1 and 3, calpain 1, and iNOS and eNOS staining compared to the control group. 5 Recently, known antioxidants, such as methionine, taurine, hesperidin, chrysin, and curcumin, have been shown to improve colistin’s antioxidant effects in animal models. 29 –32 The majority of these studies focused on an additive antioxidant agent for protection. Limited studies investigated possible nephroprotection against colistin nephrotoxicity via inhibiting renal tubular reuptake of colistin. Takahiro et al. studied a potential ligand of megalin receptors (cytochrome c) as a possible competitive inhibitor of colistin’s tubular reabsorption and found the ligand’s coadministration could be preventive in rats. 33
In this study, we aimed to evaluate the possibility of nephroprotection through enhanced excretion of colistin in urine and diminished reuptake from the renal tubuli. Urinary alkalinization enhances urinary excretion of weak acids, traps weak acids ionized state (ion trapping), and prevents reabsorption by renal tubules. 34 Urinary alkalinization against weak acid toxic drugs (e.g. methotrexate and salicylates) is widely used. 15 –18 Colistin is also a weak acid. In this study, we evaluated the effect of urine alkalinization, compared with hyper-hydration as a result of excess NaCl intake. We explored whether urinary dilution of the drug with enhanced thirst or urine alkalinization with NaHCO3 is protective against nephrotoxicity.
Our results demonstrate that colistin nephrotoxicity can be prevented through NaCl hydration and urine alkalinization. Glomerulopathy is not prominent in colistin nephropathy, neither in previous reports nor in our study under light microscopy. Thus, histopathologic scoring systems primarily focus on tubular changes. Light microscopy scores of the renal tubule epithelium did not reveal any difference between intervention groups (groups III and IV). Although we do not have a reliable scoring system for electron microscopy findings, our results are compatible with favorable ultrastructural outcomes in group III (alkaline urine) compared with group IV (saline hydration). Group II showed an increase in the mesangial matrix, a pathologic reaction seen in various glomerular diseases. Mesangial changes seen following a glomerular injury lead to production of chemo-attractants for inflammatory cells, proliferation of mesangial cells, and excessive production of extracellular mesangial matrix. Contrary to light microscopy results, colistin exposure affected renal glomeruli, which were heralded by both light and electron microscopic findings. Both the urine alkalinization and the saline hydration group showed a decrease in urinary space and congestion in capillaries, which may be attributed to glomerular edema secondary to increased thirst and hydration. The alkaline urine group had podocyte hyperplasia and podocyte proliferation, which may be considered as regenerative efforts. The alkaline urine group had better microvilli integrity compared with the NaCl hydration group. NaCl hydration group had more mitochondrial swelling and had mitochondrial cristae lysis, which were not seen in the alkaline urine group. Significant autophagic vacuoles in the tubular epithelium of the alkaline urine group suggest a protective process because of its ability to prevent cell toxicity by autophagy. Autophagy is a lysosomal degradation pathway and is an important survival mechanism during short-term stress on mammalian cells. By degrading some nonessential components, cells produce nutrients for vital reactions. This is an important cell homeostasis mechanism for development, growth regulation, longevity, and previously implicated in colistin-induced nephrotoxicity, and neurotoxicity. 7,8,35,36 Urine alkalinization seems to generate favorable conditions for tubular cell survival. Our results do confirm and provide ultrastructural benefits for previous findings.
Our results also suggested other remarkable findings. Urinary density scores were directly related to tubular degeneration scores independent of groups or urinary pH results. All rats with a daily urine density below 1010 had minimal to slight tubular degeneration, and all rats with a daily urine density above 1010 had mild to severe tubular degeneration. Low urinary density, which suggests adequate hydration, is remarkably protective independent of urinary pH results. None of the rats with low urinary density (below 1010) had significant tubular toxicity. These findings may suggest that the dilution of the colistin molecule in the tubular lumen may prevent its contact with the tubular epithelium and protect against detergent effects. In clinical practice, it is not usually appropriate to intervene with large quantities of fluids, especially in septic patients in intensive care units: that is, the majority of patients infected by antimicrobial-resistant bacteria. On the other hand, excessive fluid intake in both protection groups in this study precipitated extracellular matrix and capillary cell edema and caused decreased urinary space. It is not known whether these changes are associated with irreversible damage. Further studies may be helpful to develop other strategies to dilute colistin in urine: for example, intravenous hydration with forced diuresis with loop diuretics.
Our study has some limitations. The short course of colistin injections hindered a significant BUN and creatinine increase. There was no correlation between the rats’ urea and creatinine values and histopathological findings. Normal rats’ creatinine measurement, on the other hand, is shown to be prone to overestimation regardless of laboratory methods and shown to range between 0.25 and 3.93 mg/dL. 37 Thus, serum urea and creatinine measurement may not be the test of choice in rats, especially when a short course is administered, as in this study. New and more sensitive biochemical markers would produce more precise findings.
To our knowledge, our study is the first study exploring the mechanisms of colistin-induced nephrotoxicity ultrastructurally. It is always challenging to translate basic research findings to the clinical setting. However, our results provide insights into easy and clinically available preventive measures for colistin nephrotoxicity. In light of the study’s findings, both saline hydration and urinary alkalinization are markedly protective against colistin nephrotoxicity when evaluated by light microscopy. When the samples are evaluated by electron microscopy, however, the urine alkalinization group had preferable findings.
In conclusion, colistin is highly nephrotoxic in rats without any protection protocol. Both saline hydration and urinary alkalinization are remarkably protective against colistin nephrotoxicity. However, alkaline urine generates preferable ultrastructural benefits. Massive hydration with any fluids aiming at low urinary densities (below 1010) is markedly protective against colistin nephrotoxicity.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: All expenditures of this study were supported by the Hacettepe University Scientific Research Project Unit.
