Determinants of preferential renal accumulation of synthetic polymers in acute kidney injury

Abstract

Acute kidney injury (AKI) is a major kidney disease associated with high mortality and morbidity. AKI may lead to chronic kidney disease and end-stage renal disease. Currently, the management of AKI is mainly focused on supportive treatments. Previous studies showed macromolecular delivery systems as a promising method to target AKI, but little is known about how physicochemical properties affect the renal deposition of polymers in ischemia-reperfusion AKI. In this study, a panel of fluorescently labeled polymers with a range of molecular weights and net charge was synthesized by living radical polymerization. By testing biodistribution of the polymers in unilateral ischemia-reperfusion mouse model of AKI, the results showed that negatively charged and neutral polymers had the greatest potential for selectively accumulating in I/R kidneys. The polymers passed through glomerulus and were retained in proximal tubular cells for up to 24 hours after injection. The results obtained in the unilateral model were validated in a bilateral ischemic-reperfusion model. This study demonstrates for the first time that polymers with specific physicochemical characteristics exhibit promising ability to accumulate in the injured AKI kidney, providing initial insights on their use as polymeric drug delivery systems in AKI.

Graphical Abstract

1. Introduction

Acute kidney injury (AKI) is characterized by an abrupt decline in the glomerular filtration rate over a period of minutes to days that results in accumulation of nitrogenous wastes. AKI is a major global health problem impacting about 13.3 million people annually (Mehta et al., 2015). About 1.7 million people die annually from AKI in the world (Lewington et al., 2013; Wen et al., 2010; Zuk and Bonventre, 2016). Long-term effects of AKI in patients may develop into chronic kidney disease and end-stage renal disease (Chawla et al., 2014; Chawla and Kimmel, 2012). Clinically, AKI is triggered by toxic responses to medications, ischemia which results from a reduction of blood flow in the kidney by decreased cardiac output (cardiac surgery), and sepsis (Alejandro et al., 1995; Bellomo et al., 2012; Rosner and Okusa, 2006). Although the initiating events may differ, similar pathways are involved in the subsequent injury responses. The coagulation system is locally activated when the kidney has energy failure or renal cells are exposed to toxic medications (Basile et al., 2012; Bellomo et al., 2012). Leucocytes infiltrate the kidney (Versteilen et al., 2011), inflammatory cytokines and chemokines are released (Akcay et al., 2010; Bonventre and Yang, 2011; Ozkok and Edelstein, 2014), and oxidative stress pathways are activated (Ratliff et al., 2016). The activation of coagulation, oxidative stress, and inflammatory infiltration lead to vascular endothelial cell damage and microvascular congestion (Arany and Safirstein, 2003; Faubel et al., 2007; Sharfuddin and Molitoris, 2011). The sustained activation of these pathogenic factors further amplifies the inflammatory cascades, leading to apoptotic cell death (Havasi and Borkan, 2011; Yao et al., 2007). In addition, renal injury exhibits a cross-talk effect with other organs in the body, emphasizing the complexity of the biological response (Doi and Rabb, 2016; Li et al., 2009).

Current management of AKI is focused on supportive treatment by achieving and maintaining hemodynamic stability and avoiding hypovolemia to assure adequate renal perfusion (Kellum and Lameire, 2013). When AKI has potentially life-threatening complications, such as uncontrolled symptomatic fluid overload and refractory hyperkalemia, renal transplantation is required (Chuasuwan and Kellum, 2011; Lameire et al., 2013). Based on recent advances in understanding the renal injury and repair signaling pathways, several pharmacological agents including antioxidants, anti-apoptosis or anti-inflammatory agents, growth factors and vasodilators have been tested (Benoit and Devarajan, 2017). However, no pharmaceutical agents have been successful in clinical trials so far (Fraga et al., 2016; Khwaja, 2012; McCullough et al., 2016).

Drug targeting to the proximal tubule cells, which are the primary injured site in AKI could provide better treatment by means of enhancing the therapeutic efficacy of drugs and lowering unwanted side effects in other organs. Synthetic polymers have been used as carriers to target proximal tubular cells by means of either physically encapsulating or covalently conjugating drugs (Duncan and Kopeček, 1984; Gunatillake and Adhikari, 2003; Li et al., 2015). Several studies have demonstrated that polymers have the potential to target proximal tubular cells and might be good candidates for drug carriers. Kamada et al. demonstrated that polyvinylpyrrolidone-co-dimethyl maleic anhydride had much higher accumulation and longer retention time in kidneys than native polyvinylpyrrolidone. The superoxide dismutase modified with these copolymers accelerated recovery from acute renal failure (Kamada et al., 2003). Mitra et al. studied the biodistribution of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with different molecular weight and charge (Mitra et al., 2004). They found that negatively charged 21 kDa HPMA copolymers showed highest renal accumulation 24 h after intravenous injection in mice. These findings proved that the type and content of anionic groups introduced to the polymers, as well as polymer molecular weight play a vital role for renal cells targeting. However, the reported studies were performed in healthy kidneys and very little is currently known about how physicochemical properties of polymers affect renal deposition in AKI. A recent study from the Pun’s lab on the effect of renal pathology on polymer accumulation suggested that in experimental focal segmental glomerulosclerosis, polymer accumulation increases with anionic monomer content but not molecular weight (Liu et al., 2018).

In this report, a panel of polymers with a range of molecular weights and different charges were synthesized. The cellular uptake and intracellular trafficking of polymers in mouse proximal tubular epithelial (MCT) cells were compared at normoxia and hypoxia condition. Additionally, by testing the biodistribution of each polymer in vivo, we explored and identified preferred polymer characteristics that lead to preferential accumulation in proximal tubular cells in AKI animal models.

2. Materials and methods

2.1. Materials

N-(3-Aminopropyl) methacrylamide hydrochloride (APMA) and N-(2-hydroxypropyl) methacrylamide (HPMA) were purchased from Polysciences, Inc. (Warrington, PA). Methacrylic acid (MAA) was obtained from Acros Organics (Belgium, NJ). 4, 4’-Azobis(4-cyanovaleric acid) (ACVA), 4-cyano-4-(phenyl carbonothioylthio) pentanoic acid (CPAD) and fluorescein O-methacrylate (FMA) were from Sigma-Aldrich (St. Louis, MO). RPMI-1640 medium and Phosphate Buffered Saline (PBS) were from Hyclone (Logan, UT). Fetal bovine serum (FBS) was from Atlanta Biologicals (Flowery Branch, GA). Mouse renal tubular epithelial cells (MCT) were a kind gift from Dr. Rick Schnellmann to Dr. Padanilam and cultured in RPMI supplemented with 10% FBS.

2.2. Synthesis of polymers

The polymerization was achieved at 70 °C, employing ACVA as the initiator and CPAD as the chain transfer agent (Scheme S1). A typical protocol was as follows: APMA (178 mg, 1 mmol) was dissolved in doubly distilled water followed by the addition of CPAD (6.24 mg, 0.02 mmol, target DP_n = 45), ACVA (2 mg, 0.007 mmol) and FMA (7.15 mg, 0.018 mmol) in 1,4-dioxane stock solution. The solution was added into a small glass vial and purged with nitrogen for at least 30 minutes. The vial was placed in an oil bath for polymerization at 70 °C for 30 min, 3 h, and 4.5 h. After the polymerization, the solvent was dialyzed against water for 3 days before final freeze-drying.

The polymerization of MAA and HPMA was conducted employing similar procedure described above. The feed ratio, reagent and reaction time was according to Table 1.

Table 1.

Polymer characterization

Polymer	Feed ratioa	Solvent	Polymerization Time (h)	Mn	Ð (Mw/Mn)b
pAPMA-6	150:3:1: 2.7	1,4-dioxane	0.5	6,110	1.17
pAPMA-16	150:3:1: 2.7	1,4-dioxane	3	16,450	1.12
pAPMA-30	150:3:1: 2.7	1,4-dioxane	4.5	30,490	1.13
pMAA-5	176:3:1:1.7	Methanol	24	5,170	1.09
pMAA-16	352:3:1:3.4	Methanol	24	16,340	1.05
pMAA-31	704:3:1:6.8	Methanol	24	31,390	1.02
pHPMA-5	103:3:1:1.4	Methanol	24	5,510	1.19
pHPMA-16	200:3:1:2.8	Methanol	24	15,790	1.02
pHPMA-36	400:3:1:5.6	Methanol	24	36,820	1.05

^aFeed molar ratio of monomer/CTA/ACVA/FMA

^bMw = weight-average molecular weight, Mn = number-average molecular weight

2.3. Polymer characterization

The molecular weights of pAPMA were analyzed by gel permeation chromatography (GPC) operated in 0.1 M sodium acetate buffer (pH 5.0) using Agilent 1260 Infinity LC system equipped with a miniDAWN TREOS multi-angle light scattering (MALS) detector and a Optilab T-rEX refractive index detector (Wyatt Technology, Santa Barbara, CA). The column used was TSKgel G5000PWXL-CP (Tosoh Bioscience LLC, King of Prussia, PA) at a flow rate of 0.5 mL/min. Results were analyzed using Astra 6.1 software from Wyatt Technology. The degree of polymerization was calculated from the GPC.

The characterization of pMAA was conducted employing the same method but using AquaGel PAA-202 (London, ON, Canada) column. The characterization of pHPMA was conducted employing the same method but using 0.3 M sodium acetate buffer (pH 5.0).

The amount of conjugated FMA was determined on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, CA). An amount of 1 mg of each dried polymer was dissolved in 1 ml PBS and the fluorescence intensity of the solution was measured at an excitation wavelength of 490 nm and an emission wavelength of 525 nm, which corresponds to a peak maximum for FMA. FMA content was determined by comparison to a FMA standard curve.

2.4. Cellular uptake and intracellular trafficking of polymers

Flow cytometry analysis was conducted to study the cellular uptake of polymers. MCT cells (3 × 10⁴) were seeded in 12-well plates and cultured to approximately 50% confluence. The cells were incubated in 37 °C at a virtual pAPMA concentration of 2 μg/mL, pMAA concentration of 300 μg/mL or pHPMA concentration of 1 mg/mL for 24 h in either normoxic or hypoxic (2% O₂) conditions. The cells were then washed with PBS twice, trypsinized and subjected to analysis using a BD FACS Calibur flow cytometer (BD Bioscience, Bedford, MA). The results were processed using FlowJo software.

Intracellular trafficking was observed by LSM 800 Laser Scanning Microscope (Zeiss, Jena, Germany). MCT cells were cultured on 24-well plates with round coverslip glass at 5 × 10⁴ cells/well. After 24 h, the medium was exchanged with fresh medium and a solution of the studied polymers was added (same concentrations as cellular uptake). After incubation for another 24 h in either normoxia or hypoxia incubator, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 min and nuclei were stained with Hoechst 33258 for another 10 min.

2.5. In vitro cytotoxicity of the polymers

Toxicity of the polymers was evaluated by Cell Titer Blue assay in MCT cells. The cells (5 X 10³ cells/well) were plated in 96-well microplates. After 24 h, the cultured cells were treated with fresh medium and different polymers respectively. After further 24 h of incubation at either normoxic or hypoxic condition, the medium was removed and replaced with a mixture of 100 μL of serum-free media and 20 μL of CellTiter-Blue reagent (CellTiter-Blue Cell Viability Assay, Promega). After 2 h of incubation, the fluorescence (560/590 nm) was measured on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, CA). The relative cell viability (%) was calculated as [fluorescence]_sample/[fluorescence]_untreated × 100%.

2.6. Induction of ischemia-reperfusion kidney injury

We used unilateral and bilateral renal ischemia-reperfusion (I/R) injury to study the polymer biodistribution as described previously (Jang et al., 2008; Jang et al., 2014). All animal experiments followed a protocol approved by the UNMC Institutional Animal Care and Use Committee. C57BL/6J male mice (~10 weeks old) were purchased from Charles River. All animals were given free access to food and water and fed non-fluorescent diet for at least 1 week before experiments. The mice were anesthetized by intraperitoneal administration of ketamine (200 mg/kg) and xylazine (16 mg/kg). For unilateral I/R injury, following left dorsal flank incision, renal pedicles were bluntly dissected and a microvascular clamp (Roboz Surgical Instrument, Gaithersburg, MD) was placed on the left renal pedicle for 30 min to induce unilateral I/R injury. During the procedure, mice were kept well hydrated with warm saline and on 37 °C heating pad. After 30 min of occlusion, the clamps were removed and kidney reperfusion was verified visually. Then wound clips were used to close skin. Sham-operated right side underwent the same surgical procedure, except for the occlusion of the renal arteries. The mice were monitored until they woke up. Similar procedure was conducted to induce bilateral I/R injury by placing microvascular clamp on both renal pedicle for 30 min. Sham-operated bilateral control animals underwent the same surgical procedure, except for the occlusion of the renal arteries. After bilateral I/R injury, concentrations of plasma creatinine and blood urea nitrogen were determined to evaluate kidney function by using QuantiChrom Creatinine or Urea Assay Kit (Bioassay Systems, Hayward, CA).

2.7. In vivo biodistribution of polymers

Biodistribution of the polymers in I/R AKI mice was analyzed by ex vivo fluorescence imaging. Polymers were administered via tail vein injection 24 h post-surgery. At 4 h and 24 h after administration, mice were sacrificed and major organs were isolated and imaged using Xenogen IVIS 200. Emission wavelength of 540 nm and excitation wavelength of 500 was used to image the organs. The fluorescence intensities from liver and kidney were quantified using Living Image^® 4.5 software. The radiant efficiency of the kidney and liver was measured as (photons/sec/cm²/sr)/(μW/cm²). Background fluorescence was subtracted prior to analysis. To study intracellular localization of the polymers, bisected kidneys were embedded in OCT compound and cut into 10-μm frozen sections. The sections were either stained with DAPI or with haematoxylin and eosin (H&E). Polymer localization was visualized by LSM 800 Laser Scanning Confocal Microscope (Zeiss, Jena, Germany).

2.8. Tissue homogenization

To quantify the amounts of polymers in I/R kidneys and sham-operated kidneys, kidneys dissected from C57BL/6J mice bodies (24 h post injection) were finely minced with scissors and placed in a homogenizer vessel. 750 μL RIPA buffer and Halt^™ protease & phosphatase inhibitor cocktail were added and tissues were subsequently homogenized. Homogenized samples were centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatant was used to quantify polymer amount in kidneys by standard addition method using a fluorescence Symergy 2 Microplate Reader (BioTek, VT).

2.9. Statistical analysis

Data are presented as the means ± SD. An unpaired t test was used to compare the means of two different groups. A P value < 0.05 was considered statistically significant.

3. Results

3.1. Polymer synthesis and characterization

Synthetic polymers have been used as carriers of various therapeutic agents because of their ability to improve stability and favorably modulate pharmacokinetic and biodistribution properties (Yang et al., 2014; York et al., 2010; Yu et al., 2016). Biodistribution of polymers is influenced by their properties such as molecular weight and charge. We thus synthesized a panel of 9 fluorescently labeled polymers, including neutral pHPMA, negatively charged pMAA, and positively charged pAPMA, with reversible addition-fragmentation chain-transfer (RAFT) polymerization method to evaluate properties both in vitro and in vivo. Table 1 shows the RAFT polymerization condition of pAPMA, pMAA and pHPMA, in which polymers were prepared by copolymerization of the corresponding monomers with a fluorescein-containing co-monomer using CTA transfer agent and ACVA initiator at 70°C. The feed ratio of monomer/CTA/ACVA/FMA was variable according to different monomers. All polymers were characterized by GPC. The molecular weight range was from 5 kDa to 36 kDa with low dispersity (Ð) values (Table 1). The fluorescence of the synthesized polymers was used to track the subcellular fate and distribution of the polymers both in vitro and in vivo. The content of FMA was quantified for each polymer in order to enable the comparison of fluorescence data obtained from the different polymers. Examples of GPC curves of pAPMA-30, pMAA-5 and pHPMA-36 were shown in Figure 1, in which well-defined, monomodal and symmetric peaks were observed.

Figure 1.

GPC curves of (A) pAPMA-30, (B) pMAA-5 and (C) pHPMA-36.

3.2. Cytotoxicity and intracellular trafficking of polymers in proximal tubule cells

Safety of synthetic polymer carriers is of paramount importance, especially in conditions like AKI. Cytotoxicity of the polymers was first assessed in mouse renal proximal tubule cells MCT using CellTiterBlue assay (Table 2). Because hypoxia is a crucial feature of the renal ischemia-reperfusion injury model, we evaluated cytotoxicity at both normoxia and hypoxia conditions. As expected, the cytotoxicity was strongly dependent on the charge of the polymers and to a lesser extent on the molecular weight. No cytotoxicity was observed with the neutral pHPMA polymers at concentration up to 1 mg/mL at both normoxia and hypoxia. HpmA copolymers have been extensively examined and proven biocompatible, non-immunogenic, and non-toxic drug carriers (Kopeček et al., 2000; Yuan et al., 2008). The cationic pAPMA showed the highest cytotoxicity of all the tested polymers with IC₅₀ in the range of 4.7-10.6 μg/mL, while the anionic pMAA showed nearly complete lack of cytotoxicity with IC50 values above 500 μg/mL with increased toxicity observed in hypoxia.

Table 2.

IC₅₀ of different polymers in MCT cells at normoxia or hypoxia. Cell viability was measured by CellTiter blue after 24 h incubation at 37 °C.

Polymer	IC50 [μg/ml]
	Normoxia	Hypoxia
pAPMA-6	10.6	4.7
pAPMA-16	7.9	7.9
pAPMA-30	8.5	7.9
pMAA-5	1000.2	890.4
pMAA-16	859.5	657.0
pMAA-31	613.8	496.1
pHPMA-5	>1000	>1000
pHPMA-16	>1000	>1000
pHPMA-36	>1000	>1000

We then studied the effect of polymer properties and hypoxia on their uptake in the MCT cells. The cells were incubated with the polymers for 24 h and cellular uptake was determined by flow cytometry and expressed as the mean fluorescence intensity (MFI) ratio between normoxia and hypoxia (Figure 2). We have observed no hypoxia selectivity for the uptake of pAPMA with the normoxia-to-hypoxia ratio around 1. Similar observations was made with pMAA, although the lowest molecular weight showed about 2.8-fold increased uptake in hypoxia than in normoxia. Interestingly, pHPMA was taken up significantly more in hypoxic conditions than in the normoxic conditions regardless of the polymer molecular weight. This observation is partly affected by the overall lowest cell uptake of the neutral pHPMA.

Figure 2.

Cellular uptake of polymers in MCT cells. Quantification of cellular uptake is shown by mean fluorescence intensity (MFI) ratio between cells incubated at hypoxia and at normoxia after 24 h. Data are shown as the mean ± SD (n = 3).

We then used confocal laser scanning microscopy to validate the cell uptake and to examine intracellular trafficking of the polymers in hypoxia and normoxia. The MCT cells were incubated with different polymers for 24 h and then imaged by confocal microscopy (Figure 3). All polymers were clearly internalized inside the MCT cells and appeared mainly localized in vesicular structures consistent with their expected endocytic uptake mechanism. Compared with normoxia, there was no difference observed for cellular trafficking of pAPMA in hypoxia. For pMAA, a slightly higher intensity was only observed for pMAA-5 after hypoxic incubation. However, the intracellular fluorescence intensities of pHPMA were significantly elevated in hypoxia. The most significant difference was observed with pHPMA-36. The results of cellular uptake and intracellular trafficking indicated that MCT cells had increased cell uptake at hypoxia for certain polymers, which prompted us to further investigate the distribution in vivo.

Figure 3.

Intracellular trafficking of polymers in MCT cells by confocal microscopy after 24 h incubation. Scale bar 20 μm.

3.3. Selective accumulation of polymers in unilateral I/R injury

Polymer size and charge play an important role in kidney deposition because glomerulus basement membrane filters small molecules by size and charge. Generally, in healthy state, molecules with hydrodynamic diameter less than 5-7 nm or molecular weight less than 68 kDa can pass this barrier. However, the barrier can be broken by podocyte effacement and large or charged molecules could accumulate in the Bowman space due to the leaky and abnormal fenestrae after renal ischemia (Kamaly et al., 2016; Sutton et al., 2003; Wagner et al., 2008). Therefore, we hypothesized that renal distribution of polymers will be impacted by AKI and that this impact will depend on the size and charge of the polymers.

To understand how AKI affects the biodistribution, especially for renal accumulation, of polymers with different sizes and charges, we established a unilateral I/R kidney injury model, where left renal artery was clamped and renal injury was induced in the left kidney while the right kidney was only exposed and remained intact. Mice were sacrificed at two designated time points, 4 h or 24 h after intravenous administration, and major organs were isolated for ex vivo fluorescence imaging (Figure 4A). Generally, we found that reduced blood perfusion associated with the I/R kidney did not lead to decreased distribution of the polymers when compared with the unaffected sham-operated kidney. To the contrary, we found selected polymers displayed enhanced accumulation in the I/R kidney. Among negatively charged pMAA and neutral pHPMA, pMAA-5 and pHPMA-36 showed the most selective accumulation in the I/R kidney at 24 h. Using region of interest (ROI) ratio between I/R and sham-operated kidneys in the same animal, we found that these two polymers had about 4-times higher distribution to the injured kidney than to the normal one (Figure 4B). The higher the molecular weight of pMAA (pMAA-16 and pMAA-31) or the lower the molecular weight of pHPMA (pHPMA-5 and pHPMA-16), the lower the selectivity for the injured kidney at 24 h. Slow and occasionally retrograde blood flow after I/R injury and delayed clearance of macromolecules from the injured kidneys may have led to the delayed and increased accumulation at 24 h (Xu et al., 2017; Yamamoto et al., 2002). Pathological changes in kidneys after I/R injury, such as endothelial dysfunction, can also contribute to changes in the interactions between macromolecules and the renal tissue at cellular level and affect macromolecular transport (Brodsky et al., 2002; Xu et al., 2017).

Figure 4.

Polymer biodistribution in mice with unilateral I/R injury. (A) Ex vivo imaging of dissected organs at 4 or 24 h after administration. From left to right and top to bottom: lung, heart, liver, spleen, left kidney (I/R), and right kidney (sham). Region-of-interest (ROI) ratios of left to right kidney (B), or ROI ratio of left kidney to liver (C) at indicated time points. Data are shown as the mean ± SD (n = 5). **, p < 0.01.

The MAA and HPMA polymers did not appear to be taken up to a significant extent by any organ other than kidneys, especially for the two major organs of reticuloendothelial system (RES), liver and spleen (Figure 4A & 4C). In contrast, the positively charged polymers pAPMA displayed no apparent selectivity for the injured kidneys but showed expected high levers of hepatic accumulation. Positively charged macromolecules are usually susceptible to rapid renal elimination and high non-specific uptake in the liver (Gustafson et al., 2015; Wang et al., 2011). These results indicated that AKI has size- and charge-dependent effect on renal uptake of synthetic polymers.

Next, the ex vivo imaging data were validated by using homogenized tissues and a fluorescence measurement for quantification. To avoid possible contribution of auto-fluorescence from the tissues on the results, we analyzed the samples using standard addition method (Figure S1). Based on these data, it is evident that the ratio of each polymers amount between I/R and sham-operated kidneys was consistent with the ROI ratio from ex vivo results shown above. The highest ratio of polymers between I/R and sham-operated kidneys was observed in pMAA-5 and pHPMA-36, which accorded with previous IVIS results.

3.4. Cellular distribution of the polymers in unilateral renal I/R injury

To further understand the selective accumulation of the polymers in the injured kidney, we conducted cellular localization and histological studies on the both I/R and sham-operated kidney with frozen sections and H&E staining (24 h post injection), respectively (Figure 5 and Figure S2). Extensive tubular dilatation and cast formation were found in cortex of the I/R kidneys. We observed that all the tested polymers could pass glomerular filtration barrier and enter into Bowman space and be taken up by proximal tubule cells. The molecular weights of the polymers are generally below the threshold for macromolecules to cross the filtration barrier. Even though basement membrane has negative charge, it had no impact on the filtration of the polymers in either I/R or sham-operated kidneys. When getting into Bowman space, majority of positively charged pAPMA may be susceptible to rapid renal clearance and be quickly eliminated from kidney, while the excretion of negatively charged pMAA and neutral pHPMA from cortex to renal pelvis might be limited in I/R kidneys (Tantawy et al., 2012; Xu et al., 2017) and such polymers can be selectively reabsorbed and sustained by renal tubule cells which are the major damaged site during AKI.

Figure 5.

Confocal microscopy images and H&E staining of kidney sections (24 h post injection) isolated from mice with unilateral I/R injury receiving pMAA-5 (A) and pHPMA-36 (B).

3.5. Validation of renal accumulation in bilateral I/R injury model

The unilateral model of AKI is convenient for the study of selective accumulation in the injured kidney because of the presence of healthy contralateral organ as an internal control and because of the reduced risk of mortality due to the functional redundancy (Zager et al.). It was nevertheless necessary to validate the findings in the more conventional bilateral model (Figure 6). Bilateral renal I/R injury model is most frequently used in experimental studies because it is a clinically relevant model of AKI. We selected two polymers, pAPMA-30 and pHPMA-16 as examples of polymers with no selectivity and with high selectivity for the injured kidney in the unilateral AKI model. At 24 h after induction of the bilateral I/R injury, concentrations of plasma creatinine and blood urea nitrogen, indicators of kidney function, increased greatly compared with those of sham-operated mice, confirming that the model was generated successfully (Jang et al., 2012; Jang et al., 2008). The polymers were then injected intravenously as in the unilateral model. As demonstrated in Figure 6B, a strong fluorescence signal was observed in the I/R kidneys after administration of pHPMA-16 at 4 h and the signal was sustained at 24 h. Similar to the results from the unilateral I/R model, pAPMA-30 show no selective accumulation in either I/R or sham-operated kidneys at both 4 h and 24 h (Figure 6A). In addition, compared with the sham-operated kidneys, the quantified fluorescence intensity of the I/R kidneys increased ~2.1-fold and 2.5-fold for pHPMA-16 at 4 h and 24 h, respectively, on the basis of the ROI ratios. Alteration in renal hemodynamics for unilateral and bilateral renal I/R injury model might contribute to the differences in renal accumulation of pHPMA-16 at 4 h in the two models.

Figure 6.

Biodistribution of pAPMA-30 and pHPMA-16 in bilateral I/R renal injuary. I/R mice or sham-operated mice were given single injection of pAPMA-30 or pHPMA-16 at 24 h postsurgery. Ex vivo imaging of pAPMA-30 (A) and pHPMA-16 (B) taken at 4 h and 24 h after administration. Isolated organs: lung, heart, liver, spleen, left kidney and right kidney (from left to right and top to bottom). (C) Ratio of ROI between kidneys from I/R mice and sham-operated mice. Data are shown as mean ± SD (n = 3).

However, the overall results from the two AKI models confirm that polymers that accumulate selectively in the injured kidney in the unilateral model also tend to show elevated accumulation in the kidneys in the bilateral model. Hence, this study validated the unilateral I/R model as a viable approach to investigate renal accumulation of polymers in AKI.

4. Conclusion

In summary, unilateral renal I/R injury model allowed us to obtain the initial understanding of how AKI alters renal accumulation of synthetic polymers with various sizes and charges. Our data suggested that achieving the most selective accumulation in the I/R kidneys requires negatively charged or neutral polymers with low or intermediate molecular weight. These findings not only advance our fundamental understanding of the transport of potential polymeric drug delivery systems in normal and injured kidneys, but also provide initial insights into improved drug delivery to AKI.