Urinary miRNAs as Biomarkers for Noninvasive Evaluation of Radiation-Induced Renal Tubular Injury.

Radiation nephropathy is one of the common late effects in cancer survivors who received radiotherapy as well as in victims of radiation accidents. The clinical manifestations of radiation nephropathy occur months after exposure. To date, there are no known early biomarkers to predict the future development of radiation nephropathy. This study focuses on the development of urinary biomarkers providing readout of acute responses in renal tubular epithelial cells. An amplification-free hybridization-based nCounter assay was used to detect changes in mouse urinary miRNAs after irradiation. After a single LD50 of total-body irradiation (TBI) or clinically relevant fractionated doses (2 Gy twice daily for 3 days), changes in urinary levels of microRNAs followed either an early pattern, peaking at 6-8 h postirradiation and gradually declining, or later pattern, peaking from 24 h to 7 days. Of 600 miRNAs compared, 12 urinary miRNAs showed the acute response and seven showed the late response, common to both irradiation protocols. miR-1224 and miR-21 were of particular interest, since they were the most robust acute and late responders, respectively. The early responding miR-1224 also exhibited good dose response after 2, 4, 6 and 8 Gy TBI, indicating its potential use as a biomarker for radiation exposure. In situ hybridization of irradiated mouse kidney sections and cultured mouse primary renal tubular cells confirmed the tubular origin of miR-1224. A significant upregulation in hsa-miR-1224-3p expression was also observed in human proximal renal tubular cells after irradiation. Consistent with mouse urine data, a similar expression pattern of hsa-miR-1224-3p and hsa-miR-21 were observed in urine samples collected from human leukemia patients preconditioned with TBI. This proof-of-concept study shows the potential translational utility of urinary miRNA biomarkers for radiation damage in renal tubules with possible prediction of late effects.

Far infrared radiation promotes rabbit renal proximal tubule cell proliferation and functional characteristics, and protects against cisplatin-induced nephrotoxicity.

Far infrared radiation, a subdivision of the electromagnetic spectrum, is beneficial for long-term tissue healing, anti-inflammatory effects, growth promotion, sleep modulation, acceleration of microcirculation, and pain relief. We investigated if far infrared radiation is beneficial for renal proximal tubule cell cultivation and renal tissue engineering. We observed the effects of far infrared radiation on renal proximal tubules cells, including its effects on cell proliferation, gene and protein expression, and viability. We also examined the protective effects of far infrared radiation against cisplatin, a nephrotoxic agent, using the human proximal tubule cell line HK-2. We found that daily exposure to far infrared radiation for 30 min significantly increased rabbit renal proximal tubule cell proliferation in vitro, as assessed by MTT assay. Far infrared radiation was not only beneficial to renal proximal tubule cell proliferation, it also increased the expression of ATPase Na+/K+ subunit alpha 1 and glucose transporter 1, as determined by western blotting. Using quantitative polymerase chain reaction, we found that far infrared radiation enhanced CDK5R1, GNAS, NPPB, and TEK expression. In the proximal tubule cell line HK-2, far infrared radiation protected against cisplatin-mediated nephrotoxicity by reducing apoptosis. Renal proximal tubule cell cultivation with far infrared radiation exposure resulted in better cell proliferation, significantly higher ATPase Na+/K+ subunit alpha 1 and glucose transporter 1 expression, and significantly enhanced expression of CDK5R1, GNAS, NPPB, and TEK. These results suggest that far infrared radiation improves cell proliferation and differentiation. In HK-2 cells, far infrared radiation mediated protective effects against cisplatin-induced nephrotoxicity by reducing apoptosis, as indicated by flow cytometry and caspase-3 assay.

Protective Effect of 10-Hz, 1-mT Electromagnetic Field Exposure Against Hypoxia/Reoxygenation Injury in HK-2 Cells.

We investigated the protective effects of electromagnetic field (EMF) on the survival of the human renal proximal tubular cell line, HK-2, using an in vitro hypoxia/reoxygenation (H/R) injury model. The survival rate of cells cultured under H/R condition declined significantly, while the intracellular reactive oxygen species (ROS) levels markedly increased. The 10 Hz/1 mT EMF exposure reversed the H/R induced reduction in cell survival and induction of intracellular ROS. Our results suggest that 10 Hz/1 mT EMF exposure could inhibit H/R-induced cell death of HK-2 via suppression of intracellular ROS production and that this treatment might be clinically useful for the amelioration of renal ischemia/reperfusion injury.

[On the issue of non-mutagenic non-targeted effects in low renewable tissues. Analysis of low dose radiation effects on the rat renal tubule epithelium].

Irradiation of rats with γ-quanta at relatively low doses induces a sustainable dose-independent increase in the occurrence of lethal cytoplasmic disorders in the renal tubules epithelium together with sustainable and as well dose-independent subcelluar compensation and restorative processes. Over the period of research (6 months) these processes led to no population recovery. The detected alterations are referred to the category of non-targeted non-mutagenic effects and they are of interest because they address the issue of the sensitivity of low renewable tissues to radiation.

Nephrotoxicity profiles and threshold dose values for [177Lu]-DOTATATE in nude mice.

INTRODUCTION: In peptide receptor radionuclide therapy for neuroendocrine tumors the main dose-limiting tissue is found in the kidneys because of tubular reabsorption and retention of radioactivity. The aim of this study was to quantify late effects in renal cortex of nude mice exposed to high amounts of [(177)Lu]-DOTA-Tyr(3)-octreotate ([(177)Lu]-DOTATATE), and to determine whether a threshold dose value exists for these findings. METHODS: Nude mice were exposed to 90, 120 or 150 MBq of [(177)Lu]-DOTATATE. Renal toxicity was evaluated up to 6 months after injection. Blood samples were collected to examine renal functional markers, and after sacrifice at 6 months changes in renal morphology were explored. Tissue damage was estimated by quantifying the relative area of the different subunits in the renal cortex using point counting. Additional morphological signs of radiation damage were also noted. The absorbed doses to the kidneys were estimated by previously determined kidney pharmacokinetics and Monte Carlo simulations for different assumptions regarding the activity distribution. RESULTS: Increased serum creatinine and urea values indicated long-term renal toxicity. The tissue area occupied by proximal tubules decreased with increasing doses of [(177)Lu]-DOTATATE, whereas the other subunits in cortex slightly increased. The mean absorbed dose in the renal cortex for [(177)Lu]-DOTATATE was estimated to be 35-58 Gy for the different groups of animals. A dose-response relationship was observed for proximal tubular damage, and a threshold dose value of 24 Gy (BED 37 Gy) was determined. CONCLUSIONS: Selective morphological changes in kidney cortex of nude mice were quantified and appeared in a dose dependent manner after injection of high amounts of [(177)Lu]-DOTATATE.

Decrease in laminin content and protein excretion rate after five sixths nephrectomy and low-dose irradiation in the rat.

The effect of low-dose irradiation on laminin distribution and urine protein excretion in the remnant rat kidney has been studied. The rat remnant kidney formed after 5/6 nephrectomy is an experimental model of chronic renal failure. In the remnant kidney, focal segmental glomerulosclerosis is developed characterized by focal or segmental sclerosis in glomeruli, alterations in the tubules and thickening of the glomerular basement membrane. Low dose irradiation has been presumed to suppress sclerotic processes. In this study 24 male Wistar rats were subdivided into the nephrectomized group, nephrectomized and irradiated groups (1 or 3 Grey), and healthy control group. Animals were sacrificed at 2, 4 and 8 weeks after beginning the experiment. Laminin immunohistochemical staining was found along the tubular and glomerular basement membranes in all experimental groups, but with varying intensity. Laminin content in the basement membranes was decreased in early stages (week 2), especially after irradiation followed by increase during the later stages with relatively high levels at the end of the experiment (week 8). Irradiation at a dose of 3 Grey decreased protein excretion compared to the nephrectomized rats at all stages, while 1 Grey dose was ineffective. Based on decreased proteinuria we conclude that moderate low-dose irradiation has beneficial effects on the rat remnant kidney and that laminin in basement membranes is probably not the most crucial component in regulating membrane permeability.

[Morphological changes in the kidney after 5/6 nephrectomy and low-dose radiation]. / Morfologicheskie izmeneniia v pochke posle 5/6 nefréktomii i vozdeistviia malykh doz radiatsii.

In the present study, we investigated the effect of low-dose irradiation of experimental nephrectomized rats. We hypothized that the low-dose irradiation may slow down the development of focal-segmented glomerulosclerosis (FSGS) after 5/6 nephrectomy. Experiments were performed with 32 male Wistar rats, divided into four groups. The first group contained only operated animals. Animals in the second and third groups were irradiated on the next day after operation with 1 and 3 Gy, respectively. The healthy animals made the forth, control group. Attention was focused on physiological and morphological changes after low-dose (1 and 3 Gy) irradiation. We measured blood pressure, proteinuria, serum creatinin and cysC. Morphological changes of glomerulus and tubules were studied. Animals of the first group had significantly thicker glomerular basement membrane, compared to animals of other groups. The morphological study demonstrated degeneration of the tubular epithelium, tubular atrophy and FSGS. Besides, it was shown that changes in the third group (3 Gy) were less than in nephrectomized (first group) and 1 Gy (second group). The animals of the third group (3 Gy) had significantly lower proteinuria and FSGS. We conclude that our hypothesis, suggesting that low-dose irradiation slows down the development of FSGS, was confirmed.

Low plasma and renal tissue levels of L-arginine in rats with obstructive nephropathy.

Rats with bilateral ureteral obstruction (BUO) of 24 hours duration had significantly lower plasma levels of L-arginine than at baseline (P < 0.0001), but no significant changes occurred in sham-operated rats (SOR). In contrast, rats with bilateral nephrectomy had greater plasma levels of L-arginine four hours (P < 0.03) and 24 hours (not significant) after nephrectomy than at baseline. Total body irradiation prior to obstruction prevented the decrease in plasma levels of L-arginine in rats with BUO but had no effect on these values in SOR. Renal tissue levels of L-arginine were 20% lower in rats with BUO than in SOR. Total body irradiation prior to BUO resulted in greater renal tissue levels of L-arginine than occurred in nonirradiated rats with BUO (P < 0.002). Total body irradiation did not effect renal tissue levels of L-arginine in SOR. Excretion of reactive nitrogen intermediates in urine (URNI), indicative of L-arginine metabolism through the nitric oxide pathway, was lower in rats with BUO than in SOR (P < 0.001). Proximal tubules from rats with BUO synthesized less L-arginine than those from SOR (P < 0.02). The results indicate that: (1) decreased levels of L-arginine in plasma and renal tissue of rats with BUO correlate with leukocyte infiltration of the kidney, and (2) decreased synthesis of L-arginine occurs in proximal tubules of rats with BUO when compared to tubules from SOR.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of extracorporeal electromagnetic shock waves on renal proximal tubular function.

In order to investigate the effect of electromagnetic shock waves on proximal tubular renal function, we measured the urine levels of the lysosomal enzyme N-acetyl-beta-glucosaminidase (NAG) prior to, 1 hour, 1 day, and 1 week after extracorporeal electromagnetic lithotripsy (ESWL) in 50 patients with urinary stone disease. Mean NAG level which was 5.18 + 0.09 U/l prior to ESWL increased to 5.29 + 0.09 U/l in the first hour and to 10.7 + 0.19 U/l on the first day after ESWL (p < 0.001). The level returned to pre-ESWL values one week after ESWL (5.27 + 0.09 U/l). These findings show that extracorporeal electromagnetic shock waves cause significant damage to the proximal tubular epithelium of the kidney; however, these changes return to normal in the first week.

Ultrastructure of convoluted proximal tubule of kidney mice before and after X-ray exposure

Ultrastructural initial changes study of convoluted proximal tubule after single dose of 154.8 mC/Kg (600R), X, ray whole body radiation was performed in 12C57BL mice. Twenty-four hours later, mitochondria evidenced proeminent changes such as cristolyses, dilatation and vacuolization. Within 72 hours, heterochromatin nuclei increased and vanished 144 hours after X-ray exposure. Such findings suggest that recovery probably happened because convoluted proximal tubules take place, regeneration occurs in aslow rate

Radiation-induced proliferation in contralateral unirradiated kidneys.

Following irradiation of a single kidney in mice, the weights and labeling indices in the irradiated and contralateral unirradiated kidneys were measured. Three to 12 months after irradiation we found that the proliferation of proximal tubule cells was increased not only in the irradiated kidney but also in the contralateral unirradiated kidney. The labeling index in the contralateral unirradiated kidney did not increase in the first month after irradiation but it did in the irradiated kidney. Contralateral kidney weight increased as the weight of the irradiated kidney decreased. We suggest that cell proliferation in the contralateral kidney was probably induced through a humoral growth factor beginning 3 months after irradiation. We postulate that induced cell proliferation in the irradiated kidney is a result of a local factor at 1 month and of local and humoral factors at later times.

Ultrastructure of convoluted proximal tubule of kidney mice before and after X-ray exposure.

Ultrastructural initial changes study of convoluted proximal tubule after single dose of 154.8 mC/kg (600R), X-ray whole body radiation was performed in 12 C57BL mice. Twenty-four hours later, mitochondria evidenced proeminent changes such as cristolyses, dilatation and vacuolization. Within 72 hours, heterochromatin nuclei increased and vanished 144 hours after X-ray exposure. Such findings suggest that recovery probably happened because convoluted proximal tubules take place, regeneration occurs in a slow rate.

Acceleration of late radiation damage of the kidney by unilateral nephrectomy.

Both increased proliferation as measured by labeling index and the appearance of abnormally large nuclei in renal proximal tubule cells, which have been observed in mouse kidneys after irradiation, were enhanced by subsequent unilateral nephrectomy. Nephrectomy alone induced only a transient increase in labeling index, lasting less than 1 month, whereas nephrectomy after irradiation induced an increase above that of the irradiated kidneys without nephrectomy lasting as long as 9 months. The incidence of large nuclei in kidneys from mice unilaterally nephrectomized 1 week after irradiation showed a rapid increase with time, peaking at 4.6% at 6 months, compared to more gradual increases with peaks at about 4.0% at 9 or 12 months in irradiated kidneys without nephrectomy or those in which nephrectomy was done prior to irradiation. This result demonstrates that nephrectomy after irradiation accelerates the appearance of this indicator of radiation damage, rather than enhancing the maximum amount of damage. Unilateral nephrectomy after irradiation also increased kidney damage 9 months later, as indicated by kidney weight loss and increased blood urea nitrogen. These results are consistent with our model for radiation damage of the kidney in which radiation induces cell proliferation and the appearance of reproductively dead, large nuclear cells that are lost at subsequent attempts to divide; the acceleration of radiation damage by unilateral nephrectomy performed after radiation could very well be a result of nephrectomy-induced enhancement in the proliferation of proximal tubule cells.

Functional unit of 30 kDa for proximal tubule water channels as revealed by radiation inactivation.

The high water permeability of kidney proximal tubules is of paramount importance for isotonic reabsorption of 70% of the glomerular filtrate, and water channels have been postulated to account for the high water permeability. Target analysis following radiation inactivation was used to probe the molecular size of the water channel. Samples of brush border membranes from rat renal cortex were subjected to 3-MeV electron pulses from the Van de Graaff accelerator at a temperature of -130 degrees C. The inactivation of the renal brush border enzymes, alkaline phosphatase, and maltase was used for internal standardization of accumulated dose measurements in target analysis of the water channel. Osmotic water permeability was measured by following the change in scattered light intensity upon rapid mixing of vesicles with a hypertonic solution using stopped-flow spectrophotometry. The vesicle shrinkage response was biphasic and the rate of the fast phase decreased dose dependently by irradiation corresponding to a target size of 30 +/- 3.5 kDa. The total change in scattered light intensity was unaltered, indicating that irradiation did not destroy the lipid barrier. Our results provide strong support for the hypothesis that the high osmotic water permeability of renal proximal tubules results from a water channel-specific protein with a functional unit of 30 kDa.

Cell proliferation and abnormal nuclei induced by radiation in renal tubule epithelium as an early manifestation of late damage.

The time of appearance and the dose response of radiation effects in the mouse kidney were assessed from the determination of increases in labeling index, the appearance of proximal tubule cells with abnormally large nuclei, and kidney weight loss. Increased labeling indices and abnormally large nuclei were observed in the irradiated proximal tubule cells before any other histological changes were seen. The labeling index increased with dose (from 3 to 15 Gy) but not with time (from 1 to 12 months after irradiation). Increased labeling was evident as soon as 1 month after irradiation. Cell depletion as measured by a decrease in kidney weights compared to those of age-matched controls was not significant until 6 or more months after 11-, 13-, or 15-Gy irradiation. The frequency of cells with large nuclei increased steadily during the first 9 months after 15 Gy and tended to decline between 9 and 12 months, coincident with accelerating renal weight loss. These findings are consistent with the hypothesis that the production of these cells is a result of an abortive mitotic division and their loss is an eventual result of such an aberration. The increased proliferation induced by irradiation increases the chance for an abortive mitosis and death, presumably at a subsequent mitosis, of radiation-damaged proximal tubule cells, which is a major factor in the appearance of late radiation damage in the kidney.

Radiation target sizes of the Na,K-ATPase and p-aminohippurate transport system in the basolateral membrane of renal proximal tubule.

Basolateral membrane vesicles made from rabbit kidney proximal tubules were frozen and irradiated with a high energy electron beam and the effects of irradiation on Na,K-ATPase activity, p-aminohippurate (PAH) transport, the membrane diffusion barrier and vesicle volume were measured. The vesicle volume and diffusion barrier were not significantly changed by radiation exposure. Na,K-ATPase activity was inactivated as a simple exponential function of radiation dose. Target size analysis of the data yielded a molecular size of 267 +/- 17 kDa, consistent with its existence as a (alpha beta)2 dimer. The carrier-mediated PAH uptake by basolateral membrane vesicles was also inactivated as a function of radiation dose. A target molecular size of 74 +/- 16 kDa was calculated for the PAH transport system. This study is the first measurement of the functional size of the organic acid transport system based directly on flux measurements.

Fluorescein transport in isolated proximal tubules in vitro: epifluorometric analysis.

An epifluorometric method was used to quantify the bidirectional fluxes of fluorescein across the basolateral surfaces of nonperfused rabbit tubule segments in vitro. Proximal S2 segments, but not cortical collecting tubules or cortical thick ascending limbs, accumulated fluorescein to levels in cytoplasm over 100-fold greater than in the external medium. The rate of intracellular fluorescein accumulation was dependent on the concentration of the ligand in the external bath. The apparent Km was 10 microM and the Vmax was 623 x 10(-6) mol.min-1.l-1. Probenecid and ouabain inhibited fluorescein accumulation. We conclude that fluorescein is transported into the cytoplasm of proximal tubules by basolateral mechanisms that share features in common with the classical organic anion system. This fluorescent compound offers some unique advantages for the study of the organic anion transport system in intact cells.

Characterization of abnormal nuclei in renal proximal tubules after irradiation.

The distribution and kinetics of proximal tubular cells with abnormally large nuclei, which were observed in irradiated mouse kidneys before any other obvious histological effects, were investigated. Six months after the administration of 13 or 15 Gy, little histopathological change was noted, in the kidneys of C3H mice; however, proliferation of proximal tubular cells was stimulated, and some of these cells had abnormally large nuclei. The relative DNA content of these large nuclei was measured with a quantitative image analysis system. Most of the large nuclear cells had more than diploid DNA content. The labeling index of the large nuclei was higher than that of unselected proximal tubular nuclei. These cells might be hyperploid cells that are dying after having gone through an abortive mitotic division. Examination and quantitation of these abnormal nuclei should be useful in elucidating the steps involved in cell loss in the proximal tubules after irradiation and as an assay for radiation damage to the kidney.

Late radiation response of kidney assayed by tubule-cell survival.

An assay for the survival of renal tubule cells was developed using mice. It is analogous to other in-situ clonogenic cell survival assays. One kidney was irradiated using a 137Cs irradiator and removed 60-68 weeks later for histological examination. In unirradiated animals there were about 370 tubules in contact with the capsule in a coronal cross section at the middle of the kidney. After irradiation, extensive tubular damage was the dominant lesion. The number of epithelialized tubules in contact with the capsule showed a dose-dependent logarithmic decline. The dose-survival relationship for the clonogenic cells responsible for the regeneration of tubule epithelium was described by a D0 value of 1.5 Gy over the dose range 11-16 Gy. This radiosensitivity resembles that of stem cells in acutely responding tissues. The lack of histological evidence of damage to the arterial vasculature at the time the tubules are initially denuded of epithelium, and the similarity of renal tubule cell radiosensitivity to that of other mammalian cells, support the hypothesis that "late" radiation injury results primarily from depletion of parenchymal cells, not indirectly from injury to blood vessels, as has been the prevailing belief.

Comparative late effects of X-rays and negative pimesons on the mouse kidney.

A system is described for comparing various modalities and fractionation schedules of radiation by means of their long-term morphologic effects upon the mouse kidney. The comparison system utilizes a grading scale for histopathologic changes in which a given histologic grade depends upon meeting defined threshold criteria, rather than quantitation of a particular measurement. Renal tubular alterations served as the basis for comparison, since they appeared more reliably defined than glomerular changes. The radiation dose that induced a specific threshold effect in kidneys from 50% of the animals at 6 months was defined as the effective dose-50%, or ED50.ED50 was found for x-rays and negative pimesons administered in 1, 2, or 5 fractions. From these data, the relative biologic effectiveness (RBE) of negative pi-mesons with respect to x-rays was determined for each fractionation schedule.