Experimental Evaluation of Furosemide and/or Tadalafil in Conventional and Nanoparticle Forms in Prevention of Chronic Renal Failure Induced in Rats


 Introduction: Chronic renal failure (CRF) is a progressive loss of renal function that lead to reduced sodium filtration and inappropriate suppression of tubular reabsorption that ultimately leads to volume expansion. To improve treatment outcomes, the aim of this study was to evaluate the possible renoprotective effect of tadalafil and furosemide, individually and in combination, in both conventional and nanoforms in adenine-induced CRF rat-model. Methods: Addition of 0.75% adenine to the diet of rats for 4 weeks gained general acceptance as a model to study kidney damage as this intervention mimicked most of the structural and functional changes seen in human chronic kidney disease Urine analysis, histopathological changes and immunohistochemical expression of caspase-3 and interleukin-1β (IL-1β) in renal tissues were performed.Results: Our results showed that the combination of tadalafil and furosemide using conventional and nanoparticle formulations revealed a beneficial therapeutic effect in the treatment of CRF. This was demonstrated by improvement of urinary, serum and renal tissue markers as indicative of organ damage. This was also reflected on the reduction of tubular expression of KIM-1 and NGAL. Immunohistochemical studies showed that significant increase in the number of apoptotic tubular cells indicated by increased expression of caspase-3 in CRF. These deteriorated renal cellular changes were improved by the treatment of rats with the investigated drugs. Results from ELISA showed that IL-1β was reduced by such treatment in kidney tissue. Conclusion: Tadalafil and furosemide improved the biochemical, histopathological and immunohistochemistry changes in adenine-induced CRF which strongly support the renopreventive effects of investigated drugs in particular the nanoparticle forms.


Introduction
Chronic renal failure (CRF) is an international and national health problem that increases the risk of mortality and the use of specialized health care. Chronic renal failure is characterized by progressive loss of renal function that lead to reduced sodium ltration and inappropriate suppression of tubular reabsorption that ultimately leads to volume expansion. Chronic renal failure is also associated with in ammation and oxidative stress leading to endothelial dysfunction, glomerular brosis, and mesangial expansion. Fluid overload frequently occurs in patients with moderate to particularly late stages of CRF and has been associated with hypertension, congestive heart failure, left ventricular hypertrophy as well as edema [1].
Phosphodiesterase-5 (PDE5) inhibitors were originally developed to treat angina pectoris. However, it is subsequently used for erectile dysfunction and pulmonary hypertension [2]. There is an increasing evidence suggests that PDE5 inhibitors including sildena l, vardena l, and tadala l have broader effects, most likely due to their ability to inhibit the breakdown of cyclic 3', 5'-guanosine monophosphate, the second messenger for nitric oxide (NO) and natriuretic peptides [3]. Previous studies have demonstrated that PDE5 inhibitors improve endothelial function and possess nephroprotective effects in renal ischemia-reperfusion injury [4,5]. In addition to their vasodilatory action, PDE5 inhibitors possess antiapoptotic and anti-oxidant properties, making them a promising therapy for ischemia-reperfusion injury of various organs [6].
Loop diuretics were traditionally used to enhance renal excretion of excess salt and water. Blockage of sodium-potassium-chloride co-transporter in the thick ascending limb of the loop of Henle by the loop diuretics decreases cellular transport and reduces energy consumption and therefore preserves cellular vitality. Loop diuretics should be kept for conditions of clinically signi cant uid overload such as heart failure and signi cant uid retention or with advanced kidney failure and can be combined with thiazidediuretics [7] . Various studies demonstrated the usefulness of furosemide in different chronic kidney diseases [7,8].
These renal targeting drugs will increase the e cacy and reduce the toxicity of new, established, and preexisting drugs. The use of bionanotechnology in therapeutics of kidney diseases have been developed recently on polymer-based nanometers, which have great attention in the eld of drug delivery applications [9].
Based on the previous information, no previous studies were conducted to evaluate the possible role of combined administration of PDE5 inhibitor and loop diuretic, particularly in nanoformulations in chronic renal failure induced chemically. Therefore, this study was aimed to evaluate the renoprotective effects of tadala l and/or furosemide loaded and unloaded in nanoparticles in adenine-induced CRF in rats.

Animals and induction of chronic renal failure (CRF)
The experimental protocol was approved by the Institutional Animal Care & Use Committee (IACUC) of the Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt. The experiment was conducted using adult male albino rats weighing 150-250 g. The animals were housed in the animal house of the Faculty of Medicine, Assiut University under standard laboratory conditions and maintained under natural light and dark cycle with free access to food and water. Animals were randomly assigned to the experimental groups, 6-8 animals each. Chronic renal failure (CRF) was induced by addition of 0.75% w/w adenine to the diet of rats for 4 weeks [1].

Preparation of chitosan (CS) /alginate (ALG) nanoparticles loaded with furosemide
Furosemide loading NPs were kindly provided by the National Research Center (NRC), Cairo, Egypt. The optimum NPs preparation procedures were performed according to Radwan et al. [10] and as follows: The pH of 10 mL ALG solution (300 mg/100 mL) was modi ed to pH 5.1 by the addition of 0.5 M HCl. A calculated amount of CS was dissolved in 1% acetic acid solution overnight followed by sonication for 10 min. The pH of CS solution was adjusted to 5.4 using 2.5 M NaOH solution. Two mL calcium chloride (CaCl2) solution (332 mg/100 mL) were added drop wise, at a rate of 1 mL/min to 10 mL ALG solution, while stirring by a magnetic stirrer at 480 rpm for 30 min. Four mL CS solution of 80, 160, 240 mg/100 mL were then added drop-wise to the calcium ALG pre-gel and stirring was continued for an additional 1 h. The formed NPs were centrifuged using high speed cooling centrifuge (Sigma 30 K, Osterode am Harz, Germany) at 14,000 rpm at 4 °C for 30 min. The supernatant was removed and the precipitate was washed and reconstituted in 15 mL ltered distilled water and sonicated for 10 min. The NPs suspension was frozen for 24 h at -30 °C, then dried in a laboratory freeze-dryer. For drug loading, one mL of furosemide in ethanol containing various amounts of the drug (5, 10, 20, 40 mg), was incorporated into the ALG solution and sonicated for 1 min before adding the CaCl2 solution [11].
2.4 Preparation of poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with tadala l Tadala l loading NPs were kindly provided by the National Research Center (NRC), Cairo, Egypt. Tadala lloaded NPs were prepared according to the solid-in-oil-in-water (s/o/w) emulsion technique [12]. PLGA (35 mg) was dissolved in dichloro-methane for 6 h to obtain a uniform PLGA solution. Normal Tadala l 15 mg was added to the PLGA solution and sonicated at 55 W for 1 min to produce the solid-in-oil primary emulsion. This emulsion was added to 20 ml of polyvinyl alcohol solution (1% w/v) and again sonicated at 55 W for 2 min to get the nal solid-in-oil-in-water emulsion. The resulted nano-sized particles were stirred in the emulsion for 3 h for solvent evaporation. The nal emulsion was centrifuged at 15,000 rpm for 15 min to remove the residual solvent. The NPs obtained were washed thrice with deionized distilled water, and nally resuspended in deionized water and dried on a lyophilizer. The NPs were stored at 4°C till further use [12].

Validation of nanoparticles by Transmission Electron Microscopy (TEM)
The size and morphology of the NPs were evaluated using a transmission electron microscope (TEM) JEM-2100 HR (Jeol, USA) by high resolution TME at an accelerating voltage of 200 kV at National Research Center (NRC), Egypt. The lyophilized drug-NPs solution (1mg/ml) were placed on copper grids covered with nitrocellulose membrane and stained with 1% (w/v) sodium phosphotungstate solution. About 15 min after NPs deposition, the grid was then loaded into TEM, and the size and morphology were assessed [13].

Measurement of Zeta potential
The measurement of Zeta potential was performed using Zeta Potential Analyzer (National Research Center, Egypt) according to Sivakumar et al. [14]. The measurement of Zeta potential was performed in double distilled water using disposable Zeta cells and the standard protocol at 25 C. The instrument was calibrated routinely with a -50 mV latex standard. The mean zeta potential was determined using phase analysis light scattering technique as previously described [15].

Determination of urine volume and uid intake
Twenty-four hour urine collection by metabolic cage twice at the rst and fourth week during the induction of CRF. The calculation of urine volume, uid intake and urine analysis were then performed according to a prior study [16].

Determination of urinary albumin
Albumin was determined using available commercial kit (Cat#DIAG-250-BioAssay Systems-U.S.A) followed the standard protocol. The intensity of the color measured at 620nm which is directly proportional to the albumin concentration in the sample as described [17].

Determination of urinary glucose
Glucose was determined using available commercial kit (Cat# EGL3-100-BioAssay Systems-U.S.A) following the manufacturer's instructions. The intensity of the color was measured at 560 nm which is directly proportional to the glucose concentration in the sample as reported [18].

Determination of urinary ketone bodies
Ketone bodies were determined using available assay kit (Cat# EKBD-100-BioAssay Systems-U.S.A) followed the standard protocol. The intensity of the color was measured at 340 nm which is directly proportional to acetoacetic acid and 3-hydroxybutyric acid concentrations in the sample [19].
2.12 Determination of speci c gravity of the urine Urine speci c gravity was measured using a reagent strips for rapid detection of speci c gravity according to a previous study [21].

Assessment of renal functions
Renal functions were monitored by measuring serum creatinine (Cat. no. 234-000), blood urea nitrogen (Cat. no. UR 21-10) and total protein (Cat. no. 310-001) using commercially available assay kits (Schiffgraben, Hannover, Germany) according to the manufacturer's instruction. The previous parameters are measured spectrophotometrically.

Selective biomarkers for evaluating chronic renal failure
Kidney injury molecule-1 (KIM-1) which is a type-1 transmembrane protein, is not normally present, but is expressed on the proximal tubule apical membrane in rodent kidneys after renal injury. KIM-1 was assayed by available commercial ELISA kit (Cat. no. E-EL-R3019, Sunlong Biotechnology, Shangyi, Hangzhou, Zhejiang, China) following the manufacturer's instructions and as reported [25]. Neutrophil gelatinase-associated lipocalin (NGAL) which is a member of the lipocalin superfamily that is highly expressed in the rodent kidneys following injury especially at proximal convoluted tubule. NGAL was assayed by available commercial ELISA kit (Cat. no. E-EL-R0662, Sunlong Biotechnology, Shangyi, Hangzhou, Zhejiang, China) [26].

Histopathological and immunohistochemical studies
Kidney tissues were xed in 10% formalin for 24h followed by dehydration and embedded in para n. About 4 µm-thick kidney sections were sectioned and stained with hematoxylin-eosin (H&E). Light microscopic analysis was performed in 20 randomly selected areas in each section by blinded observation. The histopathological examination was performed to determine the extent of tubulointerstitial tissue and glomerular alterations. The tubulointerstitial damage involved tubular necrosis, atrophy, lumen dilation and in ammatory cell in ltration. Tubular damaged was scored on a scale from 0 to 4 (no necrosis cored 0, focal necrotic areas of ≤25% of the kidney scored 1, necrotic area was about 26-50% of kidney scored 2, necrotic area was 51-75% of kidney scored 3 and with the necrotic area forming about 76-100% of kidney scored 4). The average score was used for comparison according to a prior study [27].
Interleukin-1 beta (IL-1β) and Caspase-3 were analyzed by immunohistochemical staining. Tissue sections (4-µm thick) were depara nized in xylene and rehydrated. The sections were immersed in 3% H 2 O 2 for 10 min to eliminate endogenous peroxidase activity then washed with PBS (2 min X 3 times).
They were then incubated with normal goat serum according to the standard manufacturing protocol (Vector Laboratories, Burlingame, CA) at 37°C for 30 min, after which the sections were incubated with primary polyclonal rabbit active anti-caspase-3 antibody in dilution 1/200 (E-AB-6602, Elabscience Biotechnology inc, USA) and polyclonal rabbit anti-IL-1 β antibody; 1/100 (E-AB-66749, Elabscience Biotechnology inc, USA) for 1 h at room temperature. Polyperoxidase-anti-Mouse/Rabbit IgG was then added for 20 min. The antigen-antibody complex was detected using a streptavidin-biotin-peroxidase kit and counterstained with Mayer's hematoxylin. Positive and negative control sections were used for each assay.
The active caspase-3, and IL-1beta immunostaining cells were identi ed by intense brown nuclear and cytoplasmic staining. The immunoreactivity of caspase-3, and IL-1beta was described as a histological score (H-SCORE) which obtained by multiplying the number of activated cells (0-100% of cells) by the intensity of staining (1=weak, 2=moderate, 3=strong). The sections were examined using light microscope (Olympus BX41, New York) at high magni cation (X400) in the 20 randomly selected areas.
Blinded fashion assessment was performed and the average score of all groups was used for comparisons. Photomicrographs were taken using digital camera (ToupCam LCMOS05100KPA).

Statistical analysis
Statistical signi cance was assessed by one way ANOVA for repeated-measures, or two-way ANOVA as appropriate. The Dunnett test and Tukey's multiple comparisons test were used for data point comparisons in each group. Data are presented as means ± SEM. Data of p ≤ 0.05 was considered statistically signi cant. Graph Pad prism® software (version 8) was used to performed these statistical analyses.

Physicochemical characteristics of furosemide-and tadala l-loaded nanoparticles
Furosemide-loaded NPs were prepared as previously reported [11] and described in the methods. In the present study, the nanomaterial chitosan /alginate was employed. As validated by TEM, Furosemideloaded NPs are less than 50 nm in diameter with a spheroidal shape and suspension form ( Figure 2). As measured by Zeta Potential Analyzer, the charge density of chitosan/alginate NPs found to be -37 Mv ( Figure 1A) and the charge dropped to -31 Mv ( Figure 1B) upon loading of furosemide into chitosan/alginate. Tadala l-loaded NPs were prepared as described by [12]. As shown by TEM photomicrograph, tadala l-loaded NPs are 200 ± 50 nm in diameter with a regular spherical shape and suspension form ( Figure 2). As measured by Zeta Potential Analyzer, the charge density of PLGA NPs found to be -0.753 Mv ( Figure 1C) and the charge dropped to -0.402 Mv ( Figure 1D) upon loading of tadala l into PLGA.
3.2. Effect of furosemide and tadala l on water intake and urinary output Table 1 shows that water intake of the adenine treated rats (+ve control) was signi cantly higher than that of control rats (-ve control). Also, the water intake was signi cantly higher in rat treated with chitosan and PLGA NPs compared to control group (-ve control). However, there was no signi cance between adenine-induced rats (+ve control) compared to rats pretreated with chitosan and PLGA NPs. In addition, in the rst week of treatment there were no differences between drugs_ treated groups either in conventional and NP forms or their combinations compared to adenine induced rats (+ve control). However, in the fourth week the water intake in the furosemide NPs and tadala l NPs treated rats was signi cantly (P< 0.01) higher than the conventional drug treated rats.
The urine volume of the adenine treated rats (+ve control) was signi cantly decreased compare to the control group, while there was no signi cant differences between the control group (-ve control) and rats pretreated with chitosan and PLGA NPs. In the rst week of treatment, there were no differences in the urine volume in drugs_ treatedgroups in conventional forms and their combinations compared to the adenine-induced CRF rats (+ve control), while the urine volume in drug -NPs and their combinations was signi cantly (P< 0.001) higher than the adenine-induced CRF rats (+ve control). In the fourth week of treatment, the urine volume in drugs_ treated groups in both conventional, NP forms and their combinations was signi cantly (P< 0.01) higher compared to the adenine-induced CRF rats (+ve contreol). In addition, the urine volume in furosemide NPs pretreated rats was signi cantly (P< 0.05) higher compared to furosemide pretreated rats 3.3. Effect of furosemide, tadala l and their nanoparticle forms on urine albumin, glucose level and ketone bodies in CRF-induced rats.
CRF-induced rats showed an increase (p< 0.0001) in urinary albumin, glucose level and ketone bodies compared to negative control group. There were no signi cant differences in chitosan and PLGA treated rats compared to control group. Conventional and NPs of tadala l, furosemide and their combinations showed a signi cant decrease (p < 0.0001) in urinary albumin, glucose level and ketone bodies compared to CRF-induced animals. In addition, each of tadala l NPs, furosemide NPs and furosemide-tadala l NPs combination showed a signi cant decrease (p< 0. 05) in the studied parameters compared to their corresponding conventional drug-treated rats as depicted in Figure 3.

Effect of furosemide, tadala l and their nanoparticle forms on urine osmolarity level and speci c gravity in CRF-induced rats.
CRF-induced rats exhibited a signi cant decrease (p< 0.0001) in urine osmolarity and speci c gravity compared to negative control group. There were no signi cant differences in chitosan and PLGA treated rats compared to negative control group. Treatment of CRF-induced rats with conventional or NPs tadala l, furosemide and their combinations showed a signi cant increase (p < 0.0001) in urine osmolarity and speci c gravity. The NP forms of tadala l showed an increase in urine osmolarity compared to their corresponding conventional drug-treated rats (Figure 4). 3.5. Effect of furosemide, tadala l and their nanoparticle forms on serum creatinine level, urea level and total protein in CRF-induced rats.
CRF-induced rats showed signi cant (p < 0.0001) increase in serum creatinine and blood urea nitrogen level whereas a marked decrease was noted in total protein compared to negative control group. However, there were no signi cant differences in chitosan and PLGA treated rats. Conventional and NPs tadala l, furosemide and their combinations showed a signi cant decrease (p < 0.05) in serum creatinine and urea levels while an increase (p < 0.05) in total protein compared to CRF-induced rats. A greater reduction in creatinine and urea was observed instead of total protein in CRF-induced rats treated with NP forms particularly with furosemide and the combination ( Figure 5).
3.6 Effect of furosemide, tadala l and their nanoparticle forms on tissue malondialdehyde, nitrite and glutathione levels in CRF-induced rats.
As shown in Figure 6, CRF-induced rats showed signi cant increase (p < 0.01) in tissue malondialdehyde but rather a reduction in nitrite (p <0.0001) and glutathione (p <0.05) levels compared to negative control group. However, there were no signi cant differences in chitosan and PLGA treated rats compared to negative control group. Conventional and NPs forms of tadala l, furosemide and their combinations showed a signi cant decrease (p < 0.001) in tissue malondialdehyde while an increase in nitrite (p <0.0001) and glutathione (p <0.05) levels compared to CRF positive control group. Treatment of ARFinduced rats with furosemide NPs and furosemide-tadala l NPs combination showed a signi cant decrease (p < 0.01) in tissue malondialdehyde and a marked increase in tissue nitrite and glutathione (p <0.05) particularly with tadala l NPs and furosemide-tadala l NPs combination compared to their corresponding conventional drugs treated rats.
Adenine-CRF induced treated rats showed a marked increase in tissue KIM-1 (p < 0.0001) and NGAL (p <0.001) compared to negative control group. However, there were no signi cant differences in chitosan and PLGA treated rats compared to control group. Conventional and NFs forms of tadala l, furosemide and their combinations showed a signi cant decrease (p < 0.0001) in tissue KIM-1 and NGAL compared to CRF-induced group. Of note, tadala l NPs, furosemide NPs and furosemide-tadala l NPs combination showed a signi cant decrease (p < 0.01) in tissue KIM-1 and NGAL compared to their corresponding conventional drug-treated rats as depicted in Figure 7 respectively) showed mild histopathological damage while NP forms of tadala l, furosemide and their combination (sections F, G and H, respectively) showed a signi cantly protection against the severity of renal damage. Thus, the tissue damage score in the NP-treated groups was lower than that of the conventional drugs-treated groups.

Immunohistochemistry changes in caspase-3 and interleukin-1 beta (IL-1β) expression in CRFinduced rats
Caspase-3 and IL-1β immunoreactivity results are presented in Figure 8. In the kidney tissues of the control groups, relatively few active caspase-3 (section Q) and IL-1β (section I) positive tubular epithelial cells were detected and. CRF-induced group showed a signi cant increase in immunoexpression of caspase-3 (section R) and IL-1β (section J). In animals treated with conventional tadala l, furosemide and their combination in CRF-induced rats showed moderate caspase-3 (sections S, T and U, respectively) and IL-1β (sections K, L and M, respectively) immunoexpression. Interestingly, nanoparticle forms of individual and combinatory drugs in treatment of CRF-induced rats showed more decrease in immunoexpression of caspase-3 (sections V, W and X, respectively) and IL-1β (sections N, O and P, respectively). Accordingly, the immunoreactivity scores of caspase-3 and IL-1β in the NP-treated groups were lower than the conventional drugs-treated groups. Table 1. Effect of furosemide (20 mg/kg,i.m.), tadala l (5mg/kg, p.o.) and its nanoparticles form pretreatment on urine volume and water intake in CRF-induced rat.

Groups
Water intake (ml/24 hr) 1 st week Data are expressed as mean ± SEM.** p < 0.01 and ***p < 0.001 as compared with the ARF-induced group (one-way ANOVA followed by Dunnett's multiple comparisons test), a denotes p < 0.001 as compared with the control group and b denotes p < 0.05 as compared with corresponding nanoparticle group (unpaired t-test).

Discussion
Chronic renal failure (CRF) is a progressive, irreversible proces, with uncertain exact aetiology, but diabetes is the most common cause in those starting dialysis [30]. Hypertension, glomerulonephritis and pyelonephritis are less frequent causes. The presence of CRF is associated with an increased mortality risk [31]. In the present study, CRF was induced by using adenine oral model. These results showed that water intake, urinary albumin, urinary glucose and total ketones were signi cantly elevated while urinary In present study, the effect of tadala l (5 mg/kg, p.o.), furosemide (20 mg/kg, i.m.) and their combination either in conventional or NP forms for 28 days to prevent the development of CRF in adenine -induced CRF model in rats. Results showed that water intake, urinary albumin, urinary glucose and total ketones were signi cantly decreased while urinary output, urine osmolarity and urine speci c gravity were signi cantly elevated by administration of the previously mentioned drugs which means that the value of these drugs in prevention of the development of CRF in adenine model. Higher enhancement was found with nanoparticulation compared to their conventional forms.
On the same way, Tomita et al. (2020) reported that tadala l could enhance protection of the glomerular structures against brosis. Tadala l at low dose 1mg/kg and high dose 10 mg/kg attenuated proteinuria caused by glomerular injury, diminished glomerulosclerosis, and maintained glomerular structure at chronic kidney disease rat model which is consistent with our results. The only difference is the different model of induction of CRF and different doses [35]. Also, our results could be supported by the opinion of In present study, we found that serum creatinine and blood urea nitrogen were signi cantly elevated while blood total proteins was signi cantly decreased in adenine -induced CRF. These ndings are in consistent with the study of Li et al. (2018) who reported that adenine treatment led to a signi cant elevation of the serum creatinine and blood urea nitrogen levels in rats [37]. Also, in present study, the use of the investigated drugs signi cantly decreased blood urea and serum creatinine levels while increased blood total protein levels in adenine -induced CRF. These results are considered as good markers in the improvement of CRF.
In this study, a signi cant elevation was seen in the tissue malondialdehyde levels and a signi cant decline in the tissue reduced glutathione and nitrite levels in adenine -induced CRF rats. These ndings are inconsistent with Nosratola et al, (2002) who reported that malondialdehyde level was increased while nitrite levels was decreaed in nephrectomy model of CRF [38]. Treatment with tadala l, furosemide and their combination either in conventional or NP forms in the presnent study improve these parameters with the superiority of nanoparticulation which could indicate that our drugs may be effective in prevention of CRF.
In this study, we reported that NGAL and KIM-1, have been established as indicator of CRF, were signi cantly elevated in adenine -induced CRF rats. These ndings is consistent with previous studies of Ali et al. (2018), who reported that NGAL was markedly increased in adenine -induced CRF and probably supported by a clinical study of Sabbisetti et al. (2014) who mentioned that KIM-1 could con rmed as biomarker of AKI and CRF [39,40] .
Furthermore, the present study found that chronic treatment with tadala l, furosemide and their combination, both in conventional and NP forms for 28 days sign cantly decreased NGAL and KIM-1 levels in adenine -induced CRF nearly to the same levels of the control group. Such treatment with the NP forms of furosemide and tadala l showed more marked improvement. Thus, the improvement of adenine -induced CRF by the using of the investigated drugs probably explained on the basis of the reduction that occurred in NGAL and KIM-1 levels.
In On the same experimental way, the previous histopathological studies in CRF showed that light and electron microscopy after adenine administration showed tubulointerstitial damage with in ltrating leukocytes, interstitial edema and widening of the bowman s space in adenine treated rats [42]. These results are consistent with the results of our study in histopathological changes in adenine -induced CRF.
Also, we can explained the improvement of CRF by chronic treatment with tadala l, furosemide and their combination either in conventional or NP forms for 28 days on the basis of improvement of histopathological ndings in the present study. Dr. Mahmoud S. Sabra contributed to data collection, biochemical analysis, writing the manuscript, interpretation of the results, methodology and analysis of the study.

Conclusion
Dr. Dalia M. Badary contributed to histopathological and immunohistochemical evaluations.

Figure 2
Transmission electron microscopy photomicrographs depicting the spheroidal forms of furosemide nanoparticles and the regular spherical shape of tadala l nanoparticles at different magni cations. Note: ****p < 0.0001 as compared to the adenine -treated group.
a P < 0.0001 as compared with the control group.
b P < 0.001 as compared with corresponding nanoparticle group (unpaired t test).
c P < 0.05 as compared with corresponding nanoparticle group (unpaired t test). Note: ****p < 0.0001 as compared to the adenine group.
a P < 0.0001 as compared with the control group.
aP < 0.01 as compared with the control group.
bP < 0.01as compared with corresponding nanoparticle group (unpaired t test).
c P < 0.01 as compared with corresponding nanoparticle group (unpaired t test). Note: ****p < 0.0001 as compared to the chronic group.
a P < 0.0001 as compared with the control group.
b P < 0.05 as compared with corresponding nanoparticle group (unpaired t test).
c P < 0.05 as compared with corresponding nanoparticle group (unpaired t test). d P < 0.05 as compared with corresponding nanoparticle group (unpaired t test).