The kidneys of the control animals showed normal renal histological components and normal nephrotic structures of the glomeruli, renal tubules, collecting tubules, and interstitial tissue (Fig. 1a,b). Compared with the control kidneys, the renal tissues of the rats exposed to HCQ demonstrated glomerular fragmentation, partial atrophy (Fig. 1c), proximal convoluted renal tubules (PCTs) hydropic degeneration, cloudy swelling (Fig. 1d), hyaline cast formation in the distal convoluted tubules (DCTs), and occasional interstitial edema next to the renal arteries (Fig. 1e). Some histopathological reports indicate that HCQ toxicity is responsible for the glomeruli size reduction, dilatation of Bowman’s space, and degeneration of renal tubules [28].

Fig. 1.

Light microphotographs sections in the kidneys of control and HCQ-treated rats stained by H&E showing: (a,b) Control kidneys, (a) Normal glomerulus (g) and renal tubules, $\times$320 (b) Normal loops of Henle (star), $\times$80; (c–f): Kidney of HCQ-treated rat, (c) Glomerular fragmentation (g,f) and glomerular atrophy (arrow), $\times$80 (d) Renal cells hydropic degeneration (arrows), $\times$320, (e) Hyaline casts formation (white arrows), $\times$160 (f) Interstitial edema (star), $\times$80.

Treated rats’ renal cells demonstrated swelling and cytolysis with occasional brush border detachment of the PCTs’ epithelial lining. Previous studies indicated that HCQ was a potential risk to the function and structure of the kidney [6, 36]. Our findings show that the PCTs cause more injury than the distal convoluted tubule. This might be associated with the high blood volume received by the cortex and the proximal tubules compared with the medulla and the distal parts, respectively [37]. However, the hyaline casts’ appearance in the renal tubules of HCQ-treated rats may suggest subsequent glomerulonephritis caused by HCQ because of the ion pump disturbance across the renal tubules [6, 36].

Some studies reported a low risk of renal disorders among patients with rheumatoid arthritis and lupus nephritis, treated with HCQ [38, 39, 40]. An et al. [39] concluded that HCQ could protect patients from lupus erythematosus immune inflammatory lesions that disrupted the lysosomal-autophagy pathway and promoted renal cell senescence [39]. Catfish exposed to HCQ also showed a reduction in glomeruli, dilatation in Bowman’s space and deterioration in renal tubules [28]. However, Pari and Murugan [36] reported renal tubule hemorrhage and necrosis with cloudy swelling, resulting in reactive free radicals induced by oxidative stress, caused by HCQ treatment [40]. Additionally, El-Shishtawy et al. [35] found that rats treated with HCQ had glomeruli hypercellularity, hydropic degeneration, hemorrhage, inflammation, tubular necrosis, and hazy swelling. Ramesh et al. [32] also reported renal cell necrosis in the renal tissues of rats subjected to HCQ. Furthermore, HCQ attenuated renal damage by CTSB downregulation and the activation of CTSL-mediated NLRP3 inflammasomes showed significant attenuation of the renal dysfunction, based on a reduction in serum creatinine [6].

The liver of the control animals demonstrated normal intact hepatic tissues with preserved zonal accentuation. The hepatic lobular architecture exhibited normal hepatocytes, hepatic portal triads, Kupffer cells, and sinusoids (Fig. 2a). The liver of HCQ-treated rats has demonstrated little hepatic histological and histochemical alteration, leading to Kupffer cells hyperplasia and occasional hepatocytes injury in form of dysplasia and pyknosis (Fig. 2b). Additionally, the hepatic tissue of HCQ-treated rats showed inter-hepatic bile duct hyperplasia (Fig. 2c). Some hepatocytes exhibited nucleomorphism, vesiculation, binucleation, multinucleation, and irregularly clumped chromatin. These findings may demonstrate DNA fragmentation and apoptosis potential [41, 42]. However, some recent studies reported that HCQ could induce dilation of the hepatic central vein, vacuolization, hepatocyte degeneration, and vacuolization with pyknotic nuclei [28]. The liver of HCQ-exposed catfish demonstrated dilation of the lobular central vein, vacuolization of hepatocytes nuclear, and degeneration of pyknotic nuclei [28]. Previous case studies also showed hepatic failure after prolonged use of HCQ [43]. However, no necrotic or degenerative alterations were noted; no glycogen storage was affected, and no hemosiderin precipitate was observed in the liver of rats subjected to HCQ. Moreover, portal vessels and central veins were not affected.

Fig. 2.

Light microphotographs of H&E stained sections in the liver of the control and HCQ treated rats demonstrating: (a,b) Sections of control rats, (a) Normal hepatic architecture and normal hepatic strands (star), $\times$80 (b) Normal hepatocytes surrounding central vein, $\times$320 (c,d) Sections in the hepatic tissue of HCQ-treated rat exhibiting, (c) Kupffer cells hyperplasia, $\times$80 and (d) Inter-hepatic bile duct hyperplasia (arrows), $\times$80.

The liver performs a vital role in the metabolism, excretion, and detoxification of chemicals, drugs, and other xenobiotic materials that can make the hepatic tissues susceptible to potential toxic effects [33]. The absence of serious abnormality in the liver of HCQ-treated rats may indicate that the toxicity of this drug does not target the liver. However, hepatocerebral degeneration, feathery degeneration, sinusoidal dilation, hydropic degeneration, and hepatocyte pyknosis have been reported in HCQ-treated albino rats [36, 41].

Control rats showed normal cardiac branched myofibers, arranged in bundles. The myocardial fibers are attached via well-preserved intercalated discs with normal striation and central oval nuclei (Fig. 3a). Moreover, the control hearts demonstrated normal cardiac interstitial tissue rich with blood capillaries.

Fig. 3.

Light microphotographs of H&E-stained sections in the heart of control and HCQ-rats demonstrate: (a) Hepatic tissue of control rat, showing normal bundles of branched myofibres, $\times$320; (b–e) Light microphotographs of sections in the heart of HCQ-rats showing: (b) myofibers necrosis (stars), $\times$160 (c) myofibers wavy appearance (arrows), $\times$320 (d) disorganization and disarray (arrows), $\times$320 (e) cardiac myolysis and nonparallel arrangement myofibers (stars), $\times$320.

The heart of rats exposed to HCQ exhibited myofiber necrosis (Fig. 3b), a wavy appearance (Fig. 3c), disorganization, and disarray (Fig. 3d). Rats exposed to HCQ also exhibited occasional cardiac myolysis and nonparallel arrangement myofibers (Fig. 3e).

The induced cardiac alterations may reveal that the cardiac myofibers are affected by exposure to HCQ. The induced wavy appearance in the cardiac tissues may reveal myocardial degeneration, indicating an impact on the pump of the heart, proceeding toward a heart attack [44]. Additionally, the induced myofiber disarray might be associated with non-contractile scar tissue formation and heart rate reduction, which are features of hypertrophic cardiomyopathy [45]. Cardiomyocyte disorganization has also been reported, indicating hypertrophic cardiomyopathy [46]. Case studies have shown that prolonged use of HCQ could induce cardiomyopathy and heart failure [44]. Some studies have demonstrated hydropic degeneration, necrosis, interstitial hemorrhage, and vascular congestion in the vital organs of HCQ-treated rats [35].

The testes of the control rats showed normal seminiferous tubule histological structures, demonstrating normal spermatogenic cells and interstitial tissues (Fig. 4a,b). In comparison with the control testes, the following histological changes were observed in the testicular tissues of HCQ-treated rats comprising, spermatogenic cell sloughing, spermatocytes degeneration, testicular interstitial edema, and occasional spermatogenic arrest (Fig. 4c–e).

Fig. 4.

Light microphotographs of H&E-stained sections in the testes of control and HCQ-treated rats showing: (a,b) Sections in the control testes, (a) demonstrating normal seminiferous tubules (sl), $\times$320 (b) Normal spermatogenic cells (star), $\times$800; (c–e) Sections in the testicular tissue of HCQ-treated rats showing: (c) Spermatid sloughing (arrow), $\times$320 (d) Testicular interstitial edema (arrows), $\times$80 (e) Spermatogenic arrest (star), $\times$720.

Spermatid sloughing may indicate spermatocyte disruption of the testicular tissue skeleton induced by HCQ toxicity [47]. However, spermatogenesis disturbance might have resulted from spermatocyte degeneration or Leydeg cell population abnormality [46].

This finding agrees with that of Kavanagh et al. [48] where chronic exposure to HCQ-depressed testosterone decreases sperm count, increases spermatozoon morphological abnormality, and increases the organ index of the testes and epididymis, seminal vesicle, and prostate. The testicular toxicity of HCQ might be related to several enzymatic activities, needed in spermatogenesis, such as succinate dehydrogenase, glucose 6-phosphate dehydrogenase, and alteration in mitochondrial NADH and cytochrome C. However, Vawda and Saade [49] reported that HCQ could be harmful at the testicular and epididymal levels.

The control spleen demonstrated normal lymphoid white pulp and hematogenous red pulp, separated by prominent marginal zones. The white pulp showed periarteriolar lymphocyte sheaths (PALS), surrounding the follicles (Fig. 5a). The splenic red pulp showed prominent RBCs and venous sinuses, associated with B cells-rich PALS, macrophages, and plasma cells. Furthermore, the white pulp of the control spleens demonstrated normal follicles with central arterioles, surrounded by T cells-rich PALS and fibrous concentric layers (Fig. 5b).

Fig. 5.

Light microphotographs of H&E stained sections in the spleen of control and HCQ-rats show: (a) Splenic tissue of control rat, showing normal red pulp (rp) and the lymphoid white pulp (wp), $\times$80 (b) Marginal zone crowded with B rich-cells PALS, $\times$80 (c–f) Sections in the spleen of HCQ-treated rats showing: (c) Decline in the number and size follicles with decline in the number of T rich-cells, $\times$120 (d) Diffuse and decline in B rich-lymphocytes compared with the marginal zone of the control rats with macrophages infiltration in the red pulp, $\times$160 (e) Bundles of collagen fibers, Mallory trichrome stain (arrows), $\times$80 and (f) High fibrocytes density, $\times$160.

The spleen of rats, subjected to HCQ, exhibited histological alterations in both red pulp and white pulp. The white pulp showed a decline in the size, the number of follicles, and a decline in T-rich-cells PALS number with apoptotic activity (Fig. 5b). Additionally, the germinal center of the follicles showed a decrease in size compared with the control rats. However, the red pulp demonstrated diffuse and a decline in B-rich-lymphocytes with macrophage infiltration and occasional multinucleated giant cells (Fig. 5d).

Collectively, the alterations in the white pulp may indicate the suppression of T-independent humoral immune response [6]. A decrease in cellularity at both the follicles and the marginal zone might have resulted from apoptosis at both T-cells and B-cells [50]. The spleen is the largest immune organ in the human body with hematopoietic and lymphatic components and is being targeted by several chemicals, toxins, and drugs, in which injury may be in both red and white pulps. This immune organ performs a critical role in removing ruptured and degenerated red blood cells.

Hydroxychloroquine has demonstrated efficacy in treating some autoimmune diseases and in decreasing immune system activity. This drug promotes CD8 T-cell stimulation but inhibits CD4 T-cell stimulation [34]. This combined effect has a beneficial action in autoimmunity and in reducing the infections opportunity.