Lemon juice has protective activity in a rat urolithiasis model

Kidney stone formation or urolithiasis is a complex process that is a consequence of an imbalance between promoters and inhibitors in the kidneys [1]. The recurrence of urolithiasis represents a serious problem as patients who have formed one stone are more likely to form another. Not all standard pharmaceutical drugs used to prevent urolithiasis are effective in all patients, and many have adverse effects that compromise their long-term use [2].

Renal calculi can be broadly classified in two large groups: tissue attached and unattached [3]. Attached calculi are mainly integrated by calcium oxalate monohydrate (COM) renal calculi, with a detectable attachment site to the renal papilla and basically consisting of a core located near to the attachment site (concave zone) and radially striated concentrically laminated peripheral layers. Unattached calculi, with no detectable site of attachment to papilla, are developed in renal cavities of low or reduced urodynamic efficacy and can exhibit diverse composition and structures. Several reports have been published since Randall's first description of papillary calcifications and their possible active role in the genesis of COM papillary calculi [4‐6]. At present, it seems clear that renal epithelial cell injuries play a decisive role in such a type of renal calculi development [7, 8], and in fact the lithogenic effect caused by ethylene glycol (EG) must be mainly attributed to the oxidative damage caused by the high level of oxalate generated by EC. Thus, although EC rat model can be questioned as a general model to study renal stone formation, it must be considered as an interesting model to evaluate renal papillary stone development, at least for those stones which genesis is linked to oxidative cell damage. Thus, the first studies on experimental EC renal lithiasis appeared in the 60' decade [9, 10] but the importance of the oxidative damage caused by hyperoxaluria was not clearly proposed until the end of the century [11]. From this last period it appeared several prophylaxis proposals on EC induced nephrolithiasis using herbal extracts and antioxidants [12‐19]. In all these papers the effects of these compounds did not seem to be mediated by diuretic or other urinary biochemical changes and positive effects on calcium oxalate lithiasis are most likely due to antioxidative effects.

To further investigate the potential of lemon juice as a therapy for lithiasis, the present study examined the effect of lemon juice on experimentally EG-induced calcium oxalate (CaOx) nephrolithiasis in rats.

Urinary lithiasis is generally the result of an imbalance between inhibitors and promoters in the kidneys. Human kidney stones are usually composed of CaOx [1], and several studies have examined the effect of the citrus juices on calcium salt crystallization [23‐27]. However, the conclusions from those studies were not consistent.

Many in vivo models have been developed to investigate the mechanisms involved in the formation of urinary stones, and to ascertain the effect of various therapeutic agents on the development and progression of the disease [28‐33]. Rats are the most frequently used animals in models of CaOx deposition in the kidneys, a process that mimics the etiology of kidney stone formation in humans [28]. Rat models of CaOx urolithiasis induced by either EG alone or in combination with other drugs such as AC, are often used to study the pathogenesis of kidney crystal deposition [30]. Using the accelerated model [32], in the present study rats were treated with 0.75% EG and 2% AC for 10 days. All positive control rats (Group 5) developed CaOx depositions during that time.

The present study examined the effect of various lemon juice concentrations on the deposition of CaOx crystals within the rat kidney. Previous studies concluded that medicinal plants had little effect on the urinary chemistry of urolithiasis [34, 35]. The current study analyzed body weight, kidney calcium level, serum concentrations of calcium, phosphorus, urea and creatinine, and the histopathology of the kidney. We found that Group 1 rats (negative controls) remained active and gained weight, while Group 2, 3, 4 and 5 rats lost weight over the 10 days of treatment. Microscopic examination using polarized light of kidney sections derived from nephrolithiasic rats showed intratubular and interstitial crystal deposits, consistent with the findings of others [36]. These crystals were intensely birefringent, polycrystalline, and arranged in a rosette characteristic of CaOx crystals. The presence of such deposits is evidence of adhesion and retention of particles within the renal tubules. These crystal deposits were observed in the kidneys of all Group 5 rats. Moreover, 33% of these rats showed major calcifications on the papillary tip. In contrast, no rats treated with lemon juice showed such papillary crystalline deposits. Rats treated with 100% or 75% lemon juice had far less kidney calcification and lower renal tissue calcium levels than the positive control rats (Group 5) (Table 1 and 2). No papillary encrustations were seen in 100%, 83% and 50% of rats treated with 100%, 75% and 50% lemon juice, respectively. Furthermore calcic parenchymatous deposits were not observed in 83% of rats treated with 100% and 75% lemon juice. These results clearly demonstrate the ability of the lemon juice to prevent the development of papillary and renal parenchymatous calcifications on the kidney, consequently preventing the development of papillary and parenchymatous calculi. All rats treated with 50% lemon juice showed fewer calcium deposits on the kidney surface than positive control rats (Group 5). While treatment with 100% and 75% lemon juice appeared to be more beneficial that treatment with 50% juice, this difference was not found to be statistically significant.

The association of crystals with renal tubular cells is considered a potential factor in the process of renal stone formation. Indeed, calculations considering the rate of crystal growth even at its maximum speed and tubular fluid flow suggest that a single crystal would not become large enough to be retained and occlude the lumen during its normal transit through the nephron [28]. Furthermore, it is established that crystals, especially calcium oxalate monohydrate crystals, can be retained by attachment to the surface of renal epithelial cells and be internalized [28].

Lemon juice has a high antioxidant capacity due to the presence of citrate, vitamin C, vitamin E and flavonoids such as eriocitrin, hesperetin [37, 38] and limonoids [39]. Vitamin E may prevent calcium oxalate crystal deposition in the kidney by preventing hyperoxaluria-induced peroxidative damage to the renal tubular membrane surface (lipid peroxidation) [40, 41], which in turn can prevent calcium oxalate crystal attachment and subsequent development of kidney stones [41, 42].

In urolithiasis, the glomerular filtration rate (GFR) decreases due to stones in the urinary system obstructing urine outflow. This leads to the accumulation of waste products in the blood, particularly nitrogenous substances such as urea, creatinine and uric acid. In addition, increased lipid peroxidation and decreased levels of antioxidant potential have been reported in the kidneys of rats supplemented with a calculi-producing diet [20]. In this context, oxalate has been reported to induce lipid peroxidation and to cause renal tissue damage by reacting with polyunsaturated fatty acids in cell membranes [20]. In the present study, the positive control calculi-induced rats (Group 5) were found to have marked renal damage, consistent with the elevated serum levels of creatinine and urea. The administration of lemon juice inhibited these changes that would otherwise promote new stone formation in the urinary system. In rats treated with lemon juice, we attribute the lower serum creatinine and urea levels to an enhanced GFR and the anti-lipid peroxidative property of lemon juice [20]. As commended, the lithogenic effects of EG must be mainly attributed to the oxidative damage caused by the high level of oxalate generated by this substance. For this reason, the presented studies were focused to evaluate the effects on renal papillary tissue through histological studies and the protective effects caused by the consumption of lemon juice. Previous studies evaluated the effects of citrate on renal lithiasis induced by EG [43, 44]. Nevertheless, to attain an increase in citrate excretion it is necessary to induce metabolic acidosis in rats and to achieve this condition it is necessary to increase the doses of EG to 2%. In such case, urinary pH of EG treated rats was clearly inferior to urinary pH of control group, the treatment with high doses of potassium citrate significantly increased the urinary pH and, as a consequence, the urinary citrate excretion notably rose. Nevertheless, EG doses of 0.75% practically did not change the urinary pH value when compared with control group [36, 44] and consequently the administration of citrate did not cause important changes in urinary citrate excretion [45].

The present study found that the administration of lemon juice effectively prevented the development of urolithiasis in rats. These findings support the use of lemon juice as an alternative medicine to prevent urolithiasis. Further research is necessary to clarify the mechanism underlying this preventative effect of lemon juice.