Ozone Therapy and CKD

by Robin Rose MD

Ozone (O3) is a reactive oxygen species (ROS) that interacts with cellular components  - causing  oxidative stress → this “stressful milieu” exerts a mediated [hormetic] antioxidant function via controlled toxicity [produced by low concentrations of O3] → this enhances the cell’s antioxidant properties without causing any further damage. O3 therapy is effective when applied before or after traumatic renal procedures [whether caused by ischemia, xenobiotics, chronic damage, or other models]. [1] 

Ozone, by means of oxidative preconditioning, preserves the cell redox state. exerting its protective effects. [4,6]

The ozone [O3] molecule consists of three unstably-bound oxygen atoms. It is formed by ultraviolet (UV) radiation naturally when the di-oxygen molecule [O2] binds to atomic oxygen.  It is artificially created, using O3 concentrators → works through the electrical discharge of gas, creating an airtight barrier that prevents O2 from passing through, forcing it to dissociate and form O3 instead. [1]

As a ROS [more reactive than O2], the O3 molecule has notable oxidizing capacity, classified as a non-radical ROS.  O3 reacts by itself with biological structures, such as amino acids (especially aromatic), enzymes, deoxyribonucleic acid (DNA), membrane glycoproteins and lipids. [1]

It is unstable - in the gaseous phase, in water solution, or in combination with other extracellular substances. But it dissolves in a chain reaction to form a variety of free radicals [such as hydroxyl radical (•OH) and superoxide radicals (O2•−)] → these acquire missing electrons by oxidizing biological components. Then the breakdown of ozone is inhibited and the chain reaction ends → because organic and inorganic substances, [such as stable carbonate ions (CO3-2−) and bicarbonate (HCO3)]  react with OH radicals to form secondary radicals that do not generate superoxide radicals. [1]

ROS created from the interactions of O3 [and O3 itself] oxidize/destroy the cysteinyl thiols of Kelch-like ECH-associated protein 1 (KEAP1) [a protein that keeps nuclear erythroid factor 2 (Nrf2) ubiquitinated].

Transcription factor Nrf2 translocates to the nucleus→ regulates oxidative damage by inducing antioxidant response elements (ARE) [including antioxidant enzymes that are protective responses - to delay, prevent, or remove oxidative damage via catalytic or scavenger activity of free radicals]. Upregulated antioxidant proteins include catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione s-transferase (GSTr), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), and heat shock protein-70 (HSP70). [1]

O3 therapy also works by inducing controlled oxidation of polyunsaturated fatty acids (PUFAs) in cell membranes., creating ozonated lipid products (LOPs) [important signal transducers and regulators of inflammation]. LOPs exacerbate Nrf2 nuclear translocation [by oxidizing cysteine residues, promoting KEAP1-Nrf2 dissociation, and activation of Nrf2-ARE pathways]. [1]

LOPs illustrate a hormetic response: 

⦿ at low concentrations, they downregulate activation of NF-κB [nuclear factor kappa B] and inflammatory cytokines,  and they increase antioxidant and anti-inflammatory compounds

⦿ at high concentrations, they stimulate potential antimicrobial and chemotactic responses → by activating phospholipase A2 and phospholipase C, which stimulate synthesis of NF-κB and pro-inflammatory cytokines [interleukins (IL) 1 and 6, and tumor necrosis factor-alpha (TNF-α), transforming growth factor beta (TGF-β),cyclooxygenase (COX)]. [1]

Ozone is a gas mixture comprising ozone/oxygen - exhibits various immune system effects: modulation of phagocytic activity of peritoneal and alveolar macrophages. Studies have shown benefit in various diseases, including peritonitis, infected wounds, and advanced ischemic diseases. Ozone induced increase in antioxidant enzyme activities allows the patient to face physiopathological conditions mediated by ROS/RNS, including beneficial effects on kidney damage induced by renal IRI.[3]

O3 and CKD

Ischemia

Therapeutic effects observed in kidney after the administration of O3 administration include amelioration/protection of renal function [measured through plasma clearance of endogenous metabolites, blood urea nitrogen (BUN), and serum creatinine (SCr)].Ozone therapy has been reported to decrease morphological damage [mainly evidenced under photon microscopy], including medullary hemorrhage, tubular necrosis, glomerular damage, collagen deposition, and fibrosis markers, such as α-smooth muscle actin (α-SMA) and TGF. [1]

O3 therapy reduces inflammation → diminished expression of cytokines [IL, TNF-α, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1), toll Like receptor 4 (TLR 4)-NFkB pathway]. [1]

Decreased lipid peroxidation represents the oxidative stress induced by polyunsaturated fatty acids is diminished by ozone→ the marker malondialdehyde (MDA) diminishes its renal expression when ozone-treated. [1]

Renal ischemic damage occurs with diminished or restricted blood supply. In contrast, oxidative damage occurs when tissue is re-oxygenated.  Embracing an array of treatments for oxidative damage is essential since ischemic-producing scenarios are highly prevalent. [1] 

Ischemia damages endothelial cells → when blood flow decreases, shearing forces increase → more ROS releases, damaging even more endothelium.  Ozone therapy increases tissue oxygenation via its hemorheological effects, increasing erythrocyte pliability, diminishing blood viscosity and erythrocyte aggregation, allowing enhanced renal blood flow in an organ previously submitted to a lack of blood. [7]

Nitric oxide - NO - is a protective mechanism favored by O3 therapy against IRI [ischemia reperfusion injury], inflammation, and vasoconstriction caused by Endothelin-1: nitrate-derived NO applied topically is an effective therapy against IRI damage 

O3 therapy improves kidney damage by decreasing markers of kidney damage, inflammation, and fibrosis → good treatment for ischemic injuries such as kidney transplantation, iatrogenic trauma, partial nephrectomy, heart failure, and hypovolemia, [among other clinical conditions that reduce renal blood flow, like those that produce AKI].

Ozone therapy may be considered as an adjuvant to insulin in the treatment of diabetes to prevent or alleviate diabetes induced nephropathy. The efficacy of ozone therapy in diabetes has been attributed to its hypoglycemic effect, induction of antioxidant enzyme activities, and control of their expression. [4] 

Ozone-induced NO synthase expression at the renal vascular endothelium level results in arteriolar vasodilation [preventing diabetic nephropathy development] Ozone may also protect against NO/ROS imbalance by preventing NO depletion and improving NO-mediated vasodilatation [goal is to restore blood pressure to normal]. [4]

Ozone treatment decreases overproduction of ROS by the mitochondrial electron transport chain in diabetics, → inhibiting polyol pathway activation and increasing concentration of angiotensin enzyme activity. Ozone induces SOD activity through stimulation of the SOD gene expression [4]

Xenobiotics 

These exogenous chemicals are not essential for its physiological functions and processes [synthetic drugs, metals, and environmental factors etc.]. 

Acetaminophen [APAP, aka Tylenol] is a common anti-inflammatory drug, can produce severe nephrotoxicity: APAP’s hepatic degradation and further enzymatic formation of [highly toxic/reactive metabolite] N-acetyl-p-benzoquinone (NAPQI) [normally neutralized by glutathione (GSH) ]. But in APAP overdose, excessive NAPQI is formed → not controlled by antioxidant enzymes→ causes oxidative damage: especially in proximal tubules.

O3 therapy is an effective antioxidant therapy [enhances antioxidant enzymes and diminishes oxidation]. O3 therapy in APAP-induced nephrotoxicity [when combined with another antioxidant therapy, N-acetylcysteine (NAC)] produces no significant changes in the kidney’s function (creatinine, urea) and inflammation (IL-6, IL-10) → significant changes against oxidative stress, lower levels of MDA, reduction of histopathologic glomerular, tubular, and interstitial damage.

Cadmium (Cd) is a heavy non-essential metal that progressively accumulates in body tissues → humans are exposed through air particles, occupational exposure, and seafood [mollusks, crustaceans, or fish]. Cd produces nephrotoxicity by various mechanisms, [DNA damage, altered gene expression, most importantly, oxidative damage - by depleting cells’ antioxidant defenses (such as selenium, which binds to Cd to neutralize it)]. 

O3 therapy can diminish Cd accumulation, augment metallothionein levels [binds Cd in others to diminish its toxicity], and reduce morphologic damage, → an effective protective mechanism against Cd2⁺ renal damage. It reduces lysosomal enzyme NAG [N-acetyl-β-D-glucosaminidase - found mainly in proximal convoluted tubules.  The NAG increase [mediated by loss of the tubular brush border] liberates this enzyme into the urine → associated with pathologic processes such as Cd-intoxication, malignancies [kidney, liver, pancreas, lung, and breast etc.], and increased kidney failure risk. 

Some antineoplastics are proven to cause nephrotoxicity : O3 therapy, in certain doses, may mediate protective effects against the morphologic damage, arterial pressure, and proteinuria. 

Radiographic contrast media (CM) is liberally used in clinical procedures → its high toxicity causes renal dysfunction [including direct oxygen-free radical damage, modified hemodynamics, and hypoxic renal medullary injury mediated by stunted blood flow and increased tubular O2 supply.] Ozone has been used in this setting to diminish CIN [contrast induced nephropathy]. 

The use of xenobiotics as drugs in medicine can lead to nephrotoxicity by accumulation, formation of free radicals, and depletion of antioxidant substances.However, during their metabolism and excretion, some might become nephrotoxic by accumulation, directing damage,  O3 can be effective against this damage.

O3 Therapy Protects the Kidney against CKD

O3 ameliorates CKD by stimulating the expression of antioxidant enzymes and reducing inflammation .

Diabetic kidney disease (DKD) is a main cause of CKD, via apoptosis, formation of free radicals, advanced glycation end-products (AGES), inflammatory cytokines, and other growth molecules. O3 has beneficial anti-apoptotic and antioxidative effects. 

O3 treatment has been effective against the morphological and oxidative damage caused by shock wave therapy for kidney stones [due to its antimicrobial capacity, it ameliorated oxidative damage caused by microorganisms in kidney infection (pyelonephritis) and septic shock in kidneys].

In a CKD  animal model, ozone rectal administration induced delayed CKD progression advancement, protecting kidneys against vascular, hemorheological, and oxidative mechanisms. Ozone therapy offers protective effects on renal tissue by down regulation of CKD oxidative stress and proinflammatory cytokine. 

Ozone was found to target important mediators of lethal renal injury by reducing oxidative stress, decreasing calcium overload, improving endothelial function, increasing adenosine levels, and attenuating renal cell apoptosis. Positive benefits  of ozone were also seen on nitric oxide and adenosine levels and for mitochondrial  preservation in hepatic ischaemia-reperfusion processes. 

The ozone group showed improved proteinuria, plasma creatinine concentration, there was antiplatelet activity, diminishing blood viscosity with decreased friction between the blood and glomerular vascular walls, with diminished endothelial injury and glomerular collapse.  Decreased tubular hypoxia, hemorrhages, and release of proinflammatory cytokines such as IL-6,IL-1 and TNFα was appreciated. Pathological ROS levels of ROS can degrade glomerular basement membrane and induce glomerular injury [characterized by impaired glomerular filtration and increased protein excretion]. 

REFERENCES

1. Delgadillo-Valero LF, Hernández-Cruz EY, Pedraza-Chaverri J. The Protective Role of Ozone Therapy in Kidney Disease: A Review. Life (Basel). 2023 Mar 10;13(3):752. doi: 10.3390/life13030752. PMID: 36983907; PMCID: PMC10057350.

2. Yu,G et al. The NLRP3 inflammasome is a potential target of ozone therapy aiming to ease chronic renal inflammation in chronic kidney disease. International Immunopharmacology Volume 43, February 2017, Pages 203-209. 

3. Oztosun, M., Akgul, E. O., Cakir, E., Cayci, T., Uysal, B., Ogur, R., … Korkmaz, A. (2012). The Effects of Medical Ozone Therapy on Renal Ischemia/Reperfusion Injury. Renal Failure, 34(7), 921–925. https://doi.org/10.3109/0886022X.2012.692752

4. Morsy, M.D., Hassan, W.N. & Zalat, S.I. Improvement of renal oxidative stress markers after ozone administration in diabetic nephropathy in rats. Diabetol Metab Syndr 2, 29 (2010). https://doi.org/10.1186/1758-5996-2-29

5. Dayani MA, Hasanpour Dehkordi A, Miraghajani M. Ozone therapy in chronic diseases; a narrative review of the literature. J Renal Inj Prev. 2019; 8(3): x-x. DOI: 10.15171/jrip.2019.xx

6. Fernandez JLC et al. Ozone Therapy on Rats Submitted to Subtotal Nephrectomy: Role of Interleukin 6 and Antioxidant System. Revista Cubana de Investigaciones Biomedicas. Vol. 38, No. 1 (2019)