Date of Award

5-15-2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Christine Metz

Abstract

Cisplatin is a valuable chemotherapeutic agent used in the treatment of a wide variety of cancers, including ovarian, testicular, breast, bladder, non-small cell lung, head and neck cancers, as well as lymphomas. However, approximately 25-30% of the patients taking this antineoplastic agent experience dose-limiting acute kidney injury (or AKI).

Advanced age and female gender are important risk factors for cisplatin-induced renal injury. Cisplatin causes kidney injury by damaging the proximal and distal tubules of the kidneys where it accumulates. Cisplatin-induced kidney injury occurs as a result of DNA damage, inflammation, oxidative stress and apoptosis/necrosis in the kidney tubules.

Kidney damage caused by cisplatin is also accompanied by electrolyte disturbances, such as hypocalcemia, hypokalemia, and hypomagnesemia. Hypomagnesemia occurs in almost all patients receiving cisplatin and is believed to contribute to cisplatin-mediated kidney injury.

Magnesium is an essential nutrient required in the amount of 300-400mg per day. It is the second most abundant intracellular cation and acts as a co-factor for over 300 enzymatic reactions in the body. For example, magnesium is required for the biologic activity of ATP, the synthesis and stability of DNA and RNA, stabilization of mitochondrial membrane, and the production of proteins, along with many other biological/metabolic processes. Magnesium is primarily absorbed in the gut and is excreted in the urine.

However, the kidneys which are responsible for magnesium reabsorption are the primary site of magnesium homeostasis. Magnesium deficiency is surprisingly not uncommon with more than 50% of the US population consuming less than the reference daily intake (RDI). Magnesium deficiency is particularly widespread among the elderly, hospitalized and critically ill patients. Unfortunately, most multivitamins provide less than 25-30% of the RDI for magnesium.

Therefore, we hypothesized that: 

  1. Mg deficiency will worsen cisplatin-induced renal injury by promoting inflammation, oxidative stress, and apoptosis/necrosis within the kidneys.
  2. Mg replacement/supplementation before and during the cisplatin-treatment period will protect the kidneys from cisplatin-induced damage and improve cisplatin tolerability by reducing renal inflammation, oxidative stress and apoptosis/necrosis.

The overall objective of this thesis was to investigate the role of magnesium in cisplatin-induced acute kidney injury with the following specific aims:

    1. Examine the effects of magnesium deficiency on cisplatin-induced acute kidney injury in physiologically relevant older female mice.
    2. Investigate the effect of magnesium supplementation on cisplatin-induced acute kidney injury in physiologically relevant older female mice.
    3. Determine the mechanisms of enhanced cisplatin-induced kidney injury associated with Mg deficiency and the renoprotective effects of Mg therapy using in vivo and in vitro models (e.g. renal cisplatin accumulation, oxidative stress, inflammation and apoptosis)

Investigate the role of magnesium status in cisplatin-induced acute kidney injury using a tumor-bearing mouse model (A2780 human ovarian carcinoma xenograft model in athymic nude mice) and the effect of Mg status on tumor growth and cisplatin’s chemotherapeutic efficacy.

Using a rodent model of cisplatin-induced acute kidney injury, we demonstrated that pre-existing magnesium deficiency significantly increases cisplatin-mediated kidney damage and magnesium replacement following deficiency significantly abrogates cisplatin-induced kidney damage. We observed enhanced kidney damage with increased inflammation, oxidative stress and apoptosis/necrosis post-cisplatin in the setting of Mg deficiency and decreased inflammation, oxidative stress and apoptosis/necrosis post-cisplatin with Mg replacement following Mg deficiency. Mg deficiency enhanced the activation of pro-inflammatory pathways (e.g. ERK1/2 and STAT3), thereby increasing renal inflammatory markers such as CXCL2, CCL2, CXCL1, IL-6 and IL-1β. Increased inflammatory markers following cisplatin were also associated with enhanced neutrophil infiltration and myeloperoxidase levels in the kidneys. Increased mRNA expression of the oxidative stress marker, Ncf1, and enhanced renal apoptosis was also observed in magnesium-deficient mice following cisplatin. Also, we found augmented platinum accumulation in the kidneys of Mg-deficient mice following cisplatin treatment, which was accompanied by decreased expression of cisplatin efflux transporters. Magnesium supplementation following deficiency reversed these adverse effects; decreased renal platinum accumulation and increased cisplatin efflux transporter expression within the kidneys.

Using a human ovarian tumor (A2780) xenograft model in athymic nude mice, we showed similar harmful effects of magnesium deficiency on cisplatin-mediated kidney injury while magnesium supplementation following magnesium deficiency protected against cisplatin-induced kidney injury without compromising the chemotherapeutic efficacy of cisplatin. Consistent with these observations, we found that while magnesium deficiency increased ovarian tumor (A2780) cell proliferation in vitro magnesium supplementation did not enhance ovarian tumor cell proliferation in vitro and that neither magnesium deficiency nor supplementation altered the efficacy of cisplatin-mediated killing of A2780 ovarian tumor cells in vitro. Similarly, magnesium supplementation did not affect cisplatin-mediated killing of MCF-7 breast cancer cells and H460 lung cancer cells in vitro.

These findings, along with previous small clinical studies show that magnesium supplementation protects against cisplatin-induced acute kidney injury. The results described in this thesis advance the field because they are the first to describe that while magnesium guards against cisplatin-induced acute kidney injury, it doesn’t compromise cisplatin’s chemotherapeutic efficacy using the ovarian tumor xenograft model. Therefore, future large scale long-term studies to better understand the renoprotective role of magnesium in cancer patients and monitor the long-term effects of magnesium supplementation on tumor-related outcomes, including metastasis and survival need to be initiated.

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