Lack of vitamin D impairs kidney function
Karina Toledo/Agência FAPESP/DICYT In addition to the well-known problems with bone mineralization, vitamin D deficiency has recently been associated with the development of cardiovascular and autoimmune diseases, high blood pressure and several types of cancer.
Now, a study at the Medical Investigation Laboratory (LIM12) at the University of São Paulo School of Medicine (FMUSP) has revealed that the lack of this nutrient may also impair kidney function and compromise that organ’s ability to recover from injury.
“One of the principal causes of acute kidney injury in humans is injury as a result of an ischemic event, which occurs when the blood flow to the kidney is obstructed for a time and then restored. During the ischemic process, the lack of oxygen leads to cell degeneration and cell death. Our objective was to discover how the lack of vitamin D influences this regenerative process,” explained biologist Rildo Aparecido Volpini, coordinator of the FAPESP-funded project, “Assessing the role of vitamin D in the development of post-ischemic acute kidney injury.”
An animal experiment indicated that the nutrient deficiency diminishes kidney function, modifies local protein expression and increases the formation of fibrosis after induced injury.
Volpini’s group developed two experimental models of ischemia and reperfusion (restoration of blood flow after a period of lack of oxygen) in rats. In the acute protocol, two-month old animals – equivalent to young adult humans – were fed a vitamin D-free diet for 30 days. On the 28th day, the researchers induced an ischemic injury and reperfusion.
“The blood flow to the kidneys was stopped for 45 minutes, which was enough time to cause significant injury. The animals were evaluated after 48 hours and then euthanized so that we could analyze the genetic and protein expression in the organ,” said the researcher.
In the chronic protocol, the rats were fed a vitamin D-free diet for 90 days. On the 28th day, they underwent induced injury from ischemia and reperfusion; 60 days later, the animals were evaluated prior to being euthanized.
“To determine the amount of vitamin D present in the bodies of the animals, we administered a dose of serum 25-hyrdroxyvitamin D (25(OH)D), a circulating form of vitamin D, routinely used to stimulate levels of this hormone in the body,” Volpini explained.
While the control group presented between 15 and 16 nanograms (ng) per milliliter (ml) of blood, the acute protocol rats fed a vitamin D-free diet presented approximately 4 ng/ml on the 30th day of consumption. The chronic protocol animals fed the same diet for 90 days presented undetectable serum levels of vitamin D. “This shows that if there had been any vitamin D synthesis through the skin, it was negligible,” commented Volpini.
In the two experimental models, the animals were divided into four groups: the first, considered the control group, was fed normal food and did not undergo ischemia and reperfusion; the second group was fed only a vitamin D-free diet and had no induced kidney injury; the third was fed normal food and underwent ischemia and reperfusion; the fourth group was fed a vitamin D-free diet and underwent induced injury.
The analysis of kidney function conducted on the acute protocol animals revealed that while the control group presented a glomerular filtration rate between 0.8 to 1 ml per minute per 100 grams of weight, the group that was fed a vitamin D-free diet alone filtered only between 0.6 and 0.7 ml/min/100 g weight – a drop of approximately 20% in kidney function.
The group of rats that underwent ischemia and was fed normal food experienced a drop of 50% (nearly 0.4 ml/min/100 g weight), and the group that had an induced injury and was fed a vitamin D-free diet experienced a 70% drop in kidney function (0.3 ml/min/100 g weight).
“We observed that the lack of vitamin D alone already compromised kidney function. We do not know exactly why, but it was most likely due to alterations in the renin-angiotensin-aldosterone system (RAAS, the set of peptides, enzymes and receptors involved in controlling blood pressure) with resulting repercussions in the pressure control. There is evidence in the literature that shows that vitamin D deficiency contributes to inappropriate activation of the RAAS and acts as a mechanism for the progression of chronic kidney disease,” said Volpini.
The next step was to analyze the electrolytes in the plasma and urine of the animals and to determine whether there had been any proteins in the urine (proteinuria).
“The presence of proteins in the urine [proteinuria] is an indication of kidney injury. It means that the glomerular filter is not functioning properly or that the renal tubules are not managing to reabsorb the filtered proteins. Normally, the process of filtration and reabsorption does not allow these important molecules to escape into the body,” Volpini explained.
The tests showed that the group of rats that was fed a vitamin D-free diet alone presented an approximately 60% increase in proteinuria when compared to the animals in the control group. The animals that were fed a vitamin D-free diet and underwent ischemia and reperfusion had an increase of over 90% in the excretion of proteins in the urine.
“Studies have shown that low levels of vitamin D can trigger proteinuria through direct and indirect factors. Directly, low levels of vitamin D induce the loss of podocytes (cells of the renal epithelium that form an important component of the glomerular filtration barrier) and the development of glomerulosclerosis, compromising the integrity of the glomerular filtration membrane, and thus allowing macromolecules to pass into the urinary space,” said Volpini.
Another possible explanation, according to the researcher, would be the appearance of proteinuria through an indirect manner. Low levels of vitamin D promote alterations in the RAAS, triggering an increase in blood pressure. This hemodynamic alteration may contribute to the increase in the excretion of proteins in the urine.
Another alteration observed in the acute study, mainly among the vitamin D-deficient animals, was that the kidneys lost the ability to concentrate urine to conserve water. The vitamin D deficiency reduced the protein expression of aquaporin 2, the molecule responsible for carrying water in the collecting ducts.
“In the animals that were fed a vitamin D-free diet, we found a 20% to 30% reduction in the urinary osmolality compared to the control group. In the other two groups – ischemia alone, and deficiency along with ischemia – the drop in the concentration was over 50%,” explained the researcher.
Protein expression analyses conducted after sacrificing the animals showed that while the expression of aquaporin 2 was at physiological levels (100%) in the control group, the rats that were fed a vitamin D-free diet expressed only 26%. In animals that underwent ischemia, aquaporin 2 expression was approximately 50%, but in animals that underwent ischemia and were vitamin D-deficient, it was 25%.
“Previous studies have already shown that vitamin D influences gene expression in the bones, brain, intestines, liver and kidneys. Our findings show that it has a direct impact on aquaporin 2 expression, p21 [a cell cycle inhibitory protein that participates in controlling cell proliferation] protein expression and klotho expression [related to cell aging]. According to our data, klotho protein expression is reduced in animals that undergo ischemic injury, and p21 protein expression is elevated in these same animals, leading us to associate these two parameters,” Volpini said.
While p21 protein expression in the control group and in the group that was fed vitamin D-free food were at 100% (physiological levels), in the group that only underwent ischemic injury, the expression increased to 290%. The group that was fed a vitamin D-free diet and underwent induced injury expressed 182% of the p21 protein.
“Previous studies have demonstrated that when kidney injury is caused by ischemia and reperfusion, the cells needs to rest, in other words, not undergo cell division, in order to conserve energy. The increase of the p21 expression in this case may be considered protective. On the other hand, the vitamin D deficiency attenuated the p21 expression when compared to the ischemic animal group, compromising the cell protection mechanism,” Volpini explained.
The findings also show damage in klotho expression, which is involved in controlling cell senescence. The group that was fed a vitamin D-free diet presented only 76% of the expression of the control group, which was at 100%. The ischemic group expressed only 22%, and the group that was both deficient and ischemic expressed only 16%.
“Although we haven’t assessed it, it is very likely that the klotho expression in other organs is also compromised by the lack of vitamin D,” Volpini explained.