Abstract:
Chronic Kidney Diseases (CKD) are characterised by unresolved fibrosis causing progressive
loss of kidney functions and Vitamin D Insufficiency (VDI). Humans synthesise Vitamin D
(VD), an anti-fibrotic factor, when exposed to Growth Hormones (GH) and Ultraviolet B (UVB)
light. However, VDI exacerbates fibrosis during Kidney Injury (KI). Many plants like Moringa
oleifera (MO) synthesise VD in little amount, however, its enhancement using plant-GH {Indole
Acetic Acid (IAA)} and exposure to UVB has not been sufficiently explored. This study was
designed to assess IAA and UVB effects on MO’s biochemical, nutritional and nephro-
protective properties.
One hundred MO seeds (NH-41) were obtained from NIHORT, Ibadan. Twenty-five seeds were
each soaked in distilled water (control), 0.4, 0.5, 0.6mM IAA and sowed in a randomised design
(n=5) in polythene bags containing 10kg of soil. Seedlings, after seven days of germination,
were exposed to UVB (315nm) at 1hour/day for 10weeks. The MO Leaves (MOL) were
harvested, dried and milled, while MOL Oil (MOLO) was extracted with n-hexane using soxhlet
apparatus. Coding sequences of MO lanosterol and cycloartenol synthases were amplified from
cDNA of fresh MOL, cloned into plasmid, and transformed into yeast cells, respectively. The
Transformed Yeast Cells (TYC) were treated with IAA (0.04mM) and exposed to UVB
(15min).Vitamins D2 and D3 contents of MOL, IAA-UVB-treated-MOL, MOLO, IAA-UVB-
treated-MOLO, and IAA-UVB-treated TYC were quantified by HPLC. Photosynthetic
pigments, calcium and phosphorus contents of fresh MOL were determined
spectrophotometrically. The MOL and MOLO were mixed with chow as experimental diets,
separately. Twenty-five male mice (13-15g) were fed with adenine-fortified-chow (0.75%w/w)
to initiate KI. Five mice without KI served as control. Mice with KI were grouped and fed with
MOL (33%w/w), IAA-UVB-treated-MOL (33%w/w), MOLO (10%w/w), IAA-UVB-treated-
MOLO (10%w/w), calcitriol-fortified chow (0.1%w/w) respectively, while control was fed with
standard chow for four weeks, then sacrificed. Serum creatinine, Uric Acid (UA), sodium,
phosphate and calcium were determined spectrophotometrically. Serum Fibroblast Growth
Factor-23 (FGF-23), Kidney Injury Marker-1 (KIM1), and klotho were determined by ELISA.
Kidney fibrosis was evaluated by H&E stains. Data were analysed using ANOVA at α0.05.
Vitamins D2 and D3 contents of 0.6mM IAA-UVB-treated-MOL and IAA-UVB-treated-MOLO
increased by 23.4, 25.3% and 29.2, 30.1%, respectively, relative to their controls. Vitamins D2
and D3 contents of IAA-UVB-treated TYC increased by 15.6, 11.7folds, respectively, relative
to control. Chlorophylls a and b contents of 0.5mM IAA-UVB-treated-MOL (6.31±0.01 and
6.65±0.03mg/gfw), and 0.6mM IAA-UVB-treated-MOL (7.02±0.03 and 7.11±0.01mg/gfw),
increased relative to control (4.90±0.01 and 4.99±0.01mg/gfw), respectively. Calcium and
phosphorus contents of 0.5, 0.6mM IAA-UVB-treated-MOL increased by 44.6, 28.5% and 57.5,
59.1%, respectively, relative to control. The IAA-UVB-treated-MOLO reduced serum
creatinine, UA, sodium, phosphate, calcium, FGF-23 and KIM1 by 25.0, 45.5, 25.0, 23.0, 26.5,
43.6 and 47.0%, respectively, relative to control. In IAA-UVB-treated-MOLO-fed mice, serum
klotho increased by 45.0% and kidney fibrosis reduced significantly, relative to control.
Indole acetic acid and ultraviolet B light enhanced vitamin D contents of Moringa oleifera
leaves and its oil, which demonstrated nephro-protective properties via anti-fibrotic
mechanisms.
