Increased Phosphaturia Accelerates The Decline in Renal Function: A Search for Mechanisms

Abstract

In chronic kidney disease (CKD), high serum phosphate concentration is associated with cardiovascular disease and deterioration in renal function. In early CKD, the serum phosphate concentration is normal due to increased fractional excretion of phosphate. Our premise was that high phosphate intake even in patients with early CKD would result in an excessive load of phosphate causing tubular injury and accelerating renal function deterioration. In CKD 2-3 patients, we evaluated whether increased phosphaturia accelerates CKD progression. To have a uniform group of patients with early CKD, 95 patients with metabolic syndrome without overt proteinuria were followed for 2.7 ± 1.6 years. The median decline in eGFR was 0.50 ml/min/1.73 m²/year. Patients with a more rapid decrease in eGFR had greater phosphaturia. Moreover, the rate of decrease in eGFR inversely correlated with the degree of phosphaturia. Additionally, phosphaturia independently predicted renal function deterioration. In heminephrectomized rats, a high phosphate diet increased phosphaturia resulting in renal tubular damage associated with inflammation, oxidative stress and low klotho expression. Moreover, in rats with hyperphosphatemia and metabolic syndrome antioxidant treatment resulted in attenuation of renal lesions. In HEK-293 cells, high phosphate promoted oxidative stress while melatonin administration reduced ROS generation. Our findings suggest that phosphate loading in early CKD, results in renal damage and a more rapid decrease in renal function due to renal tubular injury.

Figure 1

Flow chart showing details of how patients were selected. UP/Cr: Urinary Phosphate/creatinine; HDL-chol: High Density Lipoprotein Cholesterol; TG: Triglycerides, UPr/Cr: Urinary Protein/Creatinine; UAlb/Cr: Urinary Albumin/creatinine.

Figure 2

Prediction of progression status depending on urinary albumin/creatinine (Alb/Cr) and phosphate/creatinine (P/Cr) (mg/mg). P = 1/(1 + EXP (2.093 − 4.197xP/Cr − 6.6xAlb/Cr).

Figure 3

Excessive phosphaturia promotes renal injury. Renal samples from Sham + LPD, Sham HPD, 1/2Nx + LPD and 1/2Nx + HPD groups of rats were analyzed with hematoxilin-eosin staining (A–D), periodic acid staining (E–H), von Kossa (I–L) and PCNA immunostaining (M–P). Arrows: inflammatory reaction and hypercellularity; Arrowhead: glomerulus lobulation; ^#Acute tubular necrosis; *presence of phosphate deposits. Magnification: x100. Scale Bar: 50 µm.

Figure 4

Burden phosphate reduces Klotho and promotes inflammation. Western blotting of protein extracts from kidney tissue. For detection of α-klotho (A) was used the cytoplasmic fraction and β-actin as loading control. For p65 fragment from NF-kB (B) detection was used the nuclear fraction of renal protein extracts and TFIIB as loading control. Quantification of western blots of Klotho (C) and p65 (D) were performed by measurement of average relative density and normalized to β-actin levels. ^ap < 0.05 vs Sham LPD; ^bp < 0.05 vs. Sham HPD and ^#p < 0.05 vs. Nx1/2 LPD.

Figure 5

Elevated phosphaturia generates renal oxidative stress. Glutathione peroxidase (GPx) activity in renal tissue. (A) GPx activity was determined in 20 µg of total protein extracts of kidney samples by a coupled reaction with glutathione reductase. Units are expressed as nanomoles of NADPH oxidized per minute and milliliter. White bars are LPD (0.2%P diet) groups and black bars are HPD (1.2%P diet) groups. t-test analysis *p < 0.05, **p < 0.01. (B) Negative correlation between renal mRNA α-klotho expression and GPx activity.

Figure 6

Treatment with Mangiferin reduces renal injury in obese Zucker rats. Kidney tissue samples from Lean, Obese Zucker rats and Obese Zucker rats plus 8 weeks of mangiferin treatment were analyzed with PAS stained sections (A–C) where tubular atrophy (asterisks) and peritubular inflammatory infiltrate (arrows) were observed. The thickening of the basement membrane was also evidenced in atrophic tubules (asterisks) with the Masson Trichrome, as well as interstitial fibrosis (arrowheads) (D–F). Mineral deposits are depicted by the brown color in von Kossa stain (G–I). Magnification: x400, scale Bar: 50 µm for PAS and Masson Trichrome, x200 and scale bar 100 µm for von Kossa.

Figure 7

Phosphate increases Reactive Oxygen Species (ROS) in HEK-293 cells. Intracellular ROS was determined using the probes (A) H2DCFDA (detects presence of ROS unspecifically) (B) DHRH (determinates peroxynitrate) (C) CMFDA (detects levels of glutathione) after 24 hours of incubation with different concentrations of inorganic phosphate. (t-test analysis. *p < 0.05 vs basal phosphate; **p < 0.01 vs basal phosphate; ***p < 0.001 vs basal phosphate. Basal phosphate is the content of phosphate in the culture medium at 0.9 mM.

Figure 8

In HEK-293 cells, the addition of melatonin prevents oxidative stress induced by high levels of inorganic phosphate. Intracellular ROS were determined by using the probes (A) DHRH and (B) CMFDA after 24 hours of incubation in a medium containing normal P concentration (0.9 mM), high levels of phosphate (5 mM P) and high phosphate plus melatonin (10⁻⁵ M). ^ap < 0.001 vs normal phosphate; ^bp < 0.001 vs 5 mM phosphate.