Intranasal Administration of Sugarcane Ash Causes Chronic Kidney Disease in Rats

Abstract

Background. Silica nanoparticles found in sugarcane ash have been postulated to be a toxicant contributing to chronic kidney disease of unknown etiology (CKDu). However, while the administration of manufactured silica nanoparticles is known to cause chronic tubulointerstitial disease in rats, the effect of administering sugarcane ash on kidney pathology remains unknown. Here we investigate whether sugarcane ash can induce CKD in rats. Methods. Sugarcane ash was administered for 13 weeks into the nares of rats (5 mg/day for 5d/week), and blood, urine and kidney tissues were collected at 13 weeks (at the end of ash administration) and in a separate group of rats at 24 weeks (11 weeks after stopping ash administration). Kidney histology was evaluated, and inflammation and fibrosis (collagen deposition) measured. Results. Sugarcane ash exposure led to the accumulation of silica in the kidneys, lungs, liver and spleen of rats. Mild proteinuria developed although renal function was largely maintained. However, biopsies showed focal glomeruli with segmental glomerulosclerosis, and tubulointerstitial inflammation and fibrosis that tended to worsen even after the ash administration had been stopped. Staining for the lysosomal marker, LAMP-1, showed decreased staining in ash administered rats consistent with lysosomal activation. Conclusion. Sugarcane ash containing silica nanoparticles can cause CKD in rats.

Keywords: Climate Change; Mesoamerican Nephropathy; Silica; chronic kidney disease.

Graphical abstract

Figure 1.

Histological findings in rats administered sugarcane ash via the nares. A–F: administration of sugarcane ash resulted in regions of local interstitial fibrosis [collagen type III staining (A–D) and quantification (E and F)]. Controls showed minimal abnormalities, and the differences with sugarcane ash-administered rats were significant at 24 wk. G–L: areas of interstitial myofibroblast accumulation at 13 and 24 wk [α-smooth actin-positive interstitial cells (G–J) and quantification (K and L)]. There was also a significant infiltration of inflammatory CD68-positive monocyte-macrophages into the interstitium at 13 and 24 wk [M–P; quantification (Q and R)] associated with high circulating NOX4 (oxidative stress biomarker) and serum MCP-1 levels (Table 1). Focal injury was found in both the tubulointerstitium and glomeruli by PAS stain (S; white arrow shows the area of tubulointerstitial injury, and black arrow shows the area of focal glomerular injury, week 13). Statistical analysis was performed by an unpaired t test. *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001. n = 6. See also Supplemental Figs. S1–S3. Magnification: ×20. Scale = 200 µm. MCP-1, monocyte chemoattractant protein-1; PAS, periodic acid-Schiff.

Figure 2.

Histological findings in rat glomeruli. Unlike control animals (A and D), the administration of sugarcane ash resulted in focal glomerular injury, with the development of segmental glomerulosclerosis in silica-treated rats (B and E), with significant differences between control and ash groups at 24 wk (C and F). Mesangial matrix expansion developed in ash-treated rats (G and I) and was significantly greater than that observed in controls (H and J). Magnification: ×40. Scale = 60 µm. Statistic test was an unpaired t test with Welch’s correction. ****P < 0.0001.

Figure 3.

Lysosomal activation in sugarcane ash-treated rats. Kidney biopsies were stained for LAMP-1, a lysosomal marker that is known to decrease in expression with lysosomal activation. Although lysosomal LAMP-1 expression was in proximal tubules in control animals, ash-treated rats showed a reduction in LAMP-1 staining at both 13 and 24 wk. A Mann–Whitney test was used (n = 6/group). Staining was suggestive of lysosomal activation. Magnification: ×20 and ×40. Scale = 200 µm and 60 µm. *P < 0.01; **P < 0.01.

Figure 4.

Distribution of silica nanoparticles in the ash-treated rats. Silica nanoparticles were identified in the kidney (A), lung (B), liver (C), and spleen (D) of ash-administered rats, whereas no silica was present in controls (not shown). There appeared to be a tendency for the silica nanoparticles to decrease in the kidney, lung, and liver over time, although the opposite trend was observed in the spleen. Blue circles represent individual silica measurements at 13 wk, and red circles represent silica measurements at 24 wk in the rats administered sugarcane ash (n = 6/group).

Figure 5.

Evaluation of sugarcane ash using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses (A) and single particle inductively coupled plasma mass spectrometry (ICP-MS) (B).