SPL treatment prevents diabetes-induced glomerular and tubular damage.

<p>(A) Schematic representation of the experimental strategy used in this study. Four groups of rats were studied: Control (CTL), Diabetic (DBT), DBT+SPL and SPL, respectively (for detail see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#sec002" target="_blank">Material and methods</a>). (B) Purity of isolated glomerular and proximal tubular segments was evaluated by IB analysis of Wilm´s tumor 1 (WT1) and dipeptidylpeptidase (DppD) expressions, respectively. (C) IB analysis of nephrin and WT1 shows that SPL treatment prevents diabetes-induced glomerular damage; densitometric analysis of nephrin (D) and WT1 (E) are shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s001" target="_blank">S1 Fig</a>). (F) IB analysis of kidney injury molecule 1 (Kim)-1 and heat shock protein of 72 KDa (Hsp72) show that SPL treatment decreases diabetes-induced proximal tubular damage, densitometric analysis of Kim-1 (G) and Hsp72 (H) are shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s001" target="_blank">S1 Fig</a>). No changes were found in SPL group compared to CTL group. (I) IF analysis confirms that SPL treatment decreases diabetes-induced Kim-1 expression (green label) in PT labeled with DppD (red label). Nuclei were marked with 4', 6-diamidino- 2-phenylindole (DAPI, blue label). No significant changes were found between SPL and CTL groups. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control for IB. Data are mean±SEM from 3 rats per group. *p<0.05; **p<0.01 and ***p<0.001. Images are representative of three different experiments performed in the four experimental groups. Bar = 50 μm.</p

SPL treatment decreases diabetes-induced co-immunoprecipitation (co-IP) of cldn-4 and -8.

<p>To evaluate direct interaction of cldn-4 and -8, co-IP analyzes from isolated DT from the four experimental groups were performed. As shown in panel A, cldn-4 co-immunoprecipitated with cldn-8; densitometric analysis (B) shows that diabetic condition increased their interaction and SPL treatment prevents this change (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s005" target="_blank">S5 Fig</a>). Also, increased co-IP of cldn-8 with cldn-4 was found under diabetic condition and SPL decreased this interaction (C), densitometric analysis show that these changes were significant (D) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s005" target="_blank">S5 Fig</a>). As shown in panels A and C, no signal was found under nonspecific conditions of IP performed with an unrelated antibody. GAPDH was used in input extract as loading control. Data are mean±SEM from 3 rats per group. *p<0.05.</p

Diabetic condition induces phosphorylation of cldn-4 and -8 in threonine but not in serine residues.

<p>Phosphorylation of cldn-4 and -8 in threonine and serine residues was evaluated by IP assays in the four experimental groups. As shown, diabetes induced cldn-4 (A) and cldn-8 (B) phosphorylation in threonine, but not in serine residues. SPL treatment decreased these changes. As shown in panels A and B, no signal was found under nonspecific conditions of IP performed with an unrelated antibody. GAPDH was evaluated in input extract as loading control.</p

Physiological and biochemical parameters of rats.

<p>Physiological and biochemical parameters of rats.</p

SPL decreases diabetes-induced oxidative stress in GL and PT.

<p>To evaluate oxidative stress, superoxide anion (O₂^●―) production, lipid peroxidation, protein carbonylation and reduced glutathione (GSH) content were measured in isolated GL and PT. SPL treatment prevents diabetes-induced increment in O₂^●― production (A) by using nicotinamide adenine dinucleotide phosphate (NADPH) as substrate and, diphenyleneiodonium (DPI) as inhibitor, lipid peroxidation (B) and protein carbonylation (C) and decreased GSH content (D) in GL (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s003" target="_blank">S3 Fig</a>). Also, SPL diminished diabetes-induced increment of O₂^●― production (E), lipid peroxidation (F) and protein carbonylation (G) and decreased GSH content (H) in PT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s003" target="_blank">S3 Fig</a>). Similar results were found between CTL and SPL groups. Data are mean±SEM from 5–6 rats per group. *p<0.05; **p<0.01 and ***p<0.001.</p

Aldosterone signaling regulates the over-expression of claudin-4 and -8 at the distal nephron from type 1 diabetic rats

<div><p>Hyperglycemia in diabetes alters tight junction (TJ) proteins in the kidney. We evaluated the participation of aldosterone (ALD), and the effect of spironolactone (SPL), a mineralocorticoid receptor antagonist, on the expressions of claudin-2, -4, -5 and -8, and occludin in glomeruli, proximal and distal tubules isolated from diabetic rats. Type 1 diabetes was induced in female Wistar rats by a single tail vein injection of streptozotocin (STZ), and SPL was administrated daily by gavage, from days 3–21. Twenty-one days after STZ injection the rats were sacrificed. In diabetic rats, the serum ALD levels were increased, and SPL-treatment did not have effect on these levels or in hyperglycemia, however, proteinuria decreased in SPL-treated diabetic rats. Glomerular damage, evaluated by nephrin and Wilm’s tumor 1 (WT1) protein expressions, and proximal tubular damage, evaluated by kidney injury molecule 1 (Kim-1) and heat shock protein 72 kDa (Hsp72) expressions, were ameliorated by SPL. Also, SPL prevented decrement in claudin-5 in glomeruli, and claudin-2 and occludin in proximal tubules by decreasing oxidative stress, evaluated by superoxide anion (O₂^●―) production, and oxidative stress markers. In distal tubules, SPL ameliorated increase in mRNA, protein expression, and phosphorylation in threonine residues of claudin-4 and -8, through a serum and glucocorticoid-induced kinase 1 (SGK1), and with-no-lysine kinase 4 (WNK4) signaling pathway. In conclusion, this is the first study that demonstrates that ALD modulates the expression of renal TJ proteins in diabetes, and that the blockade of its actions with SPL, may be a promising therapeutic strategy to prevent alterations of TJ proteins in diabetic nephropathy.</p></div

SPL treatment ameliorated diabetes-induced increased expression of SGK1, WNK4 and co-IP of WNK4 with SGK1.

<p>SGK1 expression was evaluated by IB analysis of isolated DT. As shown in panel A, DBT induced SGK1 expression. Densitometric analysis is shown in panel B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s007" target="_blank">S7 Fig</a>). Diabetic condition also induced co-IP of WNK4 with SGK1, and serine, but not threonine phosphorylation of WNK4 (C). SPL treatment prevented these changes. GAPDH was used as loading control in isolated DT and input extracts. As shown in panel C, no signal was found under nonspecific conditions of IP performed with an unrelated antibody. Data are mean±SEM from 3 rats per group. **p<0.01.</p

SPL treatment blunts diabetes-induced increment of protein and mRNA expressions of cldn-4 and -8.

<p>SPL decreased diabetes-induced cldn-4 and -8 expressions evaluated by IF, IB and qRT- PCR. IF analysis shows that 3 weeks after diabetes induction, increased expressions of (A) cldn-4 (green label) and (E) cldn-8 (green label) were found in the TJs of DT labeled with desmoplakin (DMPK, red label). Nuclei were marked with DAPI (blue label). SPL treatment significantly decreased these changes. To confirm IF findings, IB and qRT-PCR analyzes were performed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s004" target="_blank">S4 Fig</a>). Diabetic condition significantly increased protein (B) and mRNA (D) levels of cldn-4; these changes were decreased by SPL treatment. Also, SPL treatment decreased diabetes-induced increased protein (F) and mRNA (H) levels of cldn-8. Similar results were found between CTL and SPL groups. GAPDH was used as loading control. Densitometric analyzes of IB from the four experimental groups are shown in panels C for cldn-4 and, G for cldn-8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s004" target="_blank">S4 Fig</a>). Data are mean±SEM from 3 rats per group. *p<0.05; **p<0.01 and ***p<0.001. Images are representative of three different experiments performed in the four experimental groups. Bar = 20 μm.</p

Schematic representation of the mechanisms involved in ALD-induced decrement of expression of cldn-5 in GL and, cldn-2 and occldn in PT, and increased expression of cldn-4 and -8 in DT.

<p>(A) In GL, diabetes-induced ALD promotes glomerular damage, evaluated by decreased expression of nephrin and WT1, and oxidative stress, which may be associated to decreased cldn-5 expression and proteinuria. (B) In PT, diabetes-induced ALD promotes tubular damage, evaluated by increased expression of Kim-1 and Hsp72, and oxidative stress, which may be associated to decrements of occldn and cldn-2 expressions, this latter may explain increased natriuresis. (C) In DT, diabetes-induced ALD induces the expression of SGK1, which in turn phosphorylates serine (pSer) residues of WNK4 promoting its localization in the TJ. This latter phosphorylates threonine residues (pThr) of cldn-4 and -8, where both proteins constitute a paracellular Cl^― channel and a Na⁺ barrier. In all nephron segments, blockade of ALD actions with SPL decreased oxidative stress in GL and PT and the expression, and phosphorylation of cldn-4 and -8 in DT.</p

SPL treatment decreases diabetes-induced decrement in protein expression of cldn-5 in glomeruli (GL) and cldn-2 and occldn in proximal tubules (PT).

<p>SPL prevents diabetes-induced decrease of cldn-5 protein expression (A and B) in GL, and mRNA (F) and protein expression of cldn-2 (A and C), and protein expression of occldn (A and D) in PT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s002" target="_blank">S2 Fig</a>). However, diabetes did not have effect in the mRNA levels of cldn-5 (E) and occldn (G) evaluated by qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177362#pone.0177362.s002" target="_blank">S2 Fig</a>). Additionally, cellular localization of cldn-2 and -5 and occldn were assessed by IF (H). SPL-treatment prevents diabetes-induced loss of cldn-5 (green label) in the cell borders of GL and, cldn-2 (green label) and occldn (green label) in the cell borders of PT labeled with DppD (red label), nuclei were marked with DAPI (blue label). No changes were found between CTL and SPL groups. GAPDH was used as loading control. Densitometric analysis of IBs from the four experimental groups are shown in panels B for cldn-5, C for cldn-2 and D for occldn. Data are mean±SEM from 3 rats per group. *p<0.05; **p<0.01 and ***p<0.001. Images are representative of three different experiments performed in the four experimental groups. Bar = 50 μm.</p