Role of Klotho in Hyperglycemia: Its Levels and Effects on Fibroblast Growth Factor Receptors, Glycolysis, and Glomerular Filtration

Abstract

Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.

Keywords: Klotho protein; diabetes mellitus; diabetic nephropathy; fibroblast growth factor receptors; hyperglycemia.

Figure 1

Klotho protein levels increase in serum in Wistar rats with streptozotocin-induced diabetes (STZ) and decrease in their whole kidney tissue, glomeruli, and urine. (A) Despite the elevation of Klotho protein levels in serum in STZ rats (*** p < 0.0001, vs. control, unpaired t-test, n = 8), (B) a significant decrease in 24-h urinary Klotho excretion was found in STZ rats (* p = 0.03, vs. control, unpaired t-test, n = 8). This may have resulted from (C) a significant decrease in Klotho protein levels (renal tissue staining in brown) in glomeruli and cells that form tubules of nephrons in the kidneys in STZ rats (*** p < 0.0001, vs. control, unpaired t-test, n = 10). This was also concordant with (D) the downregulation of Klotho protein expression in glomeruli in STZ rats (*** p < 0.0001, vs. control, unpaired t-test, n = 18). (−) ctrl, negative control; MW, molecular weight.

Figure 2

mRNA and total protein levels of Klotho were not downregulated in immortalized human podocytes that were cultured under high glucose (HG) conditions, but the levels of the membrane form of Klotho protein decreased under HG conditions, together with an increase in soluble Klotho shedding. (A) The mRNA expression of Klotho was detected in immortalized human podocytes. (B) The mRNA expression of Klotho was measured under standard glucose (SG; 11 mM) and high glucose (HG; 30 mM, 5 days) conditions, which did not significantly differ (p = 0.96, Mann–Whitney test, n = 20–21). (C) The total protein expression of Klotho did not differ between podocytes that were cultured under standard glucose and high glucose conditions (p = 0.48, unpaired t-test, n = 14). (D) A significant decrease in the levels of the membrane form of Klotho protein was detected in podocytes under HG conditions (* p = 0.013, vs. SG, Mann–Whitney test, n = 5–6). (E) The immunofluorescent labeling of human podocytes for Klotho (red) confirmed the decrease in membrane-bound Klotho protein levels under HG conditions. These results were explained by (F) a significant increase in the levels of the soluble form of Klotho under HG conditions (** p = 0.007, vs. SG, Mann–Whitney test, n = 8), that was cut off from the cell membrane and shed extracellularly. bp, base pairs; MW, molecular weight.

Figure 3

Expression of FGFRs decreases under diabetic conditions in rat kidney tissue and human podocytes. (A) FGFR1, FGFR2, FGFR3, and FGFR4 gene expression was detected in immortalized human podocytes. (B) The immunofluorescent labeling of human podocytes for FGFR1-4 revealed their presence in the nucleus, the cytoplasm, and the cell membrane (FGFR1-2 in red; FGFR3-4 in green; DAPI-blue). (C) The mRNA expression of FGFR1 and FGFR2 genes in human podocytes significantly decreased under high glucose conditions (HG; 30 mM, 5 days) compared with standard glucose conditions (SG; 11 mM; FGFR1: *** p = 0.0004, unpaired t-test, n = 19–20; FGFR2: ** p = 0.0014, unpaired t-test, n = 20). No significant difference in mRNA expression of the FGFR3 and FGFR4 genes was found in human podocytes between SG and HG conditions. (D) A decrease in the levels of FGFR1, FGFR2, FGFR3, and FGFR4 was found in renal tissue (stained in brown) in STZ rats compared with healthy control rats (FGFR1-4: *** p < 0.0001, unpaired t-test, n = 9–10). bp, base pairs; (−) ctrl, negative control; MW, molecular weight.

Figure 4

Klotho increases the mRNA and protein expression of FGFRs under standard glucose conditions and recovers it after its decrease under high glucose concentration. (A) mRNA expression of the FGFR1 and FGFR2 genes increased in human podocytes after 24-h incubation with Klotho (0.5 nM; +KL24h) in the cell medium under standard glucose (SG; 11 mM; FGFR1: ** p = 0.0075, vs. SG, unpaired t-test, n = 11–19; FGFR2: ** p = 0.0038, vs. SG, unpaired t-test, n = 12–20). A trend was also found toward the rescue of FGFR1 and FGFR2 gene expression by 24-h incubation with Klotho after its initial drop under high glucose conditions (HG, 30 mM glucose, 5 days; HG+KL24h vs. HG). Detailed statistical data for the SG vs. HG comparisons are presented in Figure 3 (FGFR1: *** p = 0.0004; FGFR2: ** p = 0.0014). (B) Protein levels of FGFR1 and FGFR2 increased in human podocytes that were incubated with Klotho for 24-h (normalized to actin, HG+KL24h vs. HG: FGFR1: * p = 0.04, unpaired t-test, n = 5; FGFR2: * p = 0.03, unpaired t-test, n = 6–7; Figure 4B) after its initial drop under hyperglycemic conditions (normalized to actin, HG vs. SG: FGFR1: * p = 0.01, unpaired t-test, n = 5; FGFR2: * p = 0.03, unpaired t-test, n = 6–7; Figure 4B). (C) An increase in the protein expression of FGFR1 and FGFR2 after 24-h incubation with Klotho under both SG and HG conditions was also observed by the immunofluorescent staining of immortalized human podocytes (FGFR1-2 in red; DAPI-blue). MW, molecular weight.

Figure 5

Klotho increases glycolysis and glycolytic capacity in immortalized human podocytes. (A) The addition of Klotho (0.5 nM) for 1 h (+KL1h) and 24 h (+KL24h) to the cell medium with standard glucose (SG; 11 mM) significantly increased glycolysis (SG+KL1h vs. SG: ** p = 0.008, unpaired t-test, n = 10–28; SG+KL24h vs. SG: * p = 0.018, unpaired t-test, n = 20–28) and glycolytic capacity (SG+KL1h vs. SG: ** p = 0.002, unpaired t-test, n = 10–28; SG+KL24h vs. SG: * p = 0.017, unpaired t-test, n = 19–28) in human podocytes. (B) The addition of Klotho for 24 h (+KL24h) to the cell medium with both standard and high glucose (HG; 30 mM, 5 days) increased glycolysis (SG+KL24h vs. SG: ut supra; HG+KL24h vs. HG: ** p = 0.009, unpaired t-test, n = 12–20) and glycolytic capacity (SG+KL24h vs. SG: ut supra; HG+KL24h vs. HG: * p = 0.05, unpaired t-test, n = 12–21) in human podocytes.

Figure 6

Klotho exerted a beneficial effect on the maintenance of proper permeability of the glomerular filtration barrier. (A) The albumin permeability of glomeruli from Wistar rats with streptozotocin-induced diabetes (STZ) significantly increased compared with glomeruli from healthy Wistar control rats (*** p = 0.0007, Mann–Whitney test, n = 7–9). Thirty minute incubation with Klotho (0.5 nM; +KL30′) significantly decreased the albumin permeability of glomeruli from STZ rats (*** p = 0.0003, Mann–Whitney test, n = 7–8). (B) Klotho decreased the albumin permeability of a human podocyte monolayer under standard glucose conditions (SG; 11 mM) after 24-h incubation (+KL24h; * p = 0.049, vs. SG, Mann–Whitney test, n = 3–4), with a less prominent effect after 1-h incubation (+KL1h); # p ~ 0.05 but above it. Twenty-four hour incubation with Klotho had a significant impact on lowering albumin permeability of the human podocyte monolayer, which significantly increased under high glucose conditions (HG; 30 mM, 5 days) compared with standard glucose conditions (HG vs. SG: * p = 0.024, Mann–Whitney test, n = 3–6; HG+KL24h vs. HG: ** p = 0.004, Mann–Whitney test, n = 5–6). (C) Klotho (0.5 nM, 24 h) reversed HG-induced changes in cytoskeletal (F-actin) reorganization in immortalized human podocytes. Scale bar = 20 μm. Digitized fluorescence images of phalloidin-stained (far red) podocytes were used to obtain the fluorescence intensity profiles of the F-actin network. The data are expressed as mean ± SEM (n = 9–16).