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. 2020 May 6;16(2):175–185. doi: 10.1007/s11302-020-09695-1

P2X7 siRNA targeted to the kidneys increases klotho and delays the progression of experimental diabetic nephropathy

A M Rodrigues 1,2, R S Serralha 1,2, D Y Lima 2,3, G R Punaro 2,3, I Visona 4, M J S Fernandes 5, E M S Higa 1,2,3,6,✉
PMCID: PMC7367965  PMID: 32377919

Abstract

Previous studies in our laboratory have suggested that P2X7 could contribute to the progression of diabetic nephropathy and modulated klotho expression. The aim of this study was to investigate if P2X7 receptor is related to the expression of klotho in the onset of diabetic nephropathy in rats. Seven-week-old male Wistar rats weighing 210 g were all uninephrectomized; two-third of the animals were induced to diabetes with 60 mg/kg streptozotocin i.v., and one-third received its vehicle (control rats). At 4th day of the fifth week of the protocol, half of the diabetic rats received a small interfering RNA targeting for P2X7 mRNA, and the other half received its vehicle. Euthanasia was made at the eighth week. Diabetic animals reproduced all classic symptoms of the disease; besides, they showed reduced renal function and low NO bioavailability; also, SOD1, SOD2, and catalase were increased, probably due to the oxidative stress which was elevated in this situation. Metabolic data of diabetic rats did not change by silencing P2X7 receptor. For the other hand, silencing P2X7 was able to contribute to balance oxidative and nitrosative profile, ultimately improving the renal function and increasing plasma and membrane forms of klotho. These findings suggest that the management of P2X7 receptor can benefit the kidneys with diabetic nephropathy. Further studies are needed to show the therapeutic potential of this receptor inhibition to provide a better quality of life for the diabetic patient.

Electronic supplementary material

The online version of this article (10.1007/s11302-020-09695-1) contains supplementary material, which is available to authorized users.

Keywords: Oxidative stress, Kidney, Diabetic nephropathy, P2X7 knockdown, Klotho

Introduction

Diabetes mellitus is a serious health problem, affecting a large number of people worldwide, being its main complication, the diabetic nephropathy [1].

There is evidence that hyperglycemia increases the release of adenosine triphosphate (ATP) by exocytosis and decreases its degradation in the extracellular environment increasing the extracellular ATP (eATP) [2].

Receptors activated by eATP and its derivatives were named purinergic receptors [3], they were subclassified as P2X and P2Y [4]. P2X are ionotropic receptors and the main member of this family is the P2X7, which under large amounts of eATP promotes the passage of bivalent cations, such as calcium. When this exposition is prolonged, it occurs the opening of pores compromising the plasma membrane [5, 6].

Studies conducted in our laboratory showed that at the eighth week of experimental diabetes, the kidney had a high concentration of P2X7 protein, and its activity as measured by calcium fluorescent probe was intense; such behavior was significantly reduced when oxidative and nitrosative stress were decreased through aerobic training and/or antioxidants [7, 8].

In another study, we showed the moment that P2X7 became significantly expressed during the course of diabetes; at this moment, high P2X7 expression corresponded to a low expression of klotho gene [8]. Klotho was initially identified as an anti-aging factor and the increase in its expression can ameliorate the perspective of life. Subsequent studies showed that klotho would have other biological functions pertaining to the kidney, such as the control of apoptosis and oxidative or nitrosative stress, and also the regulation of phosphate metabolism [9–12]. The aim of the present study was to investigate the role of P2X7 knockdown on klotho and in the onset of diabetic nephropathy in rats.

Material and methods

Animals

Total n of 80 male Wistar rats with 7 weeks of age were kept at a controlled temperature of 22 ± 2 °C in an environment with a regular period of light and dark cycle of 12:12 h, with standard chow and water ad libitum. The protocol was approved by the Ethics Committee in Research of Universidade Federal de Sao Paulo under protocol #2056100314.

Surgical procedure

At 8 weeks of age, the animals were anesthetized with ketamine chloridrate (67 mg/kg, i.m.; Sesp, Sao Paulo, Brazil) and xylazine chloridrate (9 mg/kg, i.m.; Rhobiofarma, Sao Paulo, Brazil), and the left kidney was removed to accelerate the diabetic nephropathy [13].

Diabetes induction

At 9 weeks of age, diabetes was induced in 2/3 of the animals (DM) through streptozotocin (60 mg/kg i.v., Sigma-Aldrich, Sao Paulo, Brazil) dissolved in 0.1 M citrate buffer at pH 4.5, while the other 1/3 (CTL animals) received the vehicle. Diabetes was confirmed 48 h after induction, as fasting glycemia above 200 mg/dL. Animals that failed this criterion were excluded.

Metabolic profile

The animals were placed in metabolic cages (Tecniplast, Buguggiate, Italy) for 24 h, receiving water and chow ad libitum; at the eighth week of diabetes; body weight (g); diuresis (mL/24 h), water (mL/24 h), and food intake (g/24h) were recorded [8].

Small interfering RNA procedure

The small interfering RNA (siRNA) targeted for P2X7 (antisense, 5′ [Phos] CUUUAACGUCGGCUUGGGCUC [dT] [dT]-3′, and sense, 5′ [Phos] GCCCAAGCCGACGUUAAAGUA [dT] [dT]-3′, both synthesized by Sigma-Aldrich Company, was designed on the basis of the Thomas Tuschl protocol [14]. Lyophilized single-stranded RNA oligonucleotides were resuspended in sterile RNase free water (100 μM), denatured (heated at 95 °C for 5 min), aligned and slowly annealed with decreasing temperature, resulting in the formation of double-stranded siRNA at 50 μM.

This siRNA was complexed with the vector, RVG-9dR (YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR) (Sigma-Aldrich, Sao Paulo, Brazil), which was prepared at a ratio of 1:10 M for the transfection [15]. At 4th day of the 5th week, the diabetic rats were anesthetized as described [8]. The remaining kidney was exposed through a lombar incision, and using a stereotaxic apparatus, the siRNA was injected in two points of renal surface using a needle of 0.30 mm diameter. The distance between the points was 10 mm, each with 1.5 mm depth. The inhibitor complex was infused at a flow rate of 1 μL/min through a perfusion pump during 1 min (Harvard Apparatus, MA, USA). The applications were made at a concentration of 0.5 μg/μL totaling 1 μg of siRNA for P2X7. The siRNA dose was chosen based on a pilot study conducted previously, in which the inhibition of P2X7 mRNA was seen to be above 60% after 3 weeks of application in the kidney.

Euthanasia

At the end of the 8th week of the protocol, the animals were euthanized with a high dose of anesthetic (90 mg/kg ketamine chloridrate and 18 mg/kg xylazine chloridrate, both i.m.), followed with an abdominal incision and an incision of the diaphragm. The kidneys were removed and stored in the freezer at − 80 °C.

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Renal function

The plasma (mg/dL) and urinary (mg/dL) levels of creatinine and urea, and proteinuria were measured by colorimetric assay [8].

Nitric oxide measurement

Nitric oxide (NO) was measured in the plasma (nmol/dL), urine (nmol/24 h), and renal cortex (nmol/mg protein) samples by chemiluminescence using the Nitric Oxide Analyzer (NOA™280, Sievers Instruments Inc., CO, USA), a high-sensitivity detector for NO [16].

Estimation of lipid peroxidation

Lipid peroxidation was estimated in the plasma (nmol/dL), urine (nmol/24 h), and renal cortex (nmol/mg protein) using the thiobarbituric acid reactive substances (TBARS) method [17, 18].

Quantitative polymerase chain reaction

qPCR was performed by a spectrophotometer (Synergy HT Biotek, VT, USA). The cDNA strands were synthesized from the RNA samples using the SuperScriptVILO MasterMix reagent (Life Technologies, CA, USA) at a ratio of 4 μL of the reagent to 2.5 μg of RNA. The reaction was conducted by TaqMan (Applied Biosystem, USA) with klotho (Rn00580123_m1) and P2X7 (Rn00570451_m1) probes, using plectin as the housekeeping gene (Rn 00673737_m1). All of the qPCR reactions were conducted in duplicate using the LineGene 9620 thermocycler (Bioer Technology, Hangzhou, China) [19].

Western blotting

The total protein was measured in kidneys’ homogenates by the Bradford method, and 40 μg of protein of each sample was prepared and run on a 10% polyacrylamide gel. The blots were incubated with anti-nitrotyrosine sc-32757 (1:500); anti-caspase and anti-cleaved caspase sc-7148 (1:500); anti-superoxide dismutase (SOD)1 sc-11407 (1:200); anti-SOD2 sc-30080 (1:500); anti-catalase sc-50508 (1:200); anti-klotho sc-22220 (1:500), anti-P2X7 sc-25698 (1:100) and anti-actin sc-1615 (1:1000). All antibodies acquired from Santa Cruz Biotechnology, CA, USA [8].

Immunohistochemistry analysis

We used a method adapted to renal tissues with primary antibody anti-P2X7 (1:100; Chemicon, CA, USA) and anti-klotho (1:80; Santa Cruz, TX, USA). Control slides were processed without primary antibody. The percentage of P2X7 and klotho in renal tissue was expressed as a ratio of the staining area to total area [7].

Soluble klotho

The soluble klotho was measured by an enzyme-linked immunosorbent assay kit from Wuhan USCN Business Co. Ltd., Cloud-Clone Corp (SEH757Ra, Houston, USA) in plasma (ng/mL) and urine (ng/24 h).

Histological analysis

At the end of the 8-week protocol, the kidneys were removed under anesthesia. Half of the kidney was fixed in 10% formaldehyde and embedded in paraffin, sectioned to a 4 μm thickness and stained with hematoxylin–eosin (HE), and periodic acid-Schiff reagent (PAS). The analysis was carried out at a magnification of × 400 and analyzed by a pathologist under blinded conditions.

Statistical analysis

The results are expressed in the table as mean ± SEM or as box plot graphics with the medians (line) and means (cross). The data were analyzed by one-way ANOVA with a Newman-Keuls post hoc test or a two-way repeated measures ANOVA when necessary. Significance was defined as P < 0.05 (GraphPad Prism 5.0 Software, San Diego, USA).

Results

In this protocol, the DM rats presented all symptoms of diabetes: polyphagia, polydipsia, polyuria, reduced body weight, and hyperglycemia compared to controls (n = 6 for all groups) (Table 1).

Table 1.

Analysis of metabolic profile and renal function at the eighth week of diabetes in rats

CTL DM DM + siP2X7
Metabolic parameters
  Water intake (mL/24 h) 32.9 ± 1.1 176.7 ± 16.7* 140.7 ± 18.6*#
  Food intake (g/24 h) 16.3 ± 1.6 30.4 ± 1.2* 27.7 ± 2.8*
  Diuresis (mL/24 h) 15.9 ± 1.7 97.9 ± 8.5* 91.4 ± 8.4*
  Body weight (g) 388.7 ± 17.9 232.6 ± 13.4* 253.1 ± 11.5*
  Glycemia (mg/dL) 102.4 ± 2.6 494.7 ± 49.4* 469.4 ± 40.9*
Renal function
  Plasmatic creatinine (mg/dL) 0.97 ± 0.05 1.3 ± 0.03* 0.81 ± 0.07#
  Creatinine clearance (mg/min) 0.99 ± 0.06 0.65 ± 0.06* 0.93 ± 0.12#
  Plasmatic urea (mg/dL) 30.1 ± 3.7 46.2 ± 3.6* 34.3 ± 2.3#
  Urea clearance (mg/min) 1.24 ± 0.09 1.02 ± 0.5* 1.28 ± 0.04#
  Proteinuria (mg/24 h) 13.9 ± 0.9 32.7 ± 3.8* 14.8 ± 1.0#
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Values expressed as mean ± SEM. One-way ANOVA with Newman-Keuls post test, n = 6 for all groups, significance defined as P < 0.05

*vs CTL

#vs DM

CTL control group, DM diabetic group, DM + siP2X7 diabetic group knockdown for P2X7 receptor

Creatinine and urea levels were significantly reduced at the 8th week of diabetes, and proteinuria was increased. With the P2X7 silencing, there was an improvement in renal function (n = 6 for all groups) (Table 1).

In the 8th week, the diabetic animals presented a higher lipid peroxidation in urine and in the renal cortex when compared to control. Urinary and renal cortex NO were decreased. The ratio of NO to oxidative stress was lower in diabetes; i.e., these animals had a reduction in NO bioavailability when compared to control. However, the lipid peroxidation was significantly reduced when P2X7 was silenced, and in consequence, NO levels were preserved, both in plasma and in renal cortex, and its urinary excretion increased (n = 6 for all groups) (Table 2).

Table 2.

Analysis of oxidative stress at the eighth week of diabetes in rats

CTL DM DM + si P2X7
Estimation of lipid peroxidation (TBARS)
  Urinary excretion (nmol/24 h) 0.74 ± 0.07 2.88 ± 0.28* 1.81 ± 0.33*#
  Renal córtex (nmol/mg of protein) 0.033 ± 0.02 0.051 ± 0.02* 0.037 ± 0.01#
NO bioavailability
  Urinary excretion (nmol/24 h) 32.1 ± 6.3 6.6 ± 0.9* 12.5 ± 0.6*
  Renal cortex (nmol/mg of protein) 0.67 ± 0.05 0.42 ± 0.03* 0.68 ± 0.09#
Plasmatic balance of oxidative stress
  NO/TBARS ratio 0.95 ± 0.05 0.64 ± 0.02* 0.99 ± 0.14#
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Values expressed as mean ± SEM. One-way ANOVA with Newman-Keuls post test, n = 6 for all groups, significance when P < 0.05

*vs CTL

#vs DM

TBARS thiobarbituric acid reactive substances, NO nitric oxide, CTL control group, DM diabetic group, DM + siP2X7 diabetic group knockdown for P2X7 receptor

For a better understanding of how oxidative stress compromised NO levels in the kidneys, the nitrotyrosine was evaluated and its concentration was higher in diabetic when compared to control (1.72 ± 0.11 vs. 0.89 ± 0.03, P = 0.0031), meaning that the diabetic kidneys produced large amounts of peroxynitrite, explaining the high consumption of renal NO (Fig. 1a). The diabetic kidney also presented higher caspase and cleaved caspase protein (1.66 ± 0.07 vs. 1.04 ± 0.05 and 0.84 ± 0.08 vs. 0.57 ± 0.04, both P < 0.05, respectively) compared to control (Fig. 1b and c). The diabetic kidneys after P2X7 inhibition presented a reduction in the nitrotyrosine (1.07 ± 0.10, P = 0.003), caspase and cleaved caspase (1.28 ± 0.06 and 0.51 ± 0.05, both P < 0.05, respectively) (Fig. 1a–c) (n = 5 for all groups above).

Fig. 1.

Fig. 1

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Representative images of Western blotting for nitrotyrosine (a), caspase (b), cleaved caspase (c), SOD1 (d), SOD 2 (e), and catalase (f) in renal cortex at the 8th week of diabetes. The values are expressed as box-plot containing the median (line) and mean (cross); significance defined as p < 0.05; n = 5 for each group in quadruplicate. One-way ANOVA with Newman-Keuls post hoc test: * vs CTL; # vs. DM. CTL = control group; DM = diabetic group; DM + siP2X7 = diabetic group knockdown for P2X7 receptor; kDa = kilodaltons

The diabetic kidneys presented high values of SOD1 (1.90 ± 0.07 vs. 1.06 ± 0.24, P = 0.0045), SOD2 (1.14 ± 0.10 vs. 0.88 ± 0.06, P < 0.05), and catalase (1.76 ± 0.11 vs. 1.46 ± 0.05, P < 0.05), when compared to control. These values were significantly reduced with P2X7 inhibition (1.27 ± 0.13, p = 0.0045; 0.85 ± 0.06, P < 0.05; 1.30 ± 0.07, P < 0.05, respectively) (Fig. 1d–f) (n = 5 for all groups above).

In this study, the efficacy of knockdown for P2X7 mRNA was assessed by qPCR; the diabetic animals presented the highest relative expression values for P2X7, and its inhibition reduced 68% of expression (1.44 ± 0.15 vs. 0.45 ± 0.06, P < 0.001). P2X7 silencing was accompanied by an increase in the klotho relative expression, above 70% compared to diabetic group without inhibition (1.48 ± 0.20 vs. 0.43 ± 0.19, P = 0.0045) (Fig. 2) (n = 5 for all groups above).

Fig. 2.

Fig. 2

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qPCR for P2X7 and klotho in renal cortex tissue at the 8th week of diabetes. The values are expressed as box-plot containing the median (line) and mean (cross); significance defined as p < 0.05; n = 5 for each group and 4 reactions. One-way ANOVA with Newman-Keuls post hoc test: * vs CTL; # vs. DM. CTL = control group; DM = diabetic group; DM + siP2X7 = diabetic group knockdown for P2X7 receptor; ∆∆ Ct = delta of delta of cycle threshold

Analysis of P2X7 protein content in the renal cortex showed that in the diabetic animals, it was increased when compared to control (1.92 ± 0.13 vs. 0.92 ± 0.09, P < 0.05), but the receptor inhibition reduced this increase (1.41 ± 0.11, P < 0.05). In renal tissue, diabetic animals had a significant klotho reduction when compared to control (0.90 ± 0.05 vs. 1.29 ± 0.03, P < 0.05). In diabetic kidneys with P2X7 silencing, klotho was increased when compared to the diabetic group (1.57 ± 0.18, P < 0.001) (Fig. 3) (n = 5 for all groups above).

Fig. 3.

Fig. 3

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Representative images of Western blotting for P2X7 and klotho in renal cortex tissue at the 8th week of diabetes. The values are expressed as box-plot containing the median (line) and mean (cross); significance defined as p < 0.05; n = 5 for each group in quadruplicate. One-way ANOVA with Newman-Keuls post hoc test: * vs CTL; # vs. DM. CTL = control group; DM = diabetic group; DM + siP2X7 = diabetic group knockdown for P2X7 receptor; kDa = kilodaltons

Immunohistochemistry analysis showed that in the control and silenced control group animals, there was no significant staining in the areas observed in the kidney (5.18 ± 0.12 vs. 5.60 ± 0.31; % of stained area); however, diabetic animals showed intense marking for the P2X7 receptor (32.3 ± 3.3; P < 0.05); this increase was reduced in the groups of animal diabetic animals that received receptor silencing (15.3 ± 1.5; P < 0.05) (Fig. 4a); when we observe klotho IHC, the control and silenced control groups were not different from each other (36.4 ± 4.4 vs. 30.9 ± 3.6; % of stained area), but diabetic animals had a significant reduction in the stained area (14.8 ± 1.0; P < 0.05); however, when the diabetic were silenced, there was increased klotho marking (23.15 ± 2.5; P < 0.05) (Fig. 4b) (n = 5 for all groups above).

Fig. 4.

Fig. 4

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Immunohistochemistry analysis of 5 slides per group of the renal sections at the 8th week of diabetes. a Reactions against P2X7 receptor in CTL and CTL + siP2X7 did not show significant alterations in the areas observed in the kidney; however, DM showed intense marking; this increase was attenuated in the DM + siP2X7 which received the silencing (×400). b The presence and intensity of klotho in the CTL and CTL + siP2X7 were not different, but the DM group had a significant reduction in the renal area observed; however, the klotho marking increases in DM + siP2X7 (× 400). c, d The relative quantification of P2X7 and klotho, respectively, both measured by ratio of stained area to total area. Significance defined as P < 0.05; two-way ANOVA, repeated measures ANOVA: * vs CTL; # vs. DM. CTL = control group; DM = diabetic group; DM + siP2X7 = diabetic group knockdown for P2X7 receptor

In diabetic animals when compared to control group, the circulating klotho was decreased in plasma (6.25 ± 0.2 vs. 7.78 ± 0.6 ng/mL; P = 0.0177) and increased in urine (846.8 ± 130.7 vs. 99.1 ± 24.9 ng/24 h; P < 0.001); however, for the diabetic animals with P2X7 knockdown, the plasma showed an increase of soluble klotho (8.0 ± 0.4; P = 0.0177) and its excretion was attenuated (508.3 ± 0.4; P = 0.04) when compared to the diabetic kidney (Fig. 5) (n = 5 for all groups above).

Fig. 5.

Fig. 5

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Analysis of protein content for soluble klotho by ELISA in plasma and urine at the 8th week of diabetes. The values are expressed as box-plot containing the median (line) and mean (cross); significance defined as p < 0.05; n = 5 for each group. One-way ANOVA with Newman-Keuls post hoc test: * vs CTL; # vs. DM. CTL = control group; DM = diabetic group; DM + siP2X7 = diabetic group knockdown for P2X7 receptor; kDa = kilodaltons

The control and control with P2X7 silenced presented no alterations in the glomeruli neither in the tubules, analyzed by HE or PAS staining. However, the DM group showed in HE staining, an increased cellularity and mesangial matrix in the glomeruli; in these animals, the focal tubular vacuolization was evidenced. With PAS staining in the diabetic kidneys, the glomeruli presented discrete areas of diffuse sclerosis. Besides, this group showed tubules with marked hyaline goticular degeneration and proximal tubules with discontinued or even absent brush border. All the alterations observed in diabetic animals were reduced at least in 30% in diabetic kidneys with silenced P2X7 receptor (× 400) (Fig. 6a) (n = 5 for all groups above). Although in this experimental model, the appearance of nodules is not common; in this particular case, we observed a glomerulus with Kimmelstiel-Wilson lesion, which allocated this animal to level III of the diabetic nephropathy (Tervaert 2010) (PAS; × 400) (Fig. 6b).

Fig. 6.

Fig. 6

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Microscopic aspect of 5 slides per group of the renal sections at the 8th week of diabetes. a On the HE staining, we did not observe any lesions in CTL nor in CTL + siP2X7 groups; however, the DM group showed glomeruli with increased cellularity and mesangial matrix (white arrows); in these animals, focal tubular vacuolization was observed (black arrow). On the PAS staining, the CTL and CTL + siP2X7 did not show alterations, but the DM group presented glomeruli with discrete areas of diffuse sclerosis (white arrows); besides, there were tubules with hyaline goticular degeneration (black circle) and absence of brush border (white arrow). All the alterations observed in those kidneys were reduced at least in 30% in the DM + siP2X7 group (× 400). b Although in this experimental model, the appearance of nodules is not common, in this particular case, we observed a glomerulus with Kimmelstiel-Wilson lesion, which allocated this animal to level III of the diabetic nephropathy (Tervaert 2010) (PAS; × 400)

Discussion

In the present study, we demonstrated that all the characteristics of diabetes were successfully reproduced; oxidative stress was presented as high levels of lipid peroxidation and this was accompanied by lower NO values, probably as a result of peroxynitrite formation as shown by high nitrotyrosine and apoptosis. The enzyme superoxide dismutase (SOD) is the main antioxidant defense against the superoxide anion; in mammals, the expression of SOD1 and SOD2 seems to be an adaptive response to oxidative stress [20], which corroborates our findings, since diabetic animals had the highest levels of these enzymes in the kidneys, associated with high levels of catalase in the kidney. There are few studies evaluating the biological role of P2X7 in modulating oxidative stress, as well as little is known about any relationship between P2X7 and the main antioxidant enzymes [21–23]. In our study, all this redox imbalance promoted damage to renal tissues, as seen by our histological findings, categorizing these kidneys in a pathological stage 2 to 3 according to Tervaert classification 2010 [24]. All these changes observed in diabetic animals were significantly reduced after P2X7 receptor silencing.

So far, our findings have shown how the P2X7 receptor acts by favoring the progression of diabetic nephropathy, and these are corroborated by the study of Menzies et al. (2017) [25]. They showed that the use of P2X7 inhibitor can reduce renal macrophage accumulation after the establishment of hyperglycemia in a diabetic nephropathy model. Subsequently, Hou et al. (2018) [26] showed how P2X7 can trigger the activation of NLRP3 inflammasome, and this activation in the podocytes may be involved in glomerular damage.

As far as we know, there are no studies that have shown that P2X7 receptor interferes with klotho gene expression. Klotho is a glycoprotein that can be presented as a transmembrane protein, known as α-klotho, which acts as a co-receptor for the FGF receptor promoting the increase in phosphaturia. Klotho can also be found in a soluble form in body fluids, it has several biological functions, including anti-apoptotic, antioxidant and anti-inflammatory effects [27]. Recent studies of diabetes, in vitro and in vivo, showed that α-klotho is the cleavage target of ADAM-like proteases, which are increased in this disease, resulting in temporary elevation of soluble klotho, both in plasma and urine [28].

Previous study by Rodrigues et al. (2018) [8] showed not only in which week of diabetes the P2X7 receptor was significantly expressed, but suggested even that the expression of this receptor could influence the genetic manisfestation of klotho.

In the present study, the results showed that the α-klotho and soluble plasmatic klotho had significantly reduced levels. Recent studies corroborated with our findings, since they found a positive correlation between the progression of chronic disease with systemic klotho deficiency [29–31].

Other research showed that urinary klotho may be an early predictor for the occurrence of AKI, and this may help to identify patients at-risk before the progression of the disease [32]. α-klotho was reduced in chronic diseases such as diabetes, suggesting that it could be used as a biomarker for this kidney disease [32–34].

It has been known for a long time that α-klotho is significantly correlated with CKD and its complications. Klotho works as co-receptor of fibroblast growth factor receptors (FGFR), promotes hyperphosphaturia, with consequent inhibition of vascular calcification; this results in protecting the integrity of vessels preserving kidney and heart function [35, 36].

In our study, the knockdown for P2X7 receptor not only presented a significant increase in gene expression for klotho, but also a relevant increase in the content of α-klotho in renal tissue.

The low levels of P2X7 also led to the increase of soluble klotho; we believe that the elevation of klotho contributed to attenuate the damage caused by the disease.

Kacso and collaborators [37] demonstrated that the values of circulating klotho varied according to the progression of renal disease; its serum and urinary levels were reduced in the early phase and with the evolution of nephropathy/CKD these concentrations increase with the loss of renal function, until the dialytic phase. According to these, our diabetic animals appeared to be in an early stage of nephropathy; we showed a lower plasmatic value of circulating klotho after 8 weeks of diabetes, compared to control rats. We believe that the high concentrations of soluble klotho in diabetic rat urine at this time could be a reflection of its high liberation of klotho by the kidneys, since these organs are the main producers.

In the diabetic group, knockdown for P2X7 receptor, klotho was maintained in the plasma, at similar levels of control group. We could then hypothesize that this circulating soluble klotho could have a protective action over the renal tissue in the absence of P2X7 receptor. That is, in this situation, it could have a role as an antioxidant and anti-apoptotic agent, as described above. Soluble klotho was related to the phosphorylation inhibition of the pathways between JNK/MAPK and p38/MAPK, which consequently raises the levels of antioxidant enzymes in tubular epithelial cells [10].

Klotho in the bloodstream was related to an anti-inflammatory agent, it promotes a downregulation of NF-κB-linked inflammatory proteins via a mechanism of phosphorylation of Ser(536) in the transactivation domain of RelA (protein involved in NF-κB heterodimer formation) [38], and in a study, klotho probably protected cardiovascular function in mice, through endothelial dependent-NO synthesis [39]. Other findings revealed a potential novel mechanism of soluble klotho to protect the glomerular filter, reducing the detachment of podocytes and thus avoiding proteinuria [40].

We believe that a novel therapeutic pathway was found, since our data suggest that the control of inflammatory factors and the oxidative stress can delay the diabetic nephropathy, independent of an improvement in metabolic parameters. In the present study, the beneficial effects provided by silencing P2X7 receptor expression and thus increasing klotho seems to be convincing for the treatment of this disease and therefore could become a useful adjuvant tool in improving the quality of life in diabetic patients.

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Acknowledgments

The authors acknowledge Margaret G Mouro for nitric oxide measurements and Professor Sergio R R Araujo for histological analysis.

Funding information

This study was supported by Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) and FAPESP (# 2014/26750-9).

Compliance with ethical standards

Conflicts of interest

I confirm that all authors read the journal’s policy on disclosure and they have no potential conflict of interest.

Ethical approval

The protocol was approved by the Ethics Committee in Research of Universidade Federal de Sao Paulo under protocol #2056100314.

Footnotes

Publisher’s note

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