With a rapid increase in the morbidity rate of diabetes mellitus (DM), diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease (ESRD) [
An epidemiological survey showed that some patients had various degrees of kidney damage before or upon the diagnosis of DM [
In addition, the pathogenesis of DKD has not yet been fully elucidated since most findings were from type 1 DM, whereas type 2 DM is far more complicated than type 1 DM. Except for hyperglycemia, a series of other metabolic abnormalities such as insulin resistance (IR), hypertension, hyperlipidemia, and obesity are common in type 2 DM. Therefore, the mechanism of renal injury is complicated in patients with type 2 DM.
Among the main pathophysiological characteristics of type 2 DM, IR and compensatory hyperinsulinemia occur long before clinical diabetes. Hyperinsulinemia impairs insulin activity and metabolic signaling pathways and also affects the structure and function of the tissues and organs including the kidney. IR/hyperinsulinemia is an independent risk factor for chronic kidney diseases [
However, whether and how hyperinsulinemia play roles in the early damages of kidney before DM remain elusive. We dynamically studied the changes of the structure and function of renal tissue in different stages of type 2 DM (NGT, IGT, DM, and DKD stages) using OLETF rats. We also studied the effects of different concentrations of the insulin and IRS-1/PI3-K/Akt pathway on albumin reabsorption in a rat proximal renal tubular epithelial cell line (NRK-52E).
This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1996) and was approved by the Institutional Animal Care and Use Committee of the Tianjin Medical University.
32 male OLETF rats, a model of spontaneous type 2 DKD, were supplied by Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan). OLETF rats were fed a high-fat diet and were housed in an air-conditioned room at 20°C-25°C with 50%-70% humidity and a 12 h light-dark cycle. Rats were allowed free access to food and water. 32 male LETO rats were used as the nondiabetic model contrasted to OLETF rats.
An oral glucose tolerance test (OGTT) was performed every 4 weeks. Before the test, the rats were fasted for 15 hours overnight and were administered to 30% glucose solution (2 g/kg) by gastric gavage. Blood samples were taken from the tail vein before glucose loading and 30, 60, and 120 min after glucose loading. Blood glucose levels were determined by an automatic blood glucose analyzer. Plasma insulin concentrations were detected by radioimmunoassay. OLETF rats were classified into three stages on the basis of the OGTT results [
The grouping steps in animal experiment.
Rats were placed in metabolic cages. 24 h urine and random urine were collected. 24 h UMA, retinol-binding protein (RBP), N-acetyl-
Arterial blood was collected from the femoral artery. Blood urea nitrogen (BUN), serum creatinine (Scr), total cholesterol (TC), triglyceride (TG), and free fatty acid (FFA) were tested by an automatic biochemical analyzer. Tumor necrosis factor-
Proximal tubular epithelial cells from a Rattus norvegicus kidney (NRK-52E) were maintained in DMEM (1 g/L glucose, Hyclone) supplemented with 10% fetal bovine serum (Hyclone)+penicillin/streptomycin (Gibco) at 37°C and 5% CO2. Cells at 3-5 passages were used for the experiments.
Cell suspensions were seeded in a 96-well plate with 4000 cells in each well. NRK-52E cells were treated with indicated concentrations of insulin (0, 1, 5, 10, 50, 100, 103,
Cell suspensions were seeded in a 24-cell plate with 3000 cells in each cell. After being treated by different concentrations of insulin, NRK-52E cells were treated with TRITC-BSA (500
We further treated cells with 5 ng/mL insulin (CN group), insulin combined with a PI3-K inhibitor (5 ng/mL insulin+LY294002, CN+LY group), and 50 ng/mL insulin (high-insulin group) to check the intake of TRITC-albumin as well as the expression of megalin and cubilin in NRK-52E cells.
Statistical analysis was performed using the IBM SPSS Statistics 20.0 software. All normally distributed data were expressed as the
Compared with that in the control group, the FBG level in the NGT and IGT groups did not change. In the DM and DKD groups, the FBG level was increased and was higher than that of the other three groups (
Alteration of biochemical markers in different stages of type 2 DM.
Group | FBG (mmol/L) | FINS (uIU/L) | FFA (mmol/L) | TG (mmol/L) | TC (mmol/L) | Weight (g) |
---|---|---|---|---|---|---|
Control | ||||||
NGT | ||||||
IGT | ||||||
DM | ||||||
DKD | ||||||
0.003 | 0.001 | 0.014 | 0.001 | 0.001 | 0.001 |
a
Compared with that in the control group, the FINS level in the NGT group did not change. However, the FINS level was increased at the IGT group and was higher than that of the control group (
Compared with the control group, the blood lipids including TG, TC and FFA did not change in the NGT group, but were increased in the IGT and DM groups (P < 0.05) (Table
Compared with that in the control group, the weight in the NGT group did not change. In the IGT group, the weight was higher than that in the control and NGT groups (
The levels of NAG and NGAL which represent the injury of tubular epithelium and the levels of
Alteration of tubular function marker in different stages of type 2 DM.
Group | NAG (U/L) | NGAL (ng/mL) | RBP (mg/L) | CysC (ng/mL) | |
---|---|---|---|---|---|
Control | |||||
NGT | |||||
IGT | |||||
DM | |||||
DKD | |||||
0.001 | 0.001 | 0.001 | 0.001 | 0.001 |
a
24 h UMA, Scr, and BUN between the NGT and IGT groups were not different. However, in the DM group, the Scr but not 24 h UMA or BUN was higher than those in the NGT and control groups. In the DKD group, 24 h UMA was higher than those in the other four groups (Table
Alteration of glomerular function marker in different stages of type 2 DM.
Group | 24 h UMA ( |
Scr ( |
BUN (mmol/L) |
---|---|---|---|
Control | |||
NGT | |||
IGT | |||
DM | |||
DKD | |||
0.001 | 0.001 | 0.008 |
a
Compared with those in the control group, the rats in the NGT group had normal tubular morphology as well as ultrastructures (Figures
Pathological changes of the renal tubule. Light microscope: (a) the control group by H&E (×400), (b) the NGT group by H&E (×400), (c) the IGT group by H&E (×400), (d) the DM group by H&E (×400), and (e) the DKD group by H&E (×400). Transmission electron microscope: (f) Basal side of the renal tubule in the control group (×30000), (g) basal side of the renal tubule in the NGT group (×30000), (h) basal side of the renal tubule in the IGT group (×30000), (i) basal side of the renal tubule in the DM group (×10000), (j) basal side of the renal tubule in the DKD group (×10000), (k) the renal interstitium in the control group (×10000), (l) renal interstitium in the NGT group (×7000), (m) the renal interstitium in the IGT group (×60000), (n) the renal interstitium in the DM group (×3000), and (o) the renal interstitium in the DKD group (3500).
Compared with those in the control group, rats in the NGT group had normal glomerular morphology as well as ultrastructures (Figures
Pathological changes of glomerulus. Light microscope: (a) the control group by H&E (×400), (b) the NGT group by H&E (×400), (c) the IGT group by H&E (×400), (d) the DM group by H&E (×400), and (e) the DKD group by H&E (×400). Transmission electron microscope: (f) the glomerular filtration membrane in the control group (×20000), (g) the glomerular filtration membrane in the NGT group (×25000), (h) the glomerular filtration membrane in the IGT group (×20000), (i) the glomerular filtration membrane in the DM group (×20000), (j) the glomerular filtration membrane in the DKD group (×20000), (k) the mesangial area in the control group (×5000), (l) the mesangial area in the NGT group (×5000), (m) the mesangial area in the IGT group (×5000), (n) the mesangial area in the DM group (×5000), and (o) the mesangial area in the DKD group (×3500).
IRS-1 was highly expressed in renal tubular epithelial cells in the NGT group, while pSer IRS-1 was expressed at a low level. Both IRS-1 and pSer IRS-1 were not different from that in the control group. In the IGT group, IRS-1 began to decrease and was lower than that in the control group and the NGT group (
Semiquantitative analysis of IRS-1, pSer IRS-1, megalin, and cubilin in renal tubule and interstitium.
Group | IRS-1 | pSer IRS-1 | Megalin | Cubilin |
---|---|---|---|---|
Control | ||||
NGT | ||||
IGT | ||||
DM | ||||
DKD | ||||
0.001 | 0.001 | 0.001 | 0.001 |
a
Expression of IRS-1 and pSer IRS-1 by immunohistochemistry in a light microscope: (a) IRS-1 in the control group (×400), (b) IRS-1 in the NGT group (×400), (c) IRS-1 in the IGT group (×400), (d) IRS-1 in the DM group (×400), (e) IRS-1 in the DKD group (×400), (f) pSer IRS-1 in the control group (×400), (g) pSer IRS-1 in the NGT group (×400), (h) pSer IRS-1 in the IGT group (×400), (i) pSer IRS-1 in the DM group (×400), and (j) pSer IRS-1 in the DKD group (×400).
In the NGT group, megalin and cubilin were highly expressed on the luminal side of the renal tubular epithelial cells and were not different from those of the control group. In the IGT group, both megalin and cubilin were decreased compared to those in the control group and the NGT group (
Expression of megalin and cubilin by immunohistochemistry in a light microscope: (a) megalin in the control group (×400), (b) megalin in the NGT group (×400), (c) megalin in the IGT group (×400), (d) megalin in the DM group (×400), (e) megalin in the DKD group (×400), (f) cubilin in the control group (×400), (g) cubilin in the NGT group (×400), (h) cubilin in the IGT group (×400), (i) cubilin in the DM group (×400), and (j) cubilin in the DKD group (×400).
Combined with physiological concentration range 0-5 ng/mL and MTT results (Figure
Effect of different concentrations of insulin on cell viability by MTT.
NRK-52E cells were treated with indicated concentrations of insulin (0, 5, 10, and 50 ng/mL). After 24 h, we found that the mRNA levels of megalin and cubilin were the highest in cells treated with 5 ng/mL insulin, which was different from that in the control group (
Different concentrations of insulin on the expression of megalin and cubilin in NRK-52E cells and their effects on TRITC-albumin reabsorption. (a) The mRNA level of megalin and cubilin of NRK-52E cells in indicated concentrations of insulin. (b) The expression of megalin and cubilin and the reabsorption of TRITC-BSA in indicated concentrations of insulin.
The reabsorption of TRITC-BSA by NRK-52E cells was the highest in cells treated with 5 ng/mL insulin, which was different from that in the control group. However, along with the increase of insulin intervention concentration, the uptake of TRITC-BSA by cells decreased and the least was in the highest insulin concentration group. Therefore, the expressions of megalin and cubilin in renal tubular epithelial cells were inconsistent with its albumin reabsorption function when treated by different levels of insulin. High concentration of insulin inhibited the expression of megalin and cubilin and TRITC-albumin reabsorption in NRK-52E cells (Figure
Effects of different concentrations of insulin on the expression of megalin and cubilin and TRITC-albumin reabsorption in NRK-52E cells.
The mRNA levels of IRS-1, PI3-K, and Akt were the highest in cells treated with 5 ng/mL insulin and were significantly different from those in the control group (
Changes of the mRNA and the protein level of IRS-1/PI3-K/Akt after being treated by different concentrations of insulin. (a) The mRNA level of IRS-1, PI3-K, and Akt of NRK-52E cells in indicated concentrations of insulin. (b) The expression of IRS-1, PI3-K, and Akt in indicated concentrations of insulin.
After the NRK-52E cells were treated with the PI3-K inhibitor (LY294002), we further detected the expression of Akt to illustrate the effect of the PI3-K inhibitor on PI3-K/Akt insulin signaling pathway. The results showed that the expression of Akt in the CN+LY group was significantly lower than that in the control group. Therefore, the PI3-K inhibitor inhibited the PI3-K/Akt signaling pathway (Figure
The inhibitory effect of the PI3-K inhibitor on the PI3-K/Akt signaling pathway.
The expression of megalin and cubilin and TRITC-albumin reabsorption in the CN+LY group and in the high insulin group were significantly lower than those of the control group (
Comparison between the PI3-K inhibitor and the high concentration of insulin on the expression of megalin and cubilin and TRITC-albumin reabsorption in NRK-52E cells.
OLETF rat, which manifests by obese, insulin resistance, spontaneous hyperglycemia, and proteinuria after 30 weeks, is an ideal animal model of DKD in human [
We also found significant renal structure damage even in the IGT group, especially renal tubules and interstitial blood vessels. Under a light microscope, the cortical brush borders of tubular epithelial cells were shed off, vacuole granules and necrosis were found in cells, inflammatory cells infiltrated in the interstitium, and mild thickening of the arterial wall was also found. Ultrastructural observation also revealed significant damage of tubular epithelial cells as well as endothelial cells. In accordance to the structure alteration, we also found a significant impairment of renal tubular function even in the IGT group of OLETF rats. NAG (or NGAL) which reflects the injury of the renal tubular epithelial cells and RBP (or CysC) which reflects the injury of renal tubular reabsorption were increased in the IGT group. In DM group, the above-mentioned renal tubular injuries were aggravated and accompanied by a series of pathophysiological changes, such as thickening of the glomerular basement membrane, widening of the mesangial area, proliferation of mesangial cell, and increasing of 24 h UMA. In contrast to the traditional opinion about DKD, kidney disease in the IGT stage manifests by renal tubular damage. Therefore, we propose this renal tubular damage which occurs in the IGT stage of diabetes as “IGT nephropathy.” Accumulating evidences have demonstrated that in the early stage of DKD, glomeruli are not changed while the structure and function of the renal tubule have been damaged [
What is the pathogenesis of nephropathy in the IGT stage? Different from type 1 DM, type 2 DM has prolonged pathophysiological progress. As a crucial prediabetes stage, IGT not only is closely related to the onset of cardiac, cerebral, and other macrovascular diseases but also is an independent risk factor for microvascular complications such as DKD [
Megalin and cubilin are multifunctional receptor glycoproteins which play important roles in mediating renal protein reabsorption. Cubilin-deficient animal models are subjected to significant proteinuria. Moreover, proteinuria occurring in patients with Fanconi syndrome can be explained by the lack of megalin and cubilin. The expressions of megalin and cubilin are affected by a variety of factors including their ligand insulin, but whether they are different under circumstances with different insulin levels remains unclear. In our study, we found that the expressions of megalin and cubilin were increased mostly by the low level of insulin. Laser confocal microscopy also displayed that NRK-52E cells treated by low-concentration insulin had the highest uptake of albumin. In the high-concentration intervention group, however, both the expressions of megalin and cubilin and the uptake of albumin were significantly decreased. Therefore, insulin at physiological concentration promotes megalin and cubilin expression and facilitates the uptake of albumin by renal tubular epithelial cells. However, with the increase of insulin concentration, the expressions of the two receptors were gradually decreased, and the reabsorption of albumin by renal tubular epithelial cells was also reduced. Therefore, insulin at high concentrations exert significant inhibitory effects.
As multifunctional receptor glycoproteins, megalin and cubilin can bind to various ligands and then mediate the absorption function. Megalin and cubilin play key roles in the renal reabsorption of albumin, RBP, CysC, and so on. The dysfunction of megalin and cubilin will cause reabsorption disorders of small-molecule proteins leading to the appearance of these proteins in urine. In our study, the urine RBP and CysC of OLETF rats in the IGT group were increased. Since the insulin level of IGT rats were increased, will it affect the reabsorption function of renal tubular epithelial cells?
We treated renal tubular epithelial cells with different concentrations of insulin to investigate its effect on megalin/cubilin expression and on albumin reabsorption. We found that in the presence of insulin at physiological concentration, the insulin signaling pathway IRS-1/PI3-K/Akt in renal tubular epithelial cells was highly activated, and the expressions of megalin and cubilin were highly upregulated which was accompanied by a maximum uptake of albumin. We found that both the inhibitor of the insulin signaling pathway and the high concentration of insulin could inhibit the expressions of megalin and cubilin as well as the reabsorption of TRITC-albumin in NRK-52E cells. Therefore, we speculated that high concentration of insulin affected the expressions of megalin and cubilin possibly via the IRS-1/PI3-K/Akt signaling pathway, which caused the disturbance of albumin endocytosis and finally led to proteinuria.
In summary, kidney damage occurs early in the IGT stage of diabetic rats. In contrast to DKD, we named this kind of renal structural damage and functional changes as “IGT kidney disease.” By inhibiting the IRS-1/PI3-K/Akt signaling pathway, high concentration of insulin downregulates the expression of megalin and cubilin, which then causes the reabsorption dysfunction of albumin by renal tubular epithelial cells leading to albuminuria. However, whether hyperinsulinemia plays a leading role in vivo remains to be proved by further studies and more inhibitors should be used.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no duality of interest associated with this manuscript.
Yi Zhang and Shaohua Yang contributed equally to this project.
This research was supported by grants from the National Natural Science Foundation of China (Nos. 81603461 and 81774043) and the Key Program of the Natural Science Foundation of Tianjin (17JCZDJC34700).
Primer sequences of target genes as supplements are shown in the table.