Streptozotocin induces alpha-2u globulin nephropathy in male rats during the end-stage of diabetic kidney disease

Background Alpha-2u globulin nephropathy mainly shows toxicological pathology only in male rats induced by certain chemicals and drugs, such as levamisole (antiparasitic and anticancer drugs). Streptozotocin (STZ) is also an anticancer-antibiotic agent that has been used for decades to induce a diabetic kidney disease model in rodents. The purpose of this study is to determine if STZ causes alpha-2u globulin nephropathy in male rats during an advanced stage of diabetic kidney disease. Methods To prove this hypothesis, the present study used a male diabetic Wistar rat model with 45 mg/kg of STZ injected intraperitoneally. Hyperglycaemic rats were divided into 2 groups: with and without end-stage kidney disease. Alpha-2u globulin nephropathy was examined by histopathological and electron microscope studies. Water absorption and filtration capacities (via aquaporin [AQP]-1, -2, -4 and -5) and mitochondrial function (through haloacid dehalogenase-like hydrolase domain-containing protein [HDHD]-3 and NADH-ubiquinone oxidoreductase 75 kDa subunit [NDUFS]-1 proteins) were determined using immunohistochemistry, immunofluorescence and immunogold labelling techniques. Results More than 80% of end-stage diabetic kidney disease induced by STZ injection simultaneously exhibited alpha-2u globulin nephropathy with mitochondrial degeneration and filtration apparatus especially pedicels impairment. They also showed significantly upregulated AQP-1, -2, -4 and -5, HDHD-3 and NDUFS-1 compared with those of the rats without alpha-2u globulin nephropathy. Conclusions STZ-induced alpha-2u globulin nephropathy during end-stage diabetic kidney disease in association with deterioration of renal filtration, renal tubular damage with adaptation and mitochondrial apoptosis.


Background
Alpha-2u globulin nephropathy, a deposition of alpha-2u globulin (a lipocalin family protein with proteolytic and hydrolytic-resistant activities) in proximal tubule lysosomes, is an important toxicological syndrome that presents only in male rats and is relevant to nephropathy and renal neoplasia [1]. Furthermore, alpha-2u globulin is synthesised in the liver of male rats under multihormonal control, especially androgen [2]. Industrial or environmental chemicals and drugs have 3 been reported to cause alpha-2u globulin nephropathy in male rats, including unleaded gasoline, 1,4dichlorobenzene, pentachloroethane, synthetic jet fuel and diesel, fuel marine, levamisole and RG7129 (β-site amyloid binding protein) [1,[3][4][5][6]. Histopathological changes in rats with alpha-2u globulin nephropathy include hyaline droplet deposition in the cytoplasm and lumen of the proximal tubule, tubular degeneration and regeneration, tubular dilatation and parenchymal inflammation [6].
Presently, it is well documented that streptozotocin (STZ) injections have been used to induce hyperglycaemia in rodents, which leads to renal injury with similarities to human diabetic nephropathy. Renal pathology in STZ-induced hyperglycaemic rats mainly consists of glomerular hypertrophy, hypercellularity, tubular dilatation and atrophy, thickening of the glomerular basement membrane and mesangial expansion [7]. However, there have not yet been reports that alpha-2u globulin nephropathy can be induced by STZ injection in male rat as shown by previously mentioned drugs. Interestingly, Sun and colleague suggested that, in an STZ-induced diabetic rat, alpha-2u globulin and its modified form are dysregulated in renal mitochondria, leading to a reduction in βoxidation of long chain fatty acids, decreased energy supply, increment in the number of fatty acid depositions and thus renal damage [8]. Along these lines of thought, histopathology, immunohistochemistry, electron microscopy and immunogold labelling techniques were performed to demonstrate the presence of alpha-2u globulin nephropathy in STZ-induced diabetic rats in relation to the alteration of (i) water reabsorption and filtration function as characterised by aquaporins (AQP)-1, -2, -4 and − 5, (ii) mitochondrial energetic maintenance protein using haloacid dehalogenase-like hydrolase domain-containing protein (HDHD)-3 and (iii) mitochondrial apoptotic marker by NADHubiquinone oxidoreductase 75 kDa subunit (NDUFS)-1. Clinico-histopathological and fine morphological appearances are also discussed.
2. Results 2.1 STZ induces alpha-2u globulin nephropathy in moribund hyperglycaemic rats As a consequence of STZ-induced hyperglycaemia in male rats, they all had a blood glucose level > 200 mg/dL ( Fig. 1A) with polyuria 7 days post-induction. Twelve of them (12/20; 60%) showed severe clinical illness, such as anorexia, depression, weight loss (≥ 20%) and moribund 10-14 days post-4 induction, while the rest had acceptable clinical manifestations with fair prognosis. Ten of the moribund rats (10/12; 83.33%) had obviously reddish urine and were microscopically diagnosed as having alpha 2u-globulin nephropathy. The blood sugar level between the rats with (N = 10) or without (N = 10) alpha 2u-globulin nephropathy was found to be not significantly different (Fig. 1A).
Histopathological changes in the liver, pancreas and kidneys from the rats with and without alpha 2uglobulin nephropathy were scored as shown in the Fig. 1B-D, respectively. Although histopathological lesions of the liver and pancreas in alpha 2u-globulin nephropathic rats tended to be higher than in the rats without alpha 2u-globulin nephropathy, the difference was not statistically significant ( Fig. 1B,C). In addition, the alpha 2u-globulin nephropathic rats exhibited a significantly higher renal histopathological score than that presented in non-alpha 2u-globulin nephropathic rats (Fig. 1D)

Altered mitochondrial proteins in alpha-2u globulin nephropathy rats
To characterise mitochondrial function in terms of energetic balance and apoptosis via HDHD-3 and NDUFS-1 expression between the rats with or without alpha 2u-globulin nephropathy, immunogold labelling was performed. The results showed that the expression levels of HDHD-3 and NDUFS-1 were significantly increased in the mitochondria of PCTs from the alpha 2u-globulin nephropathic kidneys when compared with those of non-alpha 2u-globulin nephropathic kidneys (Fig. 5).

Discussion
Sex-and species-specific diseases have been reported for decades since the discovery of unleaded gasoline was shown to lead to kidney tumours in male rats but not in females and both sexes of mice [3]. Likewise, the characterisation of alpha 2u-globulin nephropathy in male rats has also been discussed for decades [1][2][3][4][5][6][8][9][10]. Several chemicals and drugs have been demonstrated to induce alpha 2u-globulin nephropathy only in mature male rats in association with neoplasia enhancement.
Alpha 2u-globulin is synthesised in the liver under the influence of androgenic hormone and is released into blood circulation. This protein is freely filtered by the glomeruli and is reabsorbed by the P2 segment of the proximal tubule [3,[11][12][13] with persistent deposition due to its resistance to hydrolytic and proteolytic enzymes in the lysosomes, and approximately half of them are excreted in the urine [11,12]. The accumulation of alpha 2u-globulin is cytotoxic and leads to single cell necrosis, a nidus for granular cast formation and reversible re-epithelialisation as presented by regenerative tubules [2,14,15]. Enhanced cellular proliferation initiates the transformation of proximal tubules to preneoplastic and neoplastic lesions [2,16]. The primary histopathological change in alpha 2uglobulin nephropathy is intracytoplasmic "hyaline droplet" or "eosinophilic body" deposition in the 6 proximal tubules with a variety of forms from spherical to polyangular [1, 4-6, 9, 10, 14, 16]. STZ, a nitrosourea alkylating agent or anticancer-antibiotic drug, has occasionally been used as a cytotoxic agent for treating some types of human tumours, e.g., lymphoma, sarcomas and Islet of Langerhans cancer [17]. It has also been extensively used for developing rodent models of diabetes and diabetic nephropathy. Interestingly, the present study demonstrates that end-stage nephropathy induced by STZ exhibits alpha 2u-globulin nephropathy in > 80% of the moribund male rats.
Levamisole is also an example of an anticancer and antiparasitic drug that causes alpha 2u-globulin nephropathy only in male rats. Similar to previous studies, intracytoplasmic hyaline droplet deposition in PCTs (Fig. 2C,D) leads to cellular degeneration as characterised by the increment in vacuolated degeneration (Fig. 2D,E) and tubular degeneration and regeneration (Fig. 2F,G, respectively). In contrast to other chemicals or drugs that induce 2u-globulin nephropathy, preneoplastic and neoplastic lesions were not observed in end-stage renal kidney disease induced by STZ. Moreover, electron micrographs also show the presence of mitochondrial degeneration and swelling in rats with alpha 2u-globulin nephropathy (Fig. 3D,E). These results clearly suggest that the cytotoxic properties of alpha 2u-globulin cause cellular and organelle damages. Additionally, considering the glomerular filtration capacity in alpha 2u-globulin nephropathic rats, this study demonstrates deterioration of the filtering apparatus, especially pedicels, as shown in the Fig. 3F,G. However, the detail mechanisms involved in this impairment caused by alpha 2u-globulin deposition require further study.
Diabetic nephropathy is a microangiopathic complication present in one-third of diabetes mellitus patients [18]. It has been claimed that dysregulation of the water channel membrane protein "aquaporin; AQP" in the kidney plays an important role in the pathogenesis of several kidney diseases including diabetic nephropathy [18][19][20]. Eight AQPs, AQP-1-7 and − 11, are expressed in the kidney to maintain normal urine concentration [20]. Several reports indicate that alterations of AQP-1, -2, -4 and − 5 expression are highly associated with renal diseases. AQP-1 functions in hypertonicity formation and is expressed in apical and basolateral membranes of proximal tubules, descending thin limbs of Henle and descending vasa recta [21]. It also localises in the β-laminin of the glomerular basement membrane [19]. AQP-2, a urine concentration regulator under anti-diuretic hormone, is 7 located at the apical membrane of the collecting duct [22]. AQP-4, a water permeability regulator, is located at the basolateral membrane of the collecting duct and exports water into the cytoplasm via AQP-2 [20]. Lastly, AQP-5 is located in type B intercalated cells of the collecting duct with unclear function [23]. Upregulation of glomerular AQP-1 is found in all forms of human renal diseases, probably due to compensation for losing cellular integrity [19]. Upregulation of AQP-2 and − 5 is closely related to the progression of diabetic nephropathy in diabetic patients and are good candidates to use for diagnosis [18,24]. Recently, Go and Zhang also reported that an increase in AQP-5 in patients with diabetic nephropathy is independently associated with a reduction in the glomerular filtration rate [25]. In addition, a STZ-induced diabetic rat model exhibits a high level of anti-diuretic hormone, leading to upregulation of AQP-2 as a compensatory mechanism [26].
Dysregulation of intrarenal AQP-4 is involved in end-stage renal disease in HIV patients with glomerulosclerosis and renal tubular dysfunction [27]. In the present study, immunohistochemical studies reveal significant upregulation of AQP-1, -2, -4 and − 5 in the alpha 2u-globulin nephropathic rats (Fig. 4). These findings likely indicate that increases in AQP-1, -2, -4 and − 5 responses are (i) compensatory during high cellular and mitochondrial degeneration due to alpha 2u-globulin deposition in the PCTs, (ii) an advanced stage of diabetic kidney disease, (iii) a depletion of glomerular filtration capacity in association with the presence of pedicels disruption and (iv) renal tubule dysfunction, particularly PCTs, DCTs and CD.
According to mitochondrial function and its architecture, mitochondrial dysfunction is a crucial factor in the pathogenesis of diabetic kidney diseases regarding reactive oxygen species overproduction, apoptosis activation and mitophagy defects [28][29][30][31][32][33][34]. The kidney is an extreme oxygen consumption organ, which renders it sensitive to mitochondrial dysfunction. A hyperglycaemic environment also contributes to direct damage of renal tubular cells [28]. Dysregulation of essential mitochondrial genes in diabetic kidney diseases has been reported in relevance to the severity of renal pathology, e.g., glomerular endothelial injury, glomerulosclerosis and podocyte defects [30]. A change in the metabolic energy source under diabetic conditions results in increased oxygen consumption in the kidney and leads to renal hypoxia, ischaemia and necrosis [8,29]. Our recent studies have 8 demonstrated that cellular power synthesis (Haloacid Dehalogenase-Like Hydrolase Domain-Containing [HDHD]-3) and a mitochondrial apoptotic marker (NADH: ubiquinone oxidoreductase core subunit S1 [NDUFS-1]) in liver mitochondria in sericin-fed rats are preserved compared to those of non-treated rats under hypercholesterolemic conditions [35,36]. In this study, the immunogold labelling technique indicates significant upregulation of HDHD-3 and NDUFS-1 in the alpha 2u-globulin nephropathic rats (Fig. 5). This suggests the high incidence of degenerative mitochondria in the alpha 2u-globulin nephropathic kidney, which attempt to increase energetic protein for the maintenance of renal function and integrity even when high levels of apoptosis were also observed.

Conclusions
During end-stage diabetic kidney disease induced by STZ injection in male rats, alpha 2u-globulin nephropathy was predominately observed in association with upregulation of renal water channel membrane proteins (AQP-1, -2, -4 and − 5), mitochondrial energetic maintenance protein (HDHD-3) and mitochondrial apoptotic protein (NDUFS-1). All of these phenomena are likely due to compensation for renal damage in advanced stages of kidney disease, particularly toward tubular and glomerular filtrate functions. These findings are useful for understanding the pathogenesis of alpha 2u-globulin nephropathy in association with diabetic kidney disease induced by STZ infection. Wistar rats were obtained from NLAC-MU. All of the rats were housed in a temperature-, humidity-and illumination-controlled room and fed ad libitum with standard diet and reverse-osmosis water.

STZ-induced hyperglycaemic rat model
Consequent to the acclimatisation period, all rats were fasted for 6 h before being intraperitoneally infected with a single dose of 45 mg/kg streptozotocin (Sigma-Aldrich, USA) in fresh 0.1 M citrate buffer, pH 4.0 to induce hyperglycaemia [34]. Fasting blood glucose was examined in all of the rats, and a blood sugar level of ≥200 mg/dL was considered diabetic stage. Clinical manifestations were carefully observed daily by trained personnel. Two weeks post-induction, all surviving rats and moribund or rats that lost ≥20% of their weight were humanely euthanised using an overdose of carbon dioxide inhalation. Their kidneys were collected, divided into two and then fixed in 10% neutral buffer formalin and 2.5% glutaraldehyde in 0.1 M sucrose phosphate buffer (SPB) for histopathologic and electron microscopic studies, respectively.

Histopathological studies
To demonstrate the presence of alpha 2u-globulin nephropathy in the STZ-induced diabetic rats and other histopathological changes in the liver and pancreas, histopathological studies were performed.

Immunohistochemical and immunofluorescence studies
To determine the pathogenesis of alpha 2u-globulin nephropathy induced by STZ injection, as relevant to water absorption and glomerular filtrate function via AQP, immunohistochemical (IHC) and immunofluorescence (IF) studies were conducted using EnVision FLEX/HRP kit (DAKO, Denmark) and VectaFluor Duet immunofluorescence double labelling kit, DyLight 488 Anti-rabbit (green)/DyLight 594 Anti-mouse (red) (VECTOR, USA), respectively. The sections were deparaffinised in xylene, hydrated in a series of graded ethanol and heat-retrieved to enhance the antigenicity in citrate buffer, pH 6.0.
Appropriate secondary antibodies matching their conjugate and visualisation system from the kit were applied to the sections. The nuclei were counterstained by either haematoxylin or VECTASHIELD Antifade mounting medium with DAPI (VECTOR, USA) for IHC and IF, respectively. Immunolocalisation was measured using the H-score as mentioned above. In addition, the area of expression as a percentage was determined using an image analysis programme (ImageJ, version 1.51J8, NIH).
Briefly, five images of labelled areas were captured and transformed to binary images.
Immunolocalisation was defined by the threshold mode and determined as an area fraction (%).

Electron microscopic studies
To demonstrate the fine morphological structure of alpha 2u-globulin nephropathy in STZ-induced diabetes, electron microscopic studies were performed. The kidneys were again fixed with 1%

Immunogold labelling technique
To clarify the immunolocalisation of HDHD-3 (energetic maintenance protein) and NDUFS-1 (apoptotic protein) in the renal mitochondria, immunogold electrons were used. After the sections were blocked with 50 mM glycine and 5% bovine serum albumin (BSA) (EMS, USA), they were incubated with the described primary antibodies for 1 h at room temperature. Immunoglobulin (Ig) G conjugated with 10 nm gold particles (EMS, USA) was then applied to the sections for 1 h. Silver enhancement was performed using the Aurion R-Gent SE-EM kit (EMS, USA). Finally, the sections were stained with lead citrate and uranyl acetate and examined under TEM, focusing on the amount of gold labelling/mitochondria.

Statistical analysis
GraphPad PRISM, version 6.05, was used for statistical analysis. Either independent t-tests or analysis of variance was performed to characterise the difference between the groups and was expressed as the mean ± SEM. The 95% confidence interval p < 0.05 was considered statistically significant.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. electron microscope studies. S.A., K.T. and N.P. interpreted the data and drafted the manuscript. All authors have read and approved of the final manuscript.   HDHD-3 and NDUFS-1 immunogold labelling in renal mitochondria from rats with and without alpha 2u-globulin nephropathy. A-C: gold labelling (arrow) of HDHD-3 in mitochondria from 2u-globulin nephropathic rats (A) and non-2u-globulin nephropathic rats (B) and presented via a bar graph (C); D-F: gold labelling (arrow) of HDHD-3 in mitochondria from 2u-globulin nephropathic rats (D) and non-2u-globulin nephropathic rats (E) and presented via a bar graph (F).

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