Nephrin Expression Research Articles

Quercetin ameliorates podocyte injury via inhibition of oxidative stress and the TGF-β1/Smad pathway in DN rats.

An increasing number of investigations have revealed that podocytes play a crucial role in the development and progression of diabetic nephropathy (DN). Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) is the most common flavonol in the diet and is one of the most prominent dietary antioxidants, which might have a protective effect on DN. The present study was designed to investigate the protective effect of quercetin on podocyte impairment in a rat model of DN, as well as underlying molecular mechanisms. All diabetic rats were induced by a single intraperitoneal injection of streptozotocin, and quercetin was administered daily at a dose of 50 mg kg−1 or 100 mg kg−1 for 12 weeks. In the present study, quercetin markedly decreased blood glucose levels, kidney-to-body weight ratio, albuminuria, creatinine clearance rate, blood urea nitrogen, and triglycerides and significantly attenuated oxidative stress. Moreover, quercetin was observed to inhibit podocyte effacement and decrease the thickness of glomerular basement membranes. Mechanistically, quercetin significantly increased the expression of podocyte-specific markers nephrin and podocin and decreased expression of the podocyte injury marker desmin in DN rats. Quercetin also inhibited activation of the TGF-β1/Smad signaling pathway in DN rats by decreasing expression of TGF-β1, p-Smad2, and p-Smad3, and increasing Smad7 expression. These findings suggest that quercetin administration ameliorated podocyte injury in DN rats, possibly by inhibiting oxidative stress and the TGF-β1/Smad signaling pathway. Thus, quercetin may be manipulated to act as a potential drug for prevention of early diabetic nephropathy.

Swiprosin-1 Promotes Mitochondria-Dependent Apoptosis of Glomerular Podocytes via P38 MAPK Pathway in Early-Stage Diabetic Nephropathy

Background/Aims: Podocyte injury, especially podocyte apoptosis, plays a major role in early-stage diabetic nephropathy (DN). Swiprosin-1, also known as EF hand domain containing 2 (EFhd2), is a Ca²⁺-binding protein in different cell types. However, the function of swiprosin-1 in podocytes remains unknown. Methods: The expression and distribution of swiprosin-1 were investigated in the mouse renal glomerulus and conditionally immortalized mouse podocyte cell line MPC-5. The expression of swiprosin-1 was also detected in streptozotocin (STZ)-treated mice and MPC-5 cells treated with high glucose (HG). Nephrin and podocin were detected by immunohistochemistry and immunofluroscence. Collagen IV, transforming growth factor-β (TGF-β) and fibronectin mRNA expressions were assayed by real-time PCR. Apoptotic proteins and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were detected by immunoblotting. Results: Swiprosin-1 was found to be expressed in podocytes of the mouse glomerulus and MPC-5 cells. Swiprosin-1 expression was increased in STZ-treated mice and MPC-5 cells treated with HG. In Swiprosin-1-/- diabetic mice, kidney/ body weight, urinary albumin, podocyte foot process effacement and glomerular basement membrane thickening were attenuated; the downregulation of nephrin and podocin expression in the glomerulus was inhibited; and the upregulation of collagen IV, TGF-β and fibronectin mRNA expression in the renal cortex was ameliorated as compared with those in diabetic swiprosin-1+/+ mice. In addition, the increased apoptosis of podocytes, proapoptotic protein expression and p38 phosphorylation in Swiprosin-1-/- diabetic mice were inhibited as compared with those in diabetic swiprosin-1+/+ mice. Knockdown of swiprosin-1 in MPC-5 cells reduced the apoptosis of podocytes, proapoptotic protein expression and p38 phosphorylation induced by HG. Targeted knockdown of p38 attenuated the increased apoptosis of MPC-5 cells over-expressing swiprosin-1. Conclusion: Swiprosin-1 expression in podocytes of the mouse glomerulus played a critical role in early-stage DN. Swiprosin-1 deficiency in early DN attenuated mitochondria-dependent podocyte apoptosis induced by hyperglycemia or HG via p38 MAPK signaling pathway.

Tiaolipiwei Acupuncture Reduces Albuminuria by Alleviating Podocyte Lesions in a Rat Model of Diabetic Nephropathy.

Background Diabetic nephropathy is a common and serious complication of diabetes and a major cause of end-stage renal disease. Tiaolipiwei acupuncture is a safe treatment approach that may be effective for lowering albuminuria in diabetic nephropathy. Yet, the exact mechanisms of this therapeutic effect are unclear. Methods A rodent model of type 2 diabetic nephropathy (T2DN) was induced by a high-fat diet combined with low-dose streptozotocin. T2DN rats were treated with Tiaolipiwei acupuncture (ACU) for 4, 8, or 12 weeks. At the end of treatment, urinary and blood samples were collected for analysis. Transmission electron microscopy was used to observe morphological changes, and protein expression levels of nephrin, CD2AP, podocalyxin, and desmin were quantified in renal tissue. Results Compared to the T2DN groups, the T2DN + ACU groups showed significant improvements in 24-hour urinary protein, serum urea, cholesterol, and triglycerides at all time points. ACU treatment also improved the density of slit diaphragms. Simultaneously, ACU promoted the renal expression of nephrin, CD2AP, and podocalyxin and decreased the expression of desmin. Conclusion Our study suggests that Tiaolipiwei acupuncture ameliorates podocyte lesions to reduce albuminuria and prevent the progression of T2DN in a rat model.

The role of abatacept in the treatment of type 2 diabetic nephropathy in rats

Objective To observe the role of abatacept in the treatment of diabetic nephropathy in rats. Methods Type 2 diabetic nephropathy rat model was established, and was randomly divided into abatacept group and non-intervention group, 15 each. 15 normal rats were served as control group (NC). The abatacept group was given abatacept for 8 weeks. The control group was set at the same time period. Then the blood biochemical indexes, blood flow parameters of renal main artery, elasticity of renal parenchymal, HE staining of renal parenchymal and ultrastructure of podocytes were all evaluated. The expression of CD31, CD34, podocin, nephrin, and B7-1 in renal parenchyma were detected. Results Compared to the NC group, the fasting blood glucose, creatinine clearance rate, urine albumin excretion rate, and kidney hypertrophy index in the non-intervention and abatacept groups were significantly increased (P abatacept>control groups (P abatacept>control groups (P the control group (P 0.05). There were significant differences in podocin and nephrin expression in kidney parenchyma of rats among these three groups, and these differences were ranked as the control>abatacept>non-intervention groups (P<0.05). There were significant differences in B7-1 expression in kidney parenchyma of rats among these three groups, and these differences were ranked as the control<abatacept<non-intervention groups (P<0.05). Conclusion Abatacept may significantly alleviate the renal injury in type 2 diabetic nephropathy rats. (Chin J Endocrinol Metab, 2017, 33: 1050-1056) Key words: Abatacept; Diabetic nephropathy; Glomerulus; Haemodynamics; Elasticity

Cannabinoid‐1 receptor deletion in podocytes mitigates both glomerular and tubular dysfunction in a mouse model of diabetic nephropathy

To determine the specific role of podocyte-expressed cannabinoid-1 receptor (CB1 R) in the development of diabetic nephropathy (DN), relative to CB1 R in other renal cell types. We developed a mouse model with a podocyte-specific deletion of CB1 R (pCB1Rko) and challenged this model with streptozotocin (STZ)-induced type-1 DN. We also assessed the podocyte response to high glucose in vitro and its effects on CB1 R activation. High glucose exposure for 48 hours led to an increase in CB1 R gene expression (CNR1) and endocannabinoid production in cultured human podocytes. This was associated with podocyte injury, reflected by decreased podocin and nephrin expression. These changes could be prevented by Cnr1-silencing, thus identifying CB1R as a key player in podocyte injury. After 12 weeks of chronic hyperglycaemia, STZ-treated pCB1Rko mice showed elevated blood glucose similar to that of their wild-type littermates. However, they displayed less albuminuria and less podocyte loss than STZ-treated wild-type mice. Unexpectedly, pCB1Rko mice also have milder tubular dysfunction, fibrosis and reduction of cortical microcirculation compared to wild-type controls, which is mediated, in part, by podocyte-derived endocannabinoids acting via CB1 R on proximal tubular cells. Activation of CB1 R in podocytes contributes to both glomerular and tubular dysfunction in type-1 DN, which highlights the therapeutic potential of peripheral CB1 R blockade.

Ste20-like kinase, SLK, a novel mediator of podocyte integrity.

SLK is essential for embryonic development and may play a key role in wound healing, tumor growth, and metastasis. Expression and activation of SLK are increased in kidney development and during recovery from ischemic acute kidney injury. Overexpression of SLK in glomerular epithelial cells/podocytes in vivo induces injury and proteinuria. Conversely, reduced SLK expression leads to abnormalities in cell adhesion, spreading, and motility. Tight regulation of SLK expression thus may be critical for normal renal structure and function. We produced podocyte-specific SLK-knockout mice to address the functional role of SLK in podocytes. Mice with podocyte-specific deletion of SLK showed reduced glomerular SLK expression and activity compared with control. Podocyte-specific deletion of SLK resulted in albuminuria at 4-5 mo of age in male mice and 8-9 mo in female mice, which persisted for up to 13 mo. At 11-12 mo, knockout mice showed ultrastructural changes, including focal foot process effacement and microvillous transformation of podocyte plasma membranes. Mean foot process width was approximately twofold greater in knockout mice compared with control. Podocyte number was reduced by 35% in knockout mice compared with control, and expression of nephrin, synaptopodin, and podocalyxin was reduced in knockout mice by 20-30%. In summary, podocyte-specific deletion of SLK leads to albuminuria, loss of podocytes, and morphological evidence of podocyte injury. Thus, SLK is essential to the maintenance of podocyte integrity as mice age.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy.Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Berberine prevents the apoptosis of mouse podocytes induced by TRAF5 overexpression by suppressing NF-κB activation.

Berberine(BBR) has previously been found to exert beneficial effects on renal injury in experimental rats. However, the mechanisms underlying these effects are not yet fully understood. Tumor necrosis factor(TNF) receptor-associated factor5(TRAF5) has been demonstrated to mediate the activation of nuclear factor-κB(NF-κB), which has been implicated in the pathogenesis of chronic kidney disease(CKD). The aim of this study was to investigate the effects of BBR on kidney injury and the activation of the NF-κB signaling pathway in mouse podocytes. TRAF5 was found to be overexpressed in patients with CKD and chronic renal failure(CRF)(data obtained from the dataset GSE48944, as well as from patients at Shuguang Hospital). TRAF5 overexpression significantly inhibited cell viability and induced the apoptosis of mouse podocytes. However, BBR prevented the decrease in cell viability and the apoptosis induced by TRAF5 overexpression. The NF-κB inhibitor, caffeic acid phenethyl ester(CAPE), mimicked the protective effects of BBR, as evidenced by the increased expression of nephrin and podocin, and the decreased the expression of caspase-3 and the ratio of Bax/Bcl-2. Moreover, BBR prevented the decrease in cell viability decrease and the apoptosis induced by TNF-α, interleukin(IL)-6 and lipopolysaccharide(LPS). Taken together, our data indicate that BBR exerts protective effects against CRF partly through the TRAF5-mediated activation of the NF-κB signaling pathway in mouse podocytes.

Glomerular filtration drug injury: In vitro evaluation of functional and morphological podocyte perturbations

The kidney is an organ that plays a major role in the excretion of numerous compounds such as drugs and chemicals. However, a great number of pharmacological molecules are nephrotoxic, affecting the efficiency of the treatment and increasing morbidity or mortality. Focusing on glomerular filtration, we propose in this study a simple and reproducible in vitro human model that is able to bring to light a functional podocyte injury, correlated with morphologic/phenotypic changes after drug exposure. This model was used for the evaluation of paracellular permeability of FITC-dextran molecules as well as FITC-BSA after different treatments. Puromycin aminonucleoside and adriamycin, compounds known to induce proteinuria in vivo and that serve here as positive nephrotoxic drug controls, were able to induce an important increase in fluorescent probe passage through the cell monolayer. Different molecules were then evaluated for their potential effect on podocyte filtration. Our results demonstrated that a drug effect could be time dependent, stable or scalable and relatively specific. Immunofluorescence studies indicated that these functional perturbations were due to cytoskeletal perturbations, monolayer disassembly or could be correlated with a decrease in nephrin expression and/or ZO-1 relocation. Taken together, our results demonstrated that this in vitro human model represents an interesting tool for the screening of the renal toxicity of drugs.

Nandrolone administration with or without strenuous exercise promotes overexpression of nephrin and podocin genes and induces structural and functional alterations in the kidneys of rats

Among the various adverse effects of nandrolone administration with or without strenuous exercise, kidney abnormalities, where there are associations between nandrolone decanoate consumption, have not been well defined yet. The aim of this study was to investigate the effect of nandrolone decanoate intake with or without strenuous exercise on nephrin and podocin gene expressions, cystatin C, oxidative DNA damage, and histological changes in the kidneys of rats. Thirty-two male wistar rats were assigned into four groups, namely control, nandrolone, nandrolone with strenuous exercise, and strenuous exercise groups. After six weeks of treatment, the results revealed a significant increase in the nephrin and podocin gene expression, plasma cystatin C, and the amount of 8-OHdG in the kidney tissue; as well as a decrease in creatinine clearance in nandrolone and nandrolone with strenuous exercise groups compared to the control group. Moreover, compared to the control group, the nandrolone and the nandrolone with strenuous exercise groups, showed histological changes such as fibrosis and kidney tissue cells proliferation. These findings indicate that nandrolone induces kidney abnormalities, which may in part be associated with overexpression of nephrin and podocin genes mediated by oxidative stress, which was manifested in increased 8-OHdG in kidney tissue.

Uric acid causes kidney injury through inducing fibroblast expansion, Endothelin-1 expression, and inflammation

BackgroundUric acid (UA) plays important roles in inducing renal inflammation, intra-renal vasoconstriction and renal damage. Endothelin-1 (ET-1) is a well-known profibrotic factor in the kidney and is associated with fibroblast expansion. We examined the role of hyperuricemia conditions in causing elevation of ET-1 expression and kidney injury.MethodsHyperuricemia was induced in mice using daily intraperitoneal injection of uric acid 125 mg/Kg body weight. An NaCl injection was used in control mice. Mice were euthanized on days-7 (UA7) and 14 (UA14). We also added allopurinol groups (UAL7 and UAL14) with supplementation of allopurinol 50 mg/Kg body weight orally. Uric acid and creatinine serum were measured from blood serum. Periodic Acid Schiff (PAS) and Sirius Red staining were done for glomerulosclerosis, tubular injury and fibrosis quantification. mRNA expression examination was performed for nephrin, podocin, preproEndothelin-1 (ppET-1), MCP-1 and ICAM-1. PDGFRβ immunostaining was done for quantification of fibroblast, while α-SMA immunostaining was done for localizing myofibroblast. Western blot analysis was conducted to quantify TGF-β1, α-SMA and Endothelin A Receptor (ETAR) protein expression.ResultsUric acid and creatinine levels were elevated after 7 and 14 days and followed by significant increase of glomerulosclerosis and tubular injury score in the uric acid group (p < 0.05 vs. control). Both UA7 and UA14 groups had higher fibrosis, tubular injury and glomerulosclerosis with significant increase of fibroblast cell number compared with control. RT-PCR revealed down-regulation of nephrin and podocin expression (p < 0.05 vs. control), and up-regulation of MCP-1, ET-1 and ICAM-1 expression (p < 0.05 vs. control). Western blot revealed higher expression of TGF-β1 and α-SMA protein expression. Determination of allopurinol attenuated kidney injury was based on reduction of fibroblast cell number, inflammation mediators and ppET-1 expression with reduction of TGF-β1 and α-SMA protein expression.ConclusionsUA induced glomerulosclerosis, tubular injury and renal fibrosis with reduction of podocyte function and inflammatory mediator elevation. ET-1 and fibroblast expansion might modulate hyperuricemia induced renal fibrosis.

Nephrin loss is reduced by grape seed proanthocyanidins in the experimental diabetic nephropathy rat model.

Diabetic nephropathy (DN) is one of the major causes of end-stage renal failure. Grape seed proanthocyanidin extracts (GSPE) are known to act as antioxidants. The current study aimed to determine the effects of GSPE on the streptozotocin (STZ)-induced diabetic rat model and to explore the underlying mechanism of its action. Wistar rats were induced into a diabetic state by injection of STZ and were treated with 250 mg·kg−1·day−1 GSPE for 24 weeks. Kidney samples were collected for observation of renal pathological changes by light microscope (periodic acid-Schiff staining) and electron microscopy. Reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical staining were used to detect the mRNA and protein expression of the receptor for advanced glycation end-products (RAGE), nephrin and podocin. The results indicated that diabetic rats treated with GSPE had markedly reduced Ccr, urinary albumin excretion, ratio of kidney weight to body weight, AGEs and ECM accumulation (P<0.01) compared with that in the diabetic rats. GSPE treatment can also reverse the renal pathological damage in diabetic rats. Further results indicated that GSPE treatment significantly decreased the RAGE expression level (P<0.01), and significantly increased the expression level of nephrin in the kidney and glomeruli of diabetic rats (P<0.01). However, no significant differences were identified in the expression of podocin following GSPE treatment (P>0.05). In conclusion, the results demonstrated that GSPE exerts a reno-protective effect by decreasing urinary albumin excretion and reversing renal pathological damage in diabetic rats. The underlying mechanism of GSPE activity is associated with the decreased expression of the AGEs/RAGE axis and the increased expression of nephrin in diabetic rats.

ShcA Adaptor Protein Promotes Nephrin Endocytosis and Is Upregulated in Proteinuric Nephropathies.

Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA, via its SH2 domain, was associated with several phosphorylated tyrosine residues on nephrin. Overexpression of ShcA promoted nephrin tyrosine phosphorylation and reduced nephrin signaling and cell surface expression in vitro In a rat model of reversible podocyte injury and proteinuria, phosphorylated nephrin temporally colocalized with endocytic structures coincident with upregulation of ShcA expression. In vivo biotinylation assays confirmed that nephrin expression decreased at the cell surface and correspondingly increased in the cytosol during the injury time course. Finally, immunostaining in kidney biopsy specimens demonstrated overexpression of ShcA in several human proteinuric kidney diseases compared with normal conditions. Our results suggest that increases in ShcA perturb nephrin phosphosignaling dynamics, leading to aberrant nephrin turnover and slit diaphragm disassembly.

Effects of chronic fructose overload on renal dopaminergic system: alteration of urinary L-dopa/dopamine index correlates to hypertension and precedes kidney structural damage

Insulin resistance induced by a high-fructose diet has been associated to hypertension and renal damage. The aim of this work was to assess alterations in the urinary L-dopa/dopamine ratio over three time periods in rats with insulin resistance induced by fructose overload and its correlation with blood pressure levels and the presence of microalbuminuria and reduced nephrin expression as markers of renal structural damage. Male Sprague–Dawley rats were randomly divided into six groups: control (C) (C4, C8 and C12) with tap water to drink and fructose-overloaded (FO) rats (FO4, FO8 and FO12) with a fructose solution (10% w/v) to drink for 4, 8 and 12 weeks. A significant increase of the urinary L-dopa/dopamine ratio was found in FO rats since week 4, which positively correlated to the development of hypertension and preceded in time the onset of microalbuminuria and reduced nephrin expression observed on week 12 of treatment. The alteration of this ratio was associated to an impairment of the renal dopaminergic system, evidenced by a reduction in renal dopamine transporters and dopamine D1 receptor expression, leading to an overexpression and overactivation of the enzyme Na+, K+-ATPase with sodium retention. In conclusion, urinary L-dopa/dopamine ratio alteration in rats with fructose overload positively correlated to the development of hypertension and preceded in time the onset of renal structural damage. This is the first study to propose the use of the urinary L-dopa/dopamine index as marker of renal dysfunction that temporarily precedes kidney structural damage induced by fructose overload.

The Beneficial Effects of Allicin in Chronic Kidney Disease Are Comparable to Losartan.

Recent studies suggest that allicin may play a role in chronic kidney disease (CKD), reducing hypertension and oxidative stress and improving renal dysfunction. In the present study, CKD was induced by 5/6 nephrectomy and the animals were divided into four treatment groups as follows: control (C), CKD, CKD+allicin (40 mg/kg pathway oral) (CKDA), and CKD+Losartan (20 mg/kg) (CKDL). After CKD induction, the rats developed hypertension from week 3 to the end of the study. This was associated with increased creatinine and blood urea nitrogen (BUN) levels in serum, increased albuminuria, increased urinary excretion of N-acetyl-β-d-glucosaminidase (NAG), increased nephrin expression, and incrased histological alterations in the cortex. The levels of angiotensin receptors and endothelial nitric oxide synthase (eNOS) were decreased in the renal cortex from the CKD group. Otherwise, lipid and protein oxidation were higher in the CKD group than in the control group. A disturbance was observed in the expression levels of the nuclear factor erythroid 2-related factor 2/Kelch ECH associating protein 1 system (Nrf2/keap1) and the antioxidant enzymes catalase, superoxide dismutase, and heme oxygenase-1. Allicin or losartan treatments relieved renal dysfunction, hypertension, and oxidative stress. In addition, both treatments showed the same efficacy on the expression of angiotensin receptors, the nephrin, Nrf2/keap1 pathway, and eNOS. Further in silico analyses suggest that allicin and losartan could have a common mechanism involving interaction with AT1 receptors. Allicin showed antihypertensive, antioxidant, and nephroprotective effects. The beneficial effects showed by allicin are similar, or even better, than those of losartan. In fact, the effect of allicin on blood pressure and renal function is comparable to reductions seen with losartan, a prescription drug commonly used as a first-line therapy.

PP.07.14] RENAL DOPAMINERGIC SYSTEM DYSFUNCTION ASSOCIATED TO HIGH BLOOD PRESSURE AND INFLAMMATION IN FRUCTOSE OVERLOAD INDUCED METABOLIC SYNDROME

Objective: The renal dopaminergic system (RDS) promotes sodium excretion and anti-inflammatory actions. Fructose overload (FO) produces in the rat hemodynamic and metabolic changes similar to the human metabolic syndrome. These changes are associated to an impairment of the RDS, leading to renal inflammation, sodium retention and blood pressure elevation. The aim of this study was to evaluate RDS state and its relation to hypertension and overexpression of renal inflammatory markers in the FO experimental model. Design and method: Six groups of male Sprague Dawley rats (six weeks at the beginning of the treatment) were studied: Control (C4, C8 and C12, tap water to drink) or FO (F4, F8 and F12, 10% w/v of fructose solution to drink), during 4, 8 and 12 weeks (n = 8/group/period). Urinary L-dopa and dopamine (DA) (HPLC), diuresis and albuminuria were determined. Systolic blood pressure (SBP) (tail cuff) and metabolic parameters were measured. Western blot analysis of renal expression of dopamine 1 receptor (D1R), NFkappa-beta, IL-6, TNF-alpha, TGF-beta1 and nephrin were performed. Results: Fructose overload increased SBP (mmHg, C4: 121 ± 8 vs. F4: 145 ± 1*; C8: 130 ± 4 vs. F8: 161 ± 10#; C12: 133 ± 5 vs. F12: 163 ± 4#), which positively correlated (R2 = 0.78; p < 0.002) to urinary L-dopa/DA index (C4: 0.49 ± 0.05 vs. F4: 1.9 ± 0.09#; C8: 0.53 ± 0.06 vs. F8: 2.35 ± 0.1#; C12: 0.54 ± 0.07 vs. F12: 2.57 ± 0.2#). A significant decrease in the D1R expression was related to a significant increase in nFkappa-beta, IL-6, TNF-alpha, TGF-beta1 expression since week 4. Microalbuminuria (C12:13.11 ± 1.4 vs F12:57.6 ± 2.5#) and a decrease in nephrin expression (C12: 1.00 ± 0.10 vs. F12: 0.73 ± 0.05#) were observed only at week 12. (*p < 0.05, #p < 0.01 vs. C). Conclusions: Fructose overload was associated to an L-dopa/DA index increase and D1R expression decrease since week 4 of treatment. The renal dopaminergic system dysfunction was accompanied by an increase in blood pressure levels and renal expression of inflammatory markers in all experimental periods studied. Alteration of L-dopa/DA index could be an earlier marker of renal dysfunction than the structural damage evidenced by microalbuminuria and decreased nephrin expression in week 12 of fructose treatment.

A differential expression of uncoupling protein-2 associates with renal damage in stroke-resistant spontaneously hypertensive rat/stroke-prone spontaneously hypertensive rat-derived stroke congenic lines.

Uncoupling protein-2 (UCP2), a mitochondrial anion transporter involved in mitochondrial uncoupling, limiting reactive oxygen species formation, is significantly downregulated in kidneys of high-salt-fed stroke-prone spontaneously hypertensive rat (SHRSP), where it associates with increased renal damage occurrence. We aimed at establishing whether UCP2 differential expression associates with renal damage in two stroke-resistant spontaneously hypertensive rat (SHRSR)/SHRSP-derived stroke congenic lines. For this purpose, SHRSR, SHRSP, and two reciprocal stroke congenic lines carrying the (D1Rat134-Mt1pa) segment of chromosome 1 were fed with Japanese style diet for 8 weeks. At 4, 6, and 8 weeks of Japanese diet, kidneys were removed and analyzed for UCP2 gene and protein expression [UCP2 maps within (D1Rat134-Mt1pa)]; nuclear factor kappa-light-chain-enhancer of activated B cells protein expression; oxidized total protein levels; mitochondrial function; gene expression of cubulin, megalin, and nephrin. At 6 and 8 weeks of Japanese diet, histological damage and percentage of high molecular weight urinary proteins excretion were assessed. Introgression of UCP2 in the SHRSP configuration within the SHRSR genome led to UCP2 downregulation upon Japanese diet, as compared with the SHRSR, with significantly reduced ATP levels, increased rate of inflammation, oxidative stress, renal damage, and excretion of high molecular weight proteins. The opposite phenomena were observed in the reciprocal congenic line, compared with the SHRSP. In vitro, high-NaCl medium led to UCP2 downregulation, increased apoptosis/necrosis, and reduced viability in primary renal proximal tubular epithelial cells isolated from SHRSP. Exposure of the SHRSP/proximal tubular epithelial cells to recombinant UCP2 rescued the high-salt-dependent deleterious effects. A differential UCP2 expression associates with different degree of renal damage upon Japanese diet in two SHRSR/SHRSP-derived stroke congenic lines through modulation of mitochondrial function, inflammation, and oxidative stress.

Abstract 139: Loss of Thymosin b4 Exacerbates Renal and Cardiac Damage in Angiotensin-II Hypertension

Angiotensin-II (Ang-II)-induced hypertension is associated with tissue damage and fibrosis in the kidney and heart. Thymosin β4 (Tβ4) regulates cell morphology, inflammation and fibrosis in several organs and administration of exogenous Tβ4 is protective in diabetic nephropathy and unilateral ureteral obstruction model. However, role of endogenous Tβ4 in hypertension-induced organ damage is unknown. We hypothesize that, loss of Tβ4 accelerates renal and cardiac fibrosis and damage in Ang-II hypertension. To test our hypothesis, Tβ4 knockout (Tβ4 -/- ) and wild-type (Tβ4 +/+ ) C57BL/6 mice (n=6-10) were infused continuously for six-weeks with either Ang-II (980 ng/kg/min) or vehicle via osmotic minipumps. All the results are presented in table 1. In Ang-II infusion, systolic blood-pressure were not different between both strains (Table 1). Interestingly, urinary albuminuria was significantly higher in Tβ4 -/- mice compared to Tβ4 +/+ mice by Ang-II. High expression of Tβ4 is found in the glomeruli along with high expression of Nephrin, an important protein in the filtration barrier of the kidney. In Ang-II infusion, nephrin protein expression was greatly reduced in mice deficient of Tβ4, suggesting that loss of nephrin is one of the mechanism for elevated urinary albumin in Tβ4 -/- mice. Additionally, renal fibrosis was higher in Tβ4 -/- mice. We also studied cardiac damage and observed that in Ang-II infusion, cardiac hypertrophy and cardiac fibrosis were much higher in Tβ4 -/- mice. These data indicate that loss of endogenous Tβ4 caused significant tissue damage in the kidney and heart in Ang-II hypertension, suggesting renal and cardiac protective role of this peptide.

Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor

Aims/hypothesisPodocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy.MethodsConditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs.ResultsPodocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-α and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton.Conclusions/interpretationTogether, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.

Ferulic acid alleviates symptoms of preeclampsia in rats by upregulating vascular endothelial growth factor.

Preeclampsia is a complication affecting pregnant women worldwide, which leads to maternal and fetal morbidity and mortality. In this study, we evaluated the efficacy of ferulic acid (FA) on an Nω -nitro-L-arginine methyl ester hydrochloride (L-NAME) induced rat model of preeclampsia. L-NAME was administered to pregnant rats to induce preeclampsia. 48 rats were divided into three experimental groups (n=16 each): control group, preeclampsia group and preeclampsia with FA treatment (preeclampsia+FA). Physiological characteristics such as urine volume, total urine protein and blood pressure were assessed. Expressions levels of urinary nephrin and podocin mRNAs were analyzed by RT-PCR. Levels of renal vascular endothelial growth factor (VEGF), renal soluble fms-like tyrosine kinase-1 (sFlt-1) and serum placenta growth factor (PlGF) were also examined. Urine volume, total urine protein and blood pressure were markedly increased in preeclampsia group rats compared to control (P<.05), which were then significantly reduced in preeclampsia+FA group (P<.05). Expressions of urinary nephrin and podocin mRNAs, levels of VEGF, sFlt-1 and PlGF were also reversed in preeclampsia+FA group compared to preeclampsia rats (P<.05). We hereby report for the first time, FA alleviates preeclampsia symptoms in a rat preeclampsia model, supporting its potential value in treating preeclampsia.