Sua Kim

Altered Expression of Tight Junction Proteins in Cyclosporine Nephrotoxicity

Background/Aims: The increased permeability of chloride in the distal cortical nephron in cyclosporine nephrotoxicity may involve the transcellular pathway mediated by the thiazide-sensitive Na+-Cl– cotransporter and/or the paracellular pathway mediated by the tight junctions (TJs). Methods: Cyclosporine was subcutaneously administered to Sprague-Dawley rats for 6 (7.5 mg/kg body weight) and 2 (25 mg/kg body weight) weeks, and immunoblot analysis and immunohistochemistry were carried out from the kidneys. Electrically tight epithelial Madin-Darby canine kidney (MDCK) I cells were exposed to cyclosporine for 72 h to measure changes in transepithelial electrical resistance (ΔTER). Results: Cyclosporine treatment induced a decrease in Na+-Cl– cotransporter in rat renal cortex. WNK4 protein was increased in both rat kidneys and MDCK I cells. Occludin was also increased in rat kidneys and MDCK I cells exposed to 100 ng/ml cyclosporine. In contrast, cyclosporine treatment induced a decrease in zonula occludens 1 protein abundance and no changes in claudin-1 and claudin-4 in both rat kidneys and MDCK I cells. As a measure of the barrier to small ions, ΔTER of MDCK monolayers was decreased by 100 ng/ml cyclosporine. Conclusion: Renal TJ proteins are affected by cyclosporine treatment. Changes in TJ protein assembly induced by altered expression of WNK4, occludin, and zonula occludens 1 may affect paracellular permeability.

The role of proximal nephron in cyclophosphamide-induced water retention: preliminary data.

Cyclophosphamide is clinically useful in treating malignancy and rheumatologic disease, but has limitations in that it induces hyponatremia. The mechanisms by which cyclophosphamide induces water retention in the kidney have yet to be identified. This study was undertaken to test the hypothesis that cyclophosphamide may produce water retention via the proximal nephron, where aquaporin-1 (AQP1) and aquaporin-7 (AQP7) water channels participate in water absorption. To test this hypothesis, we gave a single dose of intraperitoneal cyclophosphamide to male Sprague-Dawley rats and treated rabbit proximal tubule cells (PTCs) with 4-hydroperoxycyclophosphamide (4-HC), an active metabolite of cyclophosphamide. In the short-term 3-day rat study, AQP1 protein expression was significantly increased in the whole kidney homogenates by cyclophosphamide administration at 48 (614 ± 194%, P < 0.005), and 96 (460 ± 46%, P < 0.05) mg/kg BW compared with vehicle-treated controls. Plasma sodium concentration was significantly decreased (143 ± 1 vs. 146 ± 1 mEq/L, P < 0.05) by cyclophosphamide 100 mg/kg BW in the long-term 6-day rat study. When primary cultured rabbit PTCs were treated with 4-HC for 24 hours, the protein expressions of AQP1 and AQP7 were increased in a dose-dependent manner. Quantitative polymerase chain reaction revealed no significant changes in the mRNA levels of AQP1 and AQP7 from cyclophosphamide-treated rat renal cortices. From these preliminary data, we conclude that the proximal nephron may be involved in cyclophosphamide-induced water retention via AQP1 and AQP7 water channels. Further studies are required to demonstrate intracellular mechanisms that affect the expression of AQP proteins.

Effects of dietary salt restriction on renal progression and interstitial fibrosis in adriamycin nephrosis.

Background/Aims: Although high salt intake is thought to accelerate renal progression in proteinuric kidney disease, it is not known whether strict dietary salt restriction could delay renal inflammation and interstitial fibrosis. Here, we sought to answer this question in a rat model of adriamycin-induced nephrotic syndrome. Methods: Adriamycin was administered via the femoral vein in a single bolus (7.5 mg/kg), and the rats were put on a sodium-deficient rodent diet. Rats with intact kidneys were studied for 5 weeks (experiment 1), and uninephrectomized rats were studied for 6 weeks (experiment 2). Results: In experiment 1, restricting salt intake improved renal tubulointerstitial histopathology in adriamycin-treated rats. Immunohistochemical and immunoblot results additionally showed that restricting dietary salt lowered adriamycin-induced expression of osteopontin, collagen III, and fibronectin. In experiment 2, salt restriction improved adriamycin-induced azotemia, although it did not affect proteinuria or blood pressure. Dietary salt restriction also reduced adriamycin-induced infiltration of ED1-positive cells and the upregulated expression of osteopontin and a-SMA. Massons trichrome and Sirius red staining revealed that salt restriction slowed Adriamycin-induced progression of renal interstitial fibrosis. Finally, qPCR revealed that adriamycin-induced expression of TNF-a, IκB-a, gp91phox, p47phox, and p67phox mRNA was blocked by salt restriction. Conclusion: Our findings demonstrate that strict dietary salt restriction delays the progress of renal inflammation and fibrosis in proteinuric kidney disease, most likely via relieving the reactive oxygen species-mediated NF-κB activation.

Acute and chronic effects of dietary sodium restriction on renal tubulointerstitial fibrosis in cisplatin-treated rats

BACKGROUND Renal interstitial fibrosis is a major complication of cisplatin (CP) treatment, and increased sodium intake may accelerate its progression by stimulating transforming growth factor (TGF)-β/Smad signaling. However, it is not clear whether a low-sodium diet has beneficial effects on the development of interstitial fibrosis because it activates the renin-angiotensin-aldosterone system. Here, we tested whether the TGF-β/Smad signaling pathway is stimulated in CP-treated rats, and whether the development of tubulointerstitial fibrosis in CP nephropathy can be checked by dietary sodium restriction. METHODS Male Sprague Dawley rats were randomly divided into controls, CP treatment and CP treatment with low-sodium diet. The acute experiment lasted 7 days with a single intraperitoneal injection (6 mg/kg) of CP, and the chronic experiment involved weekly injections (2 mg/kg) for 7 weeks. RESULTS In both sets of experiments, CP treatment produced pronounced tubulointerstitial injury, increased infiltration of ED1-positive cells and increased expression of monocyte chemotactic protein-1 (MCP-1), α-smooth muscle actin (SMA), TGF-β1, phosphorylated Smad3, fibronectin and collagen III proteins. In the acute experiment, the increases in expression of osteopontin, MCP-1, α-SMA, TGF-β and collagen III were significantly reduced by dietary sodium restriction. In the chronic experiment, however, none of the measurements were improved by a low-sodium diet. Examination of CP-treated rat kidneys revealed de novo vimentin expression in tubular epithelial cells and invasion of α-SMA-positive tubular epithelial cells through the basement membrane into the interstitium. CONCLUSIONS The pro-fibrotic effect of TGF-β in CP nephropathy appears to be associated with the epithelial-mesenchymal transition and is ameliorated by dietary sodium restriction only during the acute phase.

Cyclophosphamide-induced vasopressin-independent activation of aquaporin-2 in the rat kidney.

Because cyclophosphamide-induced hyponatremia was reported to occur without changes in plasma vasopressin in a patient with central diabetes insipidus, we hypothesized that cyclophosphamide or its active metabolite, 4-hydroperoxycyclophosphamide (4-HC), may directly dysregulate the expression of water channels or sodium transporters in the kidney. To investigate whether intrarenal mechanisms for urinary concentration are activated in vivo and in vitro by treatment with cyclophosphamide and 4-HC, respectively, we used water-loaded male Sprague-Dawley rats, primary cultured inner medullary collecting duct (IMCD) cells, and IMCD suspensions prepared from male Sprague-Dawley rats. In cyclophosphamide-treated rats, significant increases in renal expression of aquaporin-2 (AQP2) and Na-K-2Cl cotransporter type 2 (NKCC2) were shown by immunoblot analysis and immunohistochemistry. Apical translocation of AQP2 was also demonstrated by quantitative immunocytochemistry. In both rat kidney and primary cultured IMCD cells, significant increases in AQP2 and vasopressin receptor type 2 (V2R) mRNA expression were demonstrated by real-time quantitative PCR analysis. Confocal laser-scanning microscopy revealed that apical translocation of AQP2 was remarkably increased when primary cultured IMCD cells were treated with 4-HC in the absence of vasopressin stimulation. Moreover, AQP2 upregulation and cAMP accumulation in response to 4-HC were significantly reduced by tolvaptan cotreatment in primary cultured IMCD cells and IMCD suspensions, respectively. We demonstrated that, in the rat kidney, cyclophosphamide may activate V2R and induce upregulation of AQP2 in the absence of vasopressin stimulation, suggesting the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis (NSIAD).

Effects of Dietary Salt Restriction on Puromycin Aminonucleoside Nephrosis: Preliminary Data

Proteinuria is a major promoter that induces tubulointerstitial injury in glomerulopathy. Dietary salt restriction may reduce proteinuria, although the mechanism is not clear. We investigated the effects of dietary salt restriction on rat kidneys in an animal model of glomerular proteinuria. Male Sprague-Dawley rats were used and divided into 3 groups: vehicle-treated normal-salt controls, puromycin aminonucleoside (PA)-treated normal-salt rats, and PA-treated low-salt rats. PA was given at a dose of 150 mg/kg BW at time 0, followed by 50 mg/kg BW on days 28, 35, and 42. Sodium-deficient rodent diet with and without additional NaCl (0.5%) were provided for normal-salt rats and low-salt rats, respectively. On day 63, kidneys were harvested for histopathologic examination and immunohistochemistry. PA treatment produced overt proteinuria and renal damage. Dietary salt restriction insignificantly reduced proteinuria in PA-treated rats, and PA-treated low-salt rats had lower urine output and lower creatinine clearance than vehicle-treated normal-salt controls. When tubulointerstitial injury was semiquantitatively evaluated, it had a positive correlation with proteinuria. The tubulointerstitial injury score was significantly increased by PA treatment and relieved by low-salt diet. ED1-positive infiltrating cells and immunostaining for interstitial collagen III were significantly increased by PA treatment. These changes appeared to be less common in PA-treated low-salt rats, although the differences in PA-treated normal-salt versus low-salt rats did not reach statistical significance. Our results suggest that renal histopathology in PA nephrosis may potentially be improved by dietary salt restriction. Non-hemodynamic mechanisms induced by low-sodium diet might contribute to renoprotection.

Effects of Increased Uric Acid Intake on the Abundance of Urate-anion exchanger and Organic Anion Transporter Proteins in the Rat Kidney

Renal handling of uric acid mainly occurs in the proximal tubule, and bidirectional transport of urate may involve apical absorption via the urate-anion exchanger (URAT1) and basolateral uptake via organic anion transporters (OAT1 and OAT3). In rat kidneys, we investigated whether the protein abundance of URAT1, OAT1, and OAT3 is affected by the increase in uric acid intake. Male Sprague-Dawley rats were randomly divided into control and uric acid-supplemented groups, and uric acid-supplemented rats were given 0.75 g of uric acid per 180 g body weight per day for 8 days. After the animal experiment, kidneys were harvested and semi-quantitative immunoblotting was carried out from cortical homogenates using polyclonal peptide-derived antibodies to URAT1, OAT1, and OAT3. Serum uric acid level showed an increasing tendency in the uric acid-supplemented rats compared with control rats, whereas urinary uric acid excretion was not significantly different between the uric acid-supplemented rats and control rats. URAT1 protein abundance in cortical homogenates was not significantly different between the uric acid-supplemented rats and control rats. However, OAT1 protein abundance was significantly increased in the uric acid-supplemented rats compared with the control rats. OAT3 protein abundance was not significantly different between the uric acid-supplemented rats and control rats. In conclusion, OAT1 may have a regulatory role in response to the increase in uric acid intake in the rat kidney. The up-regulation of OAT1 would exert stimulation of urinary uric acid excretion and might contribute to protection from hyperuricemia.

Anti-Inflammatory Action of Sitagliptin and Linagliptin in Doxorubicin Nephropathy

Background/Aims: Dipeptidyl peptidase-4 (DPP4) inhibitors are known to have a protective effect on diabetic kidney disease, possibly via reduction of oxidative stress and inflammation in the kidney. However, whether these potential mechanisms play a role in non-diabetic proteinuric kidney diseases is not clear. Methods: Two different animal experiments were carried out using sitagliptin and linagliptin for DPP4 inhibition. In each experiment, male Sprague-Dawley rats were uninephrectomized and randomly divided into vehicle-treated and doxorubicin-treated rats, with or without DPP4 inhibition. Administration of a DPP4 inhibitor was performed daily by oral gavage over six weeks. Results: A single intravenous injection of doxorubicin resulted in hypertension and remarkable proteinuria. Linagliptin, but not sitagliptin, lowered systolic blood pressure in rats with doxorubicin nephropathy. By contrast, sitagliptin ameliorated tubulointerstitial injury, inflammatory cell infiltration, and interstitial fibrosis in rat kidneys with doxorubicin nephropathy. Quantitative polymerase chain reaction analysis revealed that mRNA expression of NLRP3, caspase-1, ASC, and IL-1β was remarkably increased in rat kidneys with doxorubicin nephropathy, and that this upregulation of the major components of the NLRP3 inflammasome was effectively suppressed by treatment with either sitagliptin or linagliptin. Additionally, upregulation of IL-6 was reversed by linagliptin, but not by sitagliptin. On the other hand, sitagliptin, but not linagliptin, reversed the increase in mRNA expression of gp91phox, p47phox, and p67phox in rat kidneys with doxorubicin nephropathy. Conclusion: NLRP3 inflammasome activation was shown in our rat model of doxorubicin nephropathy. DPP4 inhibitors can suppress the activity of NLRP3, with or without relieving NADPH oxidase 2-related oxidative stress.

Effect of mycophenolic acid on cyclosporin A-induced fibronectin expression in rat mesangial cells.

This study was undertaken to determine if mycophenolic acid (MPA) inhibits the profibrotic action of cyclosporin A (CsA) and, if so, to determine the molecular mechanisms involved. The effect of MPA treatment on CsA-induced signaling through the transforming growth factor-β (TGF-β)/Smad pathway was evaluated by immunoblot analysis in cultured primary rat mesangial cells. Treatment of cells with 1 µmol/l MPA did not significantly decrease the CsA-induced expression of TGF-β1, but partially reversed the increases in Smad3 phosphorylation and fibronectin (FBN) production, and increased Smad7 expression. These results suggest that MPA may ameliorate CsA-induced FBN production by modulating the Smad signaling pathway. This study provides evidence that MPA can attenuate CsA-induced renal injury after kidney transplantation.

Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells

Background Claudin-2, ZO-1, and occludin are major components of tight junctions (TJs) in the proximal tubule. However, their roles in maintaining paracellular permeability as leaky epithelia have yet to be defined. Methods To investigate the contributory role of TJ proteins in the leaky proximal tubule, we xamined the effect of inhibiting claudin-2, occludin, and ZO-1 expression on transepithelial electrical resistance (TER) and paracellular permeability using the immortalized human proximal tubule epithelial cell line HK-2. For this, small-interfering RNAs (siRNAs) against claudin-2, occludin and ZO-1 were transfected into HK-2 cells. TER and transepithelial flux rates of dextrans (4 and 70 kDa) were determined after 24 h. Results Transfection of siRNAs (25 nM) knocked down TJ protein expression. Control HK-2 monolayers achieved a steady-state TER of 6–8 Ω·cm2 when grown in 12-well Transwell filters, which are compatible with leaky epithelia. Knockdown of claudin-2 decreased in TER and increased occludin expression. Transfection with siRNA against either occludin or ZO-1 increased TER and decreased claudin-2 expression. TER was decreased by co-inhibition of claudin-2 and ZO-1 but increased by co-inhibition of claudin-2 and occludin. TER was suppressed when claudin-2, occludin, and ZO-1 were all inhibited. Dextran flux rate was increased by claudin-2, occludin, or ZO-1 siRNA transfection. Increased dextran flux was enhanced by co-transfection of claudin-2, ZO-1, and occludin siRNA. Conclusions The depletion of claudin-2, occludin and ZO-1 in HK-2 cells had differential effects on TER and macromolecule flux. We demonstrated that integration of claudin-2, occludin and ZO-1 is necessary for maintaining the function of the proximal tubular epithelium.