Chor Ho Jo

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.

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.

EFFECTS OF EMPAGLIFLOZIN ON SALT-SENSITIVE HYPERTENSION AND RENAL INFLAMMATION IN RAT

Objective: Renal inflammation may have a role in salt-sensitive hypertension. Although sodium-glucose cotransporter-2 (SGLT2) inhibitors were reported to exert blood pressure lowering in type 2 diabetes mellitus, whether they have a role in non-diabetic kidney diseases is not clear. This study was undertaken to investigate whether salt-sensitive hypertension and its accompanying renal inflammation are ameliorated by SGLT2 inhibition. Design and method: The animal model of salt-sensitive hypertension was established by salt loading in uninephrectomized rats. Male Sprague-Dawley rats were randomly divided into 3 groups: sham controls (SC, n = 4), uninephrectomized controls (UC, n = 4), and empagliflozin-treated rats (ET, n = 5). All rats were fed a rodent diet with 8% NaCl throughout the study period. Empagliflozin (20 mg/kg/d) was orally administered for 3 weeks after uninephrectomized rats were stabilized over 2 weeks. Systolic blood pressures (SBPs) were weekly measured, and kidneys were harvested for qPCR at the end of animal experiment. Results: At baseline, SBPs were 122 ± 4, 127 ± 1, and 125 ± 3 mmHg in SC, UC, and ET, respectively. At the end of animal experiment, SBP in UC was higher than that in SC (167 ± 4 vs. 137 ± 6 mmHg, P < 0.01). However, ET had a lower SBP (146 ± 3 mmHg) compared with UC (P < 0.05). As expected, urinary glucose excretion was remarkable in ET (2.61 ± 0.59 mmol/d/100 g BW versus 0 in controls). Whereas natriuresis was not different between groups, urinary excretion of osmoles in ET (29.8 ± 3.6 mmol/d/100 g BW, P < 0.01) was higher than that in SC (20.6 ± 2.3 mmol/d/100 g BW) or UC (19.3 ± 1.4 mmol/d/100 g BW). Compared with SC, the mRNA expression level of IL-1&bgr; (268 ± 91%, P < 0.05), RANTES (167 ± 20%, P < 0.05), and gp91phox (300 ± 36%, P < 0.05) were increased in UC but ameliorated in ET (IL-1&bgr;, 159 ± 29%; RANTES, 87 ± 34%; gp91phox, 142 ± 74%, all P < 0.05). Conclusions: Empagliflozin was effective in controlling salt-sensitive hypertension induced by renal mass reduction, via glycosuria-driven osmotic diuresis rather than natriuresis. The upregulation of renal inflammation in salt-sensitive hypertension may be relieved by empagliflozin treatment.

Alteration of Tight Junction Protein Expression in Dahl Salt-Sensitive Rat Kidney

Background/Aims: Altered pressure natriuresis is an important mechanism of hypertension, but it remains elusive at the molecular level. We hypothesized that in the kidney, tight junctions (TJs) may have a role in pressure natriuresis because paracellular NaCl transport affects interstitial hydrostatic pressure. Methods: To assess the association of salt-sensitive hypertension with altered renal TJ protein expression, Dahl salt-sensitive (SS) and salt-resistant (SR) rats were put on an 8% NaCl-containing rodent diet for 4 weeks. Systolic blood pressure (SBP) and urine NaCl excretion were measured weekly, and kidneys were harvested for immunoblotting and quantitative PCR analysis at the end of the animal experiments. Results: SBP was significantly higher in SS rats than in SR rats during the first to fourth weeks of the animal experiments. During the first and second week, urinary NaCl excretion was significantly lower in SS rats as compared with SR rats. However, the difference between the two groups vanished at the third and fourth weeks. In the kidney, claudin-4 protein and mRNA were significantly increased in SS rats as compared with SR rats. On the other hand, occludin protein and mRNA were significantly decreased in SS rats as compared with SR rats. The expression of claudin-2, claudin-7, and claudin-8 did not vary significantly between the two groups. Conclusions: In SS rats, SS hypertension was associated with differential changes in renal TJ protein expression. Both upregulation of claudin-4 and downregulation of occludin might increase paracellular NaCl transport in the kidney, resulting in impaired pressure natriuresis in SS rats.

[BP.02.04] THE ANTIHYPERTENSIVE EFFECT OF EMPAGLIFLOZIN IN RATS WITH NON-DIABETIC NEPHROSIS

Objective: The effects of sodium-glucose cotransporter-2 (SGLT2) inhibiton on lowering blood pressure are well characterized in diabetic animals and humans. However, it is unclear whether SGLT2 inhibitors may have antihypertensive effects in non-diabetic kidney disease. This study was undertaken to investigate whether the hypertension in non-diabetic proteinuric kidney disease may be controlled by empagliflozin. Design and method: Male Sprague-Dawley rats were randomly divided into uni-nephrectomized controls (NX, n = 5), uni-nephrectomy plus doxorubicin-treated rats (NXD, n = 5), and uni-nephrectomy plus doxorubicin/empagliflozin-cotreated rats (NXDE, n = 5). Doxorubicin was administered via femoral vein in a single bolus (5 mg/kg) after 7 days of right nephrectomy. Empagliflozin (20 mg/kg/d) was daily given in food slurry. After 5 weeks of empagliflozin administration, kidneys were harvested for immunoblotting of sodium transporters. Results: At baseline (Day 0, immediately before doxorubicin treatment), systolic blood pressures were not different between group: NX, 120 ± 1; NXD, 120 ± 1; and NXDE, 119 ± 3 mmHg. From Day 7 (NX, 127 ± 1; NXD, 163 ± 3; and NXDE, 155 ± 1 mmHg) through Day 35 (NX, 130 ± 1; NXD, 172 ± 1; and NXDE, 160 ± 1 mmHg), remarkable hypertension was induced by doxorubicin and significantly relieved by empagliflozin cotreatment (P < 0.01). Significant proteinuria was produced from Day 14 and increased thereafter by doxorubicin but not decreased by empagliflozin cotreatment. Osmotic diuresis was evidently induced by empagliflozin administration from Day 7 (NXDE 40.7 ± 1.3 vs. NXD 21.7 ± 1.1 mosmoles/d, P < 0.01) through Day 35 (NXDE 52.3 ± 4.0 vs. NXD 27.9 ± 1.7 mosmoles/d, P < 0.01), in parallel with natriuresis and glycosuria. Immunoblot analysis from the kidney showed that compared with NXD, NXDE had decreased protein abundance of Na-K-2Cl cotransporter-2 (100 ± 31 vs. 40 ± 8%, P < 0.05) and Na-K-ATPase alpha1 subunit (100 ± 13 vs. 51 ± 13%, P < 0.05) but no change in Na/H exchanger-3. Conclusions: In our uni-nephrectomized rat model, doxorubicin-induced hypertension was significantly ameliorated by empagliflozin administration. This antihypertensive effect was associated with decreased expression of renal sodium transporters and resultant increased natriuresis, but not with proteinuria reduction.