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Renal venous congestion by increased central venous pressure in congestive heart failure is responsible for renal dysfunction. We have created a novel rat renal congestion model presenting increased renal interstitial hydrostatic pressure and vasa recta expansion, which leads to renal congestion-mediated fibrosis. Current clinical trials suggest the renoprotective effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors in heart failure patients; however, the underlying mechanisms are still discussing. Thus, we investigated the effect of phlorizin, a commercially available SGLT1/2 inhibitor, in our renal congestion model. All animal procedures were performed in accordance with the policies and guidelines of the “Position of the American Heart Association on Research Animal Use” and were approved by the Tohoku Medical and Pharmaceutical University Animal Experiment Committee (registration number: A18019-a, A19039-cn and A20005-cn). The inferior vena cava between the renal veins was ligated, which induced congestion only in the left kidney. Phlorizin (400 mg/kg body weight) or saline were injected subcutaneously daily from 1 day before the operation. Both the right control kidney and left congested kidney were collected 3 days after the surgery. Increased kidney weight and renal fibrosis were observed in the congested kidneys. Phlorizin attenuated the increased kidney weight and suppressed renal fibrosis staining. Molecules related to kidney injury (Kim1) and fibrosis (Fn1 and αSma) were also suppressed by Phlorizin in the congested kidney. Low-vacuum electron microscopy revealed the vasa recta dilatation in the congested kidney. This morphological change was alleviated by Phlorizin. These results suggest that SGLT1/2 inhibitor could suppress renal congestion-mediated fibrosis. This mechanism could be a potential therapeutic target for renoprotection against heart failure.

Increased central venous pressure in congestive heart failure is responsible for renal dysfunction; however, the underlying mechanisms are unclear. We hypothesized that renal interstitial hydrostatic pressure (RIHP) and expansion pressures of the vasa recta are responsible for pericyte detachment resulting renal congestion-mediated fibrosis. We created a novel rat renal congestion model and investigated the effect of renal congestion on hemodynamics and its molecular mechanisms. The inferior vena cava (IVC) between the renal veins was ligated by suture in male Sprague-Dawley rats to increase upstream IVC pressure and induce congestion in the left kidney only. Left kidney congestion reduced renal blood flow in cortex (33.6%, 28.3 to 16.0 mL/min) and in medulla (41.8%, 11.9 to 6.9 mL/min) and glomerular filtration rate (1.16 to 0.20 mL/min/kg BW), and increased RIHP (12.6 to 17.6 mm Hg). Hypoxia was observed in the medullary thick ascending limb of Henle, only in the congestive kidneys. Tubulointerstitial injury, podocyte injury, albuminuria, and reduced creatinine clearance were observed in the congestive kidneys. Molecules related to extracellular matrix expansion, tubular injury, and focal adhesion were upregulated in microarray analysis. Renal decapsulation ameliorated the tubulointerstitial injury (mRNA expression of Kim1 15.3 to 4.3 A.U., αSma 5.3 to 2.8 A.U.). Electron microscopy captured pericyte detachment in the congestive kidneys. Transgelin and platelet-derived growth factor receptors (PDGFRs), as indicators of pericyte-myofibroblast transition, were upregulated in the pericytes and the adjacent interstitium. Imatinib, a PDGFR inhibitor, ameliorated the interstitial injury. Our results reveal a novel mechanism of worsening renal function associated with congestive heart failure, and provide a new therapeutic strategy based on a better understanding of the pericyte-myofibroblast transition.

Background: Due to the functional linkage between cardiac function and renal function, a decline in renal function exacerbates cardiac function and a decline in cardiac function exacerbates renal function. Renal congestion is reported to contribute to the decline of renal function in heart failure patients. Regarding the mechanism of kidney failure caused by congestion, although it is suggested that ischemia is involved due to an increase in venous pressure and a decrease in blood flow, it has not been sufficiently elucidated. Since overexpression of glyoxylose-1 (GLO1), a methylglyoxal (MG) metabolizing enzyme, which is a type of carbonyl stress, has been reported to attenuate impairment due to renal ischemia reperfusion (I/R). In this study, we aimed to investigate the effect of carbonyl stress on kidney damage due to renal congestion using GLO1 overexpressing rats. Methods: Male Wistar wild type (WT) rat and GLO1 transgenic (GLO1) rat were used for experiment. Left kidney was congested by placing silver clip on left renal vein under anesthesia. Two weeks after surgery, renal cortical and outer medullary mRNA markers of fibrosis [transforming growth factor-β (TGF-β) and fibronectin (FN)], tubular injury [osteopontin (OPN) and kidney injury molecule 1 (KIM-1)] and inflammations [monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α)] were measured. Results; Relative mRNA expression of cortical TGF-β (contralateral kidney; 1.00 + 0.09, congested kidney; 2.44 + 0.33, respectively), FN (1.00 + 0.11, 2.71 + 0.46), OPN (1.00 + 0.18, 6.41 + 1.47), KIM-1 (1.00 + 0.59, 21.20 + 6.31), MCP-1 (1.00 + 0.24, 2.93 + 0.56) and TNF-α (1.00 + 0.14, 1.62 + 0.18) were significantly increased in congested kidney compared to contralateral kidney. Relative mRNA expression of cortical TGF-β (GLO1tg; 1.69 + 0.20, WT; 2.44 + 0.33, respectively), FN (1.53 + 0.33, 2.71 + 0.46), TNF-α (1.00 + 0.14, 1.62 + 0.18) in GLO1tg rat were significantly decreased compared to WT rat. No significant change was observed between renal medullary area of WT rat and GLO1tg rat. Conclusion: Renal congestion induces renal fibrosis, tubular injury and inflammation, and carbonyl stress which could contribute to the pathophysiological mechanism on renal injury in cardiac failure.

We recently reported that high salt (HS) intake increased the (pro)renin receptor ((P)RR) expression by 3-5 fold in several nephron segments of Sprague-Dawley rats (Peptides 63: 156-162, 2015). The preset study examined the effects of HS intake on the renal (P)RR expression in Dahl-Salt sensitive (DS) rats. Male DS rats were fed a normal salt (NS) diet (0.6%NaCl) and a HS diet (8%NaCl) for 4weeks. A part of the rats fed the HS diet were treated orally with angiotensin II type 1 receptor (AT 1 R) antagonist, candesartan (Can,3mg/kg/day) or mineralocorticoid receptor (MR) antagonist, spironolactone (Spi, 100mg/kg/day). The (P)RR expression in nephron segments was examined by immunoblot and immunohistochemical analyses. HS intake increased the blood pressure, which did not significantly affected by Can or Spi. (P)RR was expressed in the all kidney sections, glomeruli, proximal tubules (PT), medullary thick ascending limbs and inner medullary collecting ducts. HS intake increased the (P)RR expression in the cortex by 22.6 fold (p1 R and MR-dependent manner in the distal tubules, but AT 1 R or MR-independent manner in the PT.

Studies were designed to determine the effects of increases of renal perfusion pressure on the production of hydrogen peroxide (H 2 O 2 ) and NO 2 − +NO 3 − within the renal outer medulla. Sprague-Dawley rats were studied with either the renal capsule intact or removed to ascertain the contribution of changes of medullary blood flow and renal interstitial hydrostatic pressure on H 2 O 2 and NO 2 − +NO 3 − production. Responses to three 30-minute step changes of renal perfusion pressure (from ≈85 to ≈115 to ≈145 mm Hg) were studied using adjustable aortic occluders proximal and distal to the left renal artery. Medullary interstitial H 2 O 2 determined by microdialysis increased at each level of renal perfusion pressure from 640 to 874 to 1593 nmol/L, as did H 2 O 2 urinary excretion rates, and these responses were significantly attenuated by decapsulation. Medullary interstitial NO 2 − +NO 3 − increased from 9.2 to 13.8 to 16.1 μmol/L, with parallel changes in urine NO 2 − +NO 3 − , but decapsulation did not significantly blunt these responses. Over the range of renal perfusion pressure, medullary blood flow (laser-Doppler flowmetry) rose ≈30% and renal interstitial hydrostatic pressure rose from 7.8 to 19.7 cm H 2 O. Renal interstitial hydrostatic pressure and the natriuretic and diuretic responses were significantly attenuated with decapsulation, but medullary blood flow was not affected. The data indicate that pressure-induced increases of H 2 O 2 emanated largely from increased tubular flow rates to the medullary thick-ascending limbs of Henle and NO largely from increased medullary blood flow to the vasa recta. The parallel pressure-induced increases of H 2 O 2 and NO indicate a participation in shaping the “normal” pressure-natriuresis relationship and explain why an imbalance in either would affect the blood pressure salt sensitivity.

Studies were conducted to determine whether the diffusion of NO from the renal medullary thick ascending limb (mTAL) to the contractile pericytes of surrounding vasa recta was reduced and, conversely, whether diffusion of oxygen free radicals was enhanced in the salt-sensitive Dahl S rat (SS/Mcwi). Angiotensin II ([Ang II] 1 μmol/L)–stimulated NO and superoxide (O 2 ·− ) production were imaged by fluorescence microscopy in thin tissue strips from the inner stripe of the outer medulla. In prehypertensive SS/Mcwi rats and a genetically designed salt-resistant control strain (consomic SS-13 BN ), Ang II failed to increase either NO or O 2 ·− in pericytes of isolated vasa recta. Ang II stimulation resulted in production of NO in epithelial cells of the mTAL that diffused to vasa recta pericytes of SS-13 BN rats but not in SS/Mcwi rats except when tissues were preincubated with the superoxide scavenger TIRON (1 mmol/L). Ang II resulted in a greater increase of O 2 ·− in the mTAL of SS/Mcwi compared with SS.13 BN mTAL. The O 2 ·− diffused to adjoining pericytes in tissue strips only in SS/Mcwi rats but not in control SS-13 BN rats. Diffusion of Ang II-stimulated O 2 ·− from mTAL to vasa recta pericytes was absent when tissue strips from SS/Mcwi rats were treated with the NO donor DETA-NONOate (20 μmol/L). We conclude that the SS/Mcwi rat exhibits increased production of O 2 ·− in mTAL that diffuses to surrounding vasa recta and attenuates NO cross-talk. Diffusion of O 2 ·− from mTAL to surrounding tissue could contribute to reduced bioavailability of NO, reductions of medullary blood flow, and interstitial fibrosis in the outer medulla of SS/Mcwi rats.

Objective: V2 receptor (V2R) antagonist increases aquaresis, and was reported to have renoprotective and natriuretic effect, although the mechanism is not fully clarified. Renal medullary hemodynamics contributes sodium retention and renal injury. Therefore, the present study was designed to evaluate the effect of V2R antagonist on renal medullary blood flow. Methods: Catheter was inserted in femoral artery and vein of anesthetized SD rats to monitor blood pressure (BP), heart rate (HR) and to infuse drugs, respectively. Renal medullary blood flow (MBF) and renal medullary oxygen pressure (pO2) were measured with laser-Doppler flowmetry or oxygen microelectrode, respectively. V2R antagonist, OPC-31260 (OPC, 0.25mg/kg bw/h) or furosemide (Furo, 0.5mg/kg bw/h) was intravenously administrated for 90min. Urine was collected in 30 min interval and urinary sodium (UNaV), hydrogen peroxide (UH2O2V) and [nitrate + nitrite] (UNOxV) excretion were measured. Results: OPC and Furo treatment did not change BP and HR. Urine volume was significantly increased by OPC (1.1+0.2 to 6.1+0.5 g/30 min) and Furo (1.4+0.6 to 4.7+0.3 g/30 min) treatment but was not different between groups. MBF was significantly decreased in Furo (12+4% decrease from baseline), while OPC did not changed MBF (1+3% increase from baseline). pO2 was significantly increased by both OPC and Furo treatment (20+6 and 27+10% increase from baseline, respectively). UNaV was significantly increased in OPC (0.10+0.02 to 0.44+0.05 mEq/30 min) and Furo (0.14+0.08 to 0.69+0.06 mEq/30 min) treatment, the increase of UNaV was significantly higher in Furo than OPC group. UH2O2V was significantly increased by Furo treatment (16+4 to 28+6 nmol/30 min), while did not change in OPC treatment (10+2 to 19+4 nmol/30 min). UNOx was significantly increased in OPC treatment (211+30 to 376+45 nmol/30 min), while did not change in Furo treatment (142+27 to 237+75 nmol/30 min). Conclusion: OPC treatment increased NO production. Increased NO could contribute to decrease of sodium reabsorption, result in increase of renal medullary pO2. This scheme could be one on the mechanisms of renal protective effect by V2R antagonist treatment.

We tested the hypothesis that blockade of angiotensin II type 1 receptors reduces oxidative stress markers in parallel with urinary albumin and type IV collagen excretions. Sixty-six diabetic patients with nephropathy were randomly assigned to either the angiotensin II receptor blocker (ARB; n=33) or trichlormethiazide (n=33) group. The majority of patients had been treated with angiotensin-converting enzyme inhibitors or calcium channel blockers for ≥1 year before the present study. Reduction of blood pressure was not different between the 2 groups, and HbA1c levels did not change over the study period (8 weeks). Treatment with ARB (candesartan 8 mg/day, n=11 or valsartan 80 mg/day, n=22) for 8 weeks reduced the levels of plasma monocyte chemoattractant protein 1, interleukin 6, urinary 8-epi-prostaglandin F2α, 8-hydroxydeoxyguanosine, albumin, and type IV collagen, whereas the levels of these markers were not altered with trichlormethiazide (2 mg/day). Significant correlation was observed between the reduction of the urinary 8-epi- prostaglandin F2α and 8-hydroxydeoxyguanosine and those of the urinary albumin and type IV collagen. Subjects with large oxidative stress had large reduction rates because of ARB administration and showed large urinary albumin suppression. These results suggest that ARBs reduce oxidative stress and inflammation in diabetic patients independent of their effects on blood pressure. In addition, increases in oxidative stress caused by angiotensin II may play an important role in the progression of diabetic nephropathy. Our results may help to explain the clinical observation that ARB reduces urinary albumin excretion very efficiently in some patients but not in others.

Renal perfusion pressure was servo-controlled chronically in rats to quantify the relative contribution of elevated arterial pressure versus angiotensin II (Ang II) on the induction of renal injury in Ang II-induced hypertension. Sprague-Dawley rats fed a 4% salt diet were administered Ang II for 14 days (25 ng/kg per minute IV; saline only for sham rats), and the renal perfusion pressure to the left kidney was continuously servo-controlled to maintain a normal pressure in that kidney throughout the period of hypertension. An aortic occluder was implanted around the aorta between the two renal arteries and carotid and femoral arterial pressure were measured continuously throughout the experiment to determine uncontrolled and controlled renal perfusion pressure, respectively. Renal perfusion pressure of uncontrolled, controlled, and sham kidneys over the period of Ang II or saline infusion averaged 152.6+/-7.0, 117.4+/-3.5, and 110.7+/-2.2 mm Hg, respectively. The high-pressure uncontrolled kidneys exhibited tubular necrosis and interstitial fibrosis, especially prominent in the outer medullary region. Regional glomerular sclerosis and interlobular artery injury were also pronounced. Controlled kidneys were significantly protected from interlobular artery injury, juxtamedullary glomeruli injury, tubular necrosis, and interstitial fibrosis as determined by comparing the level of injury. Glomerular injury was not prevented in the outer cortex. Transforming growth factor (TGF)-beta and active NF-kappaB proteins determined by immunohistochemistry were colocalized in the uncontrolled kidney in regions of interstitial fibrosis. We conclude that the preferential juxtamedullary injury found in Ang II hypertension is largely induced by pressure and is probably mediated through the TGF-beta and NF-kappaB pathway.

The effects of NaCl, glucose, and thyroid hormone on the production of superoxide (O 2 ·− ) within the renal medulla of Sprague-Dawley rats were examined. Responses of intracellular superoxide [O 2 ·− ] i in isolated medullary thick ascending limbs (mTALs) were studied using real-time fluorescent microscopy with measurement of the dehydroethidium (DHE) to ethidium (Eth) conversion ratio (Eth/DHE ratio unit). The results demonstrated that elevations of extracellular NaCl (from 152 to 252 mmol/L), d -glucose (from 5 to 25 mmol/L), and triiodo-thyronine (T3; 10 μmol/L) significantly increased [O 2 ·− ] i levels. Preincubation with superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (1 mmol/L) significantly inhibited these responses. Stimulation with equamolar amounts of choline chloride or l -glucose failed to increase [O 2 ·− ] i , indicating that these O 2 ·− responses were not determined by changes in osmolality. The responses to NaCl, d -glucose, and T3 were abolished by pretreatment with the Na + /K + -ATPase pump inhibitor ouabain (4 mmol/L) and with Na + /H + -exchanger inhibitor dimethylamiloride (100 μmol/L). We conclude that elevations of extracellular NaCl, d -glucose, or T3 levels can activate both the Na + /K + -ATPase pump and Na + /H + exchanger in mTAL, which, in turn, is associated with increased intracellular concentrations of superoxide.

The present study was designed to determine whether arginine vasopressin (AVP) stimulates NO production in the epithelial collecting duct cells of the inner medulla (IMCDs) and if this is mediated through Ca 2+ signaling. Thin tissue layers containing IMCDs were dissected from Sprague-Dawley rats. Intracellular Ca 2+ concentration ([Ca 2+ ] i ) and NO production were measured in IMCDs by a fluorescence imaging system with the use of fura 2-AM and the cell-permeable form of the NO-sensitive dye 4,5-diaminofluorescein (DAF-2), respectively. AVP (100 nmol/L) produced a rapid peak increase in [Ca 2+ ] i of 320±70 nmol/L within a few seconds and a sustained increase of 120±62 nmol/L. The peak increase in [Ca 2+ ] i was followed by a significant increase of NO production (34±7 U). This was similar to that produced by 20 μmol/L of the NO donor DETA-NONOate (42±11 U). The NO scavenger carboxy-PTIO (100 μmol/L) or depletion of [Ca 2+ ] i by preincubation with 5 μmol/L of the Ca 2+ -ATPase inhibitor thapsigargin in Ca 2+ -free buffer abolished the NO response to AVP. We conclude that AVP mobilizes Ca 2+ to produce NO in IMCDs.

Dahl salt-sensitive (SS) rats fed a high salt diet exhibit increased blood pressure and renal damage. Epithelial sodium channels (ENaC) in the aldosterone-sensitive distal nephron (ASDN) are responsible for the fine tuning of Na + absorption in the kidney and are regulated by various stimuli including hormones and growth factors. Members of the epidermal growth factor (EGF) family are important for maintaining transepithelial Na + transport. A combination of electrophysiological, immunohistochemical, biochemical in vitro and chronic in vivo studies were used to provide mechanistic insights into ENaC regulation by EGF and how changes in this pathway may contribute to salt-induced hypertension in SS rats. It was found that the ENaC inhibitor benzamil, administered through the drinking water (15 mg/L), significantly reduced the MAP from 173.3±15.0 to 127.0±10.1 mm Hg. ENaC expression (as measured by Western blotting and immunohistochemistry) and activity (single channel analysis in freshly isolated split open ASDN) were significantly enhanced in SS rats fed a high salt diet compared to SS rats fed a low salt diet and consomic SS.13 BN rats fed a high salt chow. Servo-controlled measurements in SS rats also revealed increased ENaC expression in the uncontrolled kidneys compared to controlled left kidneys. As assessed by ELISA assay, EGF level in the kidney cortex of SS rats fed a high salt diet was lower compared to rats on a low salt diet. To directly evaluate the role of EGF in the development of hypertension and its effect on ENaC activity, EGF (10 μg/h/kg) was intravenously infused and blood pressure was continuously monitored. Chronic infusion of EGF decreased ENaC activity ( NP o ; from 1.5±0.2 to 0.4±0.1), prevented the development of hypertension (from 184.0±8.9 to 149.0±9.6 mm Hg) and attenuated renal glomerular and tubular damage. Thus, in controversy to existing dogma, we provide an evidence of a critical role of ENaC in the ASDN in the development of salt-sensitive hypertension. Furthermore, these findings indicate that EGF deficiency in the renal cortex contributes to salt-sensitive hypertension and that in vivo EGF infusion prevents development of hypertension and kidney damage via downregulation of ENaC.