No description available.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Acute kidney injury (AKI) is a major health burden associated with a 50% mortality rate. Of particular concern, the incidence of AKI has increased dramatically over the last decade. Yet, there is a paucity of available treatments to prevent AKI or to reduce the high rate of AKI-associated mortality. A common cause of AKI, especially in hospital settings, is prolonged decreases in renal blood flow (i.e., renal ischemia). Recent studies have demonstrated that activating the cholinergic anti-inflammatory pathway via vagal stimulation can mitigate AKI severity in rodent models of renal ischemia-reperfusion (IR) injury. While vagal stimulation is not a practical approach to prevent AKI in patients due its invasive nature and numerous side effects, recent studies have identified non-neuronal cholinergic cells within the kidney that could be targeted to reduce the severity of AKI. The overarching goal of this project is to examine the potential role of the renal cholinergic system in modulating the severity of and recovery from AKI in transgenic mice expressing green fluorescent protein (GFP) under control of the choline acetyl-transferase (ChAT) promoter, a protein involved in the synthesis of acetylcholine. The objectives of this study were to develop a clinically relevant model of renal IR-induced AKI in mice by identifying the duration of ischemia required for manifestation of the effects of AKI and to determine whether differences in susceptibility to AKI exists between male and female mice. Initially, male mice underwent 20 (n=3), 22 (n=3), or 25 (n=4) minutes of bilateral renal IR under isoflurane anesthesia with body temperature controlled at 37°C. Ischemia was achieved by careful placement of vascular clamps on the renal artery and vein supplying each kidney. The severity of AKI was determined by measuring serum creatinine (SCr) at 3 days post-AKI. Compared to SCr of mice that were 3 days post-sham AKI (SCr = 0.47 mg/dl, n=2), SCr of male mice from all three ischemia time categories was substantially elevated (SCr > 3 mg/dl, n=10). However, mortality associated with 22 and 25 minutes IR was striking (>90%) making studies of long-term AKI effects difficult. In contrast, 20 minutes IR resulted in AKI manifest by elevated SCr (3.43±0.7 mg/dl, n=3), widespread acute tubular necrosis and a clinically relevant mortality rate of 50%. Next, male (n=10) and female (n=5) mice were subjected to 20 minutes of IR. The mortality rate in male mice (n=10) was 50% (n=10) through 7 days post-AKI; however, all female mice survived. Additional studies showed that female mice had lower SCr 3 days post-AKI (0.63±0.1 mg/dl, n=2) with very modest levels of acute tubular necrosis as compared to the higher SCr (1.92±0.1 mg/dl, n=2) and extensive acute tubular necrosis observed in male mice. The differences observed in AKI severity and mortality rates suggest that female mice are protected against AKI as compared to male mice and future studies will explore the potential role of the renal cholinergic system in contributing to these sex differences in AKI.

No description available.

Renal hypertrophy occurs early in the natural history of human and experimental diabetes and may be a manifestation of the same pathophysiological process which ultimately results in diabetic nephropathy. The precise biological events which stimulate and regulate this growth process remain incompletely understood. We postulated that renal eicosanoids contribute to the development of renal hypertrophy in diabetes. We elected to test the effects of suppression of dienoic eicosanoid metabolism (arachidonic acid metabolism) on renal hypertrophy in diabetic rats by feeding fish oil. Diabetic rats fed fish oil had markedly reduced insulin requirements compared to control rats pair-fed a beef tallow-rich diet. The concentrations of prostaglandin E2, 6-keto-prostaglandin F1α, and thromboxane B2 were depressed in the renal cortex of diabetic rats fed fish oil. This alteration in eicosanoid metabolism was associated with a substantial enhancement of diabetic renal hypertrophy. These results indicate that dietary fish oil has profound effects on renal eicosanoid metabolism in experimental diabetes and that these autocoids may participate in the biological events which regulate diabetic renal hypertrophy.

Chronic arthritis adversely affects glomerular function and nonsteroidal anti-inflammatory drugs (NSAIDs) reduce electrolyte urinary excretion. In addition, both acute and chronic inflammations may alter clearance of drugs. We studied (a) the effects of inflammation on the renal function and pharmacokinetics of rofecoxib and meloxicam; (b) whether inflammation could exacerbate reduced electrolytes excretion changes observed with NSAIDs; and (c) the influence of inflammation on distribution of these drugs into the kidney. Single oral doses of rofecoxib (10 mg kg-1), meloxicam (3 mg kg -1) or placebo were administered to normal or pre-adjuvant arthritic rats. Blood and urine samples were collected for the measurement of plasma nitrite, BUN and creatinine. The urinary excretion of sodium and potassium was also determined. Nitrite, BUN and plasma creatinine were increased starting on day 9 in the groups with inflammation. Sodium and potassium excretion rates were not affected by inflammation. Meloxicam did not alter the electrolyte excretion in any of the groups. Rofecoxib significantly decreased sodium and potassium excretion in normal rats and potassium excretion in inflamed rats. Inflammation significantly increased plasma concentrations of rofecoxib, but not meloxicam. The ratios of the kidney:plasma concentrations were not significantly altered by inflammation following either drug. Inflammation altered kidney function, demonstrated by increases in BUN and plasma creatinine. However, it did not influence the urinary electrolytes excretion. Since we have observed similar patterns of the effect of NSAIDs on kidney under healthy and inflammatory conditions, one may conclude that inflammation does not exacerbate the adverse effect.

INTRODUCTION : Acute kidney injury (AKI) is a disorder that is defined by rising serum creatinine and reduced urine output. It occurs in approximately 1-7% of hospitalized patients and is a major predictor of morbidity and mortality. It increases the costs and duration of hospital stay. AKI has been extensively studied post cardiac surgery, but there has been little attention on AKI occurring after non cardiac surgery . There have been few studies on AKI from developing countries and a paucity of data of post non cardiac surgery AKI. OBJECTIVE : To identify which known risk factors for AKI are commonly encountered at Groote Schuur Hospital, to document 30 and 90 day mortality, length of hospital stay, recovery of renal function at 90 days and identify factors associated with outcome post non-cardiac surgery. DESIGN: Prospective observational study. SETTING: Surgical Wards and ICU. PARTICIPANTS: Patients with AKI post non-cardiac surgery admitted between July 2012 and July 2013, who were 18 years and above without underlying stage 5 chronic kidney disease. OUTCOME MEASURES: Mortality, identification of risk factors, length of hospital stay and recovery of renal function. RESULTS: Of 367 patients referred to renal unit with AKI, 60 patients met inclusion criteria. Patients had an average age of 52.8 years (standard deviation 16.6) and 70% (42/60) were male. 61.7% (37 /60) were Coloured, 20% (12/60) were White and 18.3% (11/60) were Black. These patients were exposed to the following risk factors: 80%(48/60) had emergency surgery, 66. 7%(40/60) had sepsis, 65%(39/60) had perioperative contrast exposure, 53.3%(32/60) had hypotension that required inotropic support in 50%(30/60). Mortality was 33.3% (20/60) at 30 days and 45% (27/60) at 90 days. Of the 33 patients who did not die, 81.8% (27 /33) recovered their renal function to normal baseline creatinine at 90 days. Of the 6 patients, whose renal function did not return to baseline, none required long term dialysis. Perioperative contrast exposure was associated with a longer median length of hospital stay compared to patients not exposed to contrast (21 vs 16 days respectively, p<0.05). Sepsis and age > 60 years was associated with poor recovery of renal function (p=0.005, p=0.01 respectively). No risk factor was identified to be associated with mortality. CONCLUSION: Risk factors for post non cardiac surgery AKI commonly encountered at Groote Schuur Hospital were emergency surgery, sepsis, hypotension, perioperative use of inotropes and perioperative contrast exposure. The latter was identified as a modifiable risk factor which significantly prolonged hospital stay. Sepsis and age > 60 years were associated with poorer recovery of renal function.

Yi-Shen-Hua-Shi (YSHS) granule is a Chinese patent drug for treating chronic glomerulonephritis (CGN). It was marketed in 2009. However, up to now, there is no report about the quality and pharmacological activities of this product. In this work，we evaluated the quality and anti-CGN effects of the drug. To evaluate the quality of the granule, a qualitative and quantitative HPLC-DAD analytical method was developed. For qualitative analysis, HPLC fingerprint of ten batches of YSHS granule was established. The fingerprints were analyzed using similarity evaluation, hierarchical cluster analysis (HCA), principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) based on 15 characteristic fingerprint peaks. Similarity values of 10-batche samples were all above 0.960, indicating a stable quality. Minor differences were observed among batches by HCA and PCA. For quantification analysis, contents of six constituents in the granule were simultaneously measured. To establish the chemical profile of the granule, a HPLC-Q-TOF- MS/MS method was developed. A total of 105 peaks were detected using HPLC-Q-TOF-MS/MS in the granule, of which, 99 were tentatively identified as terpenoids, flavonoids, coumarins, alkaloids, phenols and other types of compounds, and 15 were further validated with reference substances. HPLC fingerprint chromatogram establishment, quantification analysis of 6 constituents and compound identification should improve the quality control of YSHS granule. To study the pharmacological activities of the granule, we investigated its anti-CGN effects and TGFβ signaling-related mechanism of action. A CGN rat model was established by injection of cationization-bovine serum albumin (C-BSA) for five weeks. After C-BSA injection, drugs were intragastrically administered to the rats once daily for four weeks. Clinical signs were recorded daily. Urine and serum biochemical parameters were analyzed using respective kits. Protein levels were examined by Western blotting. Pathological changes of renal tissues were evaluated using HE and Masson's trichrome staining. No significant differences in body weights and clinical signs were found among normal, model and drug treatment groups. Proteinuria; albuminuria; increased urine volume; elevated creatinine, urea nitrogen, triglyceride levels and total cholesterol in serum; decreased serum total protein and albumin; as well as renal pathological damages and fibrosis were observed in CGN model rats. YSHS granule ameliorated all the abnormal behavioral and biochemical changes in the model rats. Mechanistic investigations revealed that YSHS granule down-regulated proteins levels of TGFβ1, phospho-Smad2/3 (Thr 8) and Smad4 in rat renal tissues. These findings indicate that the drug has anti-CGN effects in rats, and inhibiting TGFβ signaling contributes to the underlying mechanisms. In summary, our chemical analytical studies will help in improving the quality control of YSHS granule. Our bioactivity and mechanistic studies provide a pharmacological basis for the clinical use of the granule in treating CGN.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrodynamic delivery is a powerful tool that allows delivery of macromolecules to the kidney culminating in gene expression. This finding is important in the fight against kidney disease. Current therapy for kidney injury, specifically acute kidney injury, is lacking. Supportive care in the form of IV fluids and medications aimed at restoring Glomerular Filtration Rate (GFR) and urine output are currently used. However, even with these treatments, prognoses of patients diagnosed with this disease remains poor. We believe that hydrodynamic delivery provides a mechanism that can be used to reverse and prevent AKI. Hydrodynamic delivery following ischemic injuries leads to reductions in serum creatinine and infiltrating mononuclear cells, as well as increased renal blood flow and survival. These changes are due to reductions in vascular congestion and inflammation typically seen following injury. To determine the underlying mechanisms of gene delivery preventing AKI, we used candidate genes identified in a proteomic screen on kidneys that recovered from AKI. We selected Isocitrate Dehydrogenase II (IDH2) and Sulfotransferase 1C2 (SULT1C2) for study and found that delivery prior to injury prevents serum creatinine increase and reduces cell death. We found that gene delivery of IDH2 prevents a glycolytic shift typically seen following ischemic injuries. The mechanism underlying the prevention of this shift are seen in increased ATP stores and spare respiratory capacity allowing the cell to remain in an oxidative state. Additionally, we show that SULT1C2 post-translationally modifies the mitochondria membrane, increasing oxidative phosphorylation providing the cell with additional energy needed in times of oxidative stress. These candidate genes allow cells to remain in an oxidative state preventing the activation of cell death pathways typically activated following injury, thereby preserving normal kidney function.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13067号 / 論医博第2122号 / 新制||医||1019(附属図書館) / 33218 / (主査)教授 長船 健二, 教授 松田 道行, 教授 岩井 一宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM