Satoshi Shimada

BACKGROUND: Salt-sensitive (SS) hypertension is associated with oxidative stress and impaired renal metabolism, but the mechanistic link remains unclear. We investigated the role of NOX4 (NADPH oxidase 4)–derived reactive oxygen species in shaping renal metabolic and vascular responses to high-salt intake in SS hypertension. METHODS: Male Dahl SS and SS ^NOX4−/− rats were maintained on a low-salt (0.4% NaCl) diet and then exposed to high salt (4.0% NaCl) for 21 days. Mean arterial pressure and renal blood flow were continuously measured, with intermittent arterial and renal venous sampling. The glomerular filtration rate was measured in separate groups of rats. Transcriptomic and metabolomic profiling of renal cortex, outer medulla, plasma, and urine was performed. RESULTS: High-salt intake led to progressive hypertension in SS rats, accompanied by transcriptomic and metabolic patterns suggestive of increased glutamate utilization, redox imbalance, and elevated reactive oxygen species. This glutamine-related profile was associated with high oxygen demand, increased proteolysis, and reduced adaptability in substrate utilization. SS ^{NOX4 −/−} rats exhibited attenuated hypertension, preserved redox balance, and increased nitric oxide bioavailability. Their kidneys exhibited metabolic patterns suggestive of greater reliance on fatty acid oxidation, with indications of improved oxygen efficiency, reduced amino acid catabolism, and lactate release without upregulating glycolytic genes. The glomerular filtration rate and renal blood flow changes were similar between strains. CONCLUSIONS: NOX4-derived reactive oxygen species were linked to less favorable metabolic responses to salt loading in SS hypertension. NOX4 deletion was associated with a more protective, fatty acid oxidation–driven metabolic profile. These findings highlight redox-sensitive metabolic reprogramming as a potential therapeutic target in hypertension and chronic kidney disease.

Sexual dimorphism profoundly influences physiology, disease susceptibility, and therapeutic responses, yet its effects on kidney mitochondrial function remain unclear. We hypothesized that sex differences in kidney mitochondrial function would parallel those in other organs, with females exhibiting greater oxidative capacity and lower oxidative stress. To test this, we measured oxidative phosphorylation (OXPHOS) kinetics and hydrogen peroxide (H2O2) emission in cortical and outer medullary (OM) mitochondria isolated from young male and female Dahl salt-sensitive (SS) rats maintained on a low-salt diet. Unexpectedly, male cortical mitochondria showed significantly higher O2 consumption during ATP synthesis (OXPHOS) than females when fueled by either complex I- or complex II-linked substrates. Cortical H2O2 emission was also greater in males, under both forward and reverse electron transport fueled by succinate. This was accompanied by increased Complex IV protein abundance without changes in mitochondrial DNA copy number or dynamics markers. In the OM, both mitochondrial respiration and H2O2 emission exceeded cortical levels, but showed no sex differences. Analysis of kidney transporter protein abundance revealed a sex-specific “downstream shift” in nephron transport, with males exhibiting greater proximal tubule (PT) sodium reabsorption potential and reduced distal transport capacity. Elevated cortical OXPHOS activity in males likely supports these higher PT energy demands. Thus, sex differences in renal mitochondrial function diverge from other organs, reflecting kidney-specific energetic priorities that override systemic maternal inheritance and sex hormone influences. Enhanced cortical H2O2 emission in males may underlie their heightened susceptibility to kidney injury and salt sensitivity.

Studies have found that blood flow to the renal medulla is an important determinant of pressure-natriuresis and the long-term regulation of arterial pressure. First, a brief review of methods developed enabling the study of the medullary circulation is presented. Second, studies performed in rats are presented showing medullary blood flow plays a vital role in the pressure-natriuresis relationship and thereby in hypertension. Third, it is shown that chronic reduction of medullary blood flow results in hypertension and that enhancement of medullary blood flow reduces hypertension hereditary models of both salt-sensitive rats and salt-resistant forms of hypertension. The key role that medullary nitric oxide production plays in protecting this region from ischemic injury associated with circulating vasoconstrictor agents and reactive oxygen species is presented. The studies cited are largely the work of my students, research fellows, and colleagues with whom I have performed these studies dating from the late 1980s to more recent years.

This study explored mechanisms underlying the natriuretic effects of mammalian target of rapamycin protein complex 2 inhibition using PP242 and revealed both epithelial Na⁺ channel and Na-Cl cotransporter in the distal tubular segments were potentially inhibited. These observations, with prior lab evidence, indicate that PP242 prevents hypertension via its potent inhibitory effects on these specific sodium transporters and by reducing renal immune responses. This dual action, coupled with potassium sparing effects, suggests an improved approach for managing hypertension and associated kidney damage.

In this study, novel methods were developed, which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O₂ and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O₂ consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague–Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to 4.0% NaCl diet. In addition, targeted mRNA expression analysis of cortical segments was performed. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O₂ consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. A novel finding was the increased expression of glycolysis-related genes in Cx and isolated proximal tubular segments in response to an HS diet, consistent with increased release of pyruvate and lactate from the kidney to the renal venous blood. Data suggests that aerobic glycolysis (eg, Warburg effect) may contribute to energy production under these circumstances. The study provides evidence that kidney metabolism responds to an HS diet enabling enhanced energy production while protecting from oxidative stress and injury. Metabolomic and transcriptomic analysis of kidneys of Sprague-Dawley rats fed a high salt diet.

In the present study, novel methods were developed which allowed continuous (24/7) measurement of blood pressure (BP) and renal blood flow (RBF) in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O ₂ and metabolites. The study determined the effects of a high salt (HS) diet upon whole kidney O ₂ consumption and the metabolomic profiles of normal Sprague Dawley (SD) rats. A separate group of rats was studied to determine changes in the cortex (Cx) and outer medulla (OM) tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to a 4.0% NaCl diet. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O ₂ consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. Increased glycolysis was evident with the elevation of mRNA expression encoding key glycolytic enzymes and release of pyruvate and lactate from the kidney in the renal venous blood. Glycolytic production of NADH is used in either the production of lactate or oxidized via the malate aspartate shuttle. Aerobic glycolysis (e.g., Warburg-effect) may account for the needed increase in cellular energy. The study provides evidence that kidney metabolism responds to a HS diet enabling enhanced energy production while protecting from oxidate stress and injury.

This study found that the immediate increases of renal blood flow (RBF) following unilateral nephrectomy (UNX) are a consequence of reduced ANG II type 1 (AT₁) receptor stimulation. The continuous monitoring of RBF and intermittent measurement of glomerular filtration rate (GFR) in conscious rats during the 1-wk period of rapid hypertrophy following UNX provided unique insights into the regulation of RBF and GFR when faced with increased metabolic loads. It was found that neither kidney hypertrophy nor the associated increase of capillaries was an AT₁-dependent phenomenon.

Background: The present study in Sprague-Dawley rats determined the effects of a rapid rise of renal perfusion pressure (RPP) upon the activation of mTOR (mechanistic target of rapamycin), and the effects upon the infiltration of CD68-positive macrophages/monocytes and CD3-positive T lymphocytes into the kidneys. Methods: RPP was elevated by 40 mm Hg for 30 minutes in male Sprague-Dawley rats while measuring renal blood flow and urine flow rate. Sham rats were studied in the same way, but RPP was not changed. Since initial studies found that the acute increase of RPP resulted in activation of mTORC1 (phosphorylation of S6 ^S235/236 ), the effects of inhibition of mTORC1 with rapamycin pretreatment were then determined. Results: It was found that a 30-minute increase of RPP (≈40 mm Hg) resulted in an 8-fold increase of renal sodium excretion which was blunted by rapamycin treatment. Renal blood flow was not affected by the elevation of RPP. Activation of mTORC1 was observed. Significant increases in CD68-positive macrophages were found in both the cortex (intraglomerular and periglomerular regions) and in the outer medullary interstitial regions of the kidney and prevented by rapamycin treatment. Increases in CD3-positive T lymphocytes were observed exclusively in the periglomerular regions and prevented by rapamycin treatment. Upregulation of several proinflammatory markers was observed. Conclusions: We conclude that elevation of RPP rapidly activates mTORC1 resulting in infiltration of immune cells into the kidney.

The physiological relationships between kidney O ₂ delivery and consumption and metabolism have been studied only under conditions of anesthesia and stressful surgical procedures. We report here techniques developed in our laboratory enabling determination of kidney O ₂ utilization over many days in instrumented unanesthetized rats in which the effects of a high salt diet were determined in normal Sprague Dawley (SD) rats. Male SD rats (10 weeks of age) fed a 0.4% NaCl diet were instrumented with a left renal artery flow probe (Transonic) for continuous (24 hr/day) measurement of renal blood flow (RBF). An indwelling intra‐renal vein catheter (MRE 025) was inserted through the femoral vein and tip fixed close to the renal pelvis. An aortic catheter inserted though the femoral artery was implanted to sample blood and continuously (24 hr/day) measure arterial blood pressure (BP). Catheters and flow probe cables which exited from the neck were attached to a custom designed swivel and a servo‐controlled circular cage system (BASi cage) enabling the rat to move freely throughout the study. Following 7 days of recovery from surgery, continuous monitoring of RBF and BP began and continued throughout the study. Following a 3 day control period with rats fed a 0.4% NaCl diet, renal venous and arterial blood were samples (250 ul) and blood gas was measured immediately by a radiometer (ABL800 FLEX). The diet was switch to 4% NaCl and blood sampling was performed at days 7, 14 and 21 of the high salt diet. O ₂ content, O ₂ consumption, and O ₂ extraction were determined as: O ₂ content (mL/dL) : 0.31 × Hemoglobin (Hb) (g/dL) × HbO­ ₂ (%) + 0.003 × PaO ₂ (mmHg). O ₂ consumption (ml/min) : RBF (ml/min) × (arterial‐renal venous O ₂ content difference). O ₂ extraction (% of arterial supply) : 1‐(renal venous O ₂ content/arterial O ₂ content) × 100. Daily average mean arterial pressure (MAP) increased moderately soon after switching to the 4% NaCl diet from 113 ± 2 to 120 ± 2 mmHg by day 3 (p=0.03, n=9) and remain elevated at this level throughout the study (120 ± 3 mmHg at day 13). Renal blood flow also rose soon after switching to the 4% salt diet from 9.4 ± 0.8 to 10.3 ± 0.8 ml/min during day 2 (p=0.03, n=9) . Thereafter, it also remained elevated throughout the study (10.7 ± 0.8 ml/min at day 13). Changes in O ₂ consumption occurred more gradually increasing from 0.21 ± 0.04 and rising to 0.27 ± 0.03 (ml/min) at day 14 of 4% salt diet (p=0.02, n=5). The calculated O ₂ extraction ratio remained unchanged ranging from 12.1 ± 0.1 (%) to 12.8 ± 0.1 (%) (p=0.48, n=5) throughout the study. It appears that in normal SD rats, the increased tubular metabolic work associated with a high salt diet is initially accommodated by increased perfusion (RBF) and a gradual increase of O ₂ consumption while extraction remains unchanged for a two‐week period.