Biomarkers of Covert Acid Stress in Patients with Chronic Kidney Disease: A Cross-Sectional Study.

INTRODUCTION: Like metabolic acidosis, earlier stages of acid (H+) stress, including an ongoing H+ challenge in the form of dietary H+, without or with steady-state H+ accumulation but with normal plasma total CO2 (PTCO2) (the latter state known as eubicarbonatemic acidosis), are associated with augmented progression of chronic kidney disease (CKD), but diagnosis of this covert H+ stress is clinically problematic. Prior published studies to identify clinically practical biomarkers of covert H+ stress did not include assessments of either dietary H+ or H+ retention. METHODS: We tested plasma pH (PpH), 8-h urine excretion of citrate (UcitV) or ammonium (UNH4+V) as biomarkers of dietary H+ assessed as potential renal acid load (PRAL), and of steady-state H+ retention by comparing observed to expected PTCO2 increase 2 h after an oral NaHCO3 bolus. We recruited 313 non-diabetic participants with PTCO2 ≥ 22 mM to exclude participants with metabolic acidosis and with eGFR (mean [SD], mL/min/1.73 m2) stages G1 (n = 62, 99.2 [7.3]), G2 (n = 167, 73.8 [6.3]), and G3 (n = 84, 39.9 [6.7]). We performed linear regressions (LR) between H+ retention or PRAL (dependent variables) and PpH, UcitV, or UNH4+V (independent variables) after adjusting for eGFR. RESULTS: Steady-state H+ retention (mean [SD], mmol) increased with stage (G1 = 3.8 [12.5], G2 = 18.2 [12.4], and G3 = 25.6 [9.0]). PpH was not significantly associated with PRAL in any group, and its association with H+ retention was significant only for G3 (p < 0.01). UcitV association with PRAL was significant only for G1 (p < 0.01) but not for G2 (p = 0.65) or G3 (p = 0.11). UcitV association with H+ retention was negative for both G2 (p < 0.01) and G3 (p < 0.01) but was not significant for G1 (p = 0.50). Adding UNH4+V to UcitV as a regressor for H+ retention increased r2 only marginally for G2 (0.61-0.63) and G3 (0.75-0.79). UNH4+V association with PRAL was positive (p < 0.01) for G1 and G2 but was not significant for G3 (p = 0.46). UNH4+V association with H+ retention was significant for both G2 (p < 0.04) and G3 (p < 0.01) but diverged directionally, being positive for G2 but negative for G3. DISCUSSION: Among patients with CKD at risk for covert H+ stress, lower UcitV better identified eubicarbonatemic acidosis than UNH4+V because the UNH4+V versus H+ retention relationship diverged between G2 and G3. Neither test identified eubicarbonatemic acidosis with certainty, indicating need for further work to establish a clinically useful test. On the other hand, UNH4+V had better utility identifying increased dietary H+ assessed as PRAL in G1 and G2.

Treatment of Chronic Kidney Disease-Related Metabolic Acidosis With Fruits and Vegetables Compared to NaHCO3 Yields More and Better Overall Health Outcomes and at Comparable Five-Year Cost.

OBJECTIVES: Current guidelines recommend treatment of metabolic acidosis in chronic kidney disease (CKD) with Na+-based alkali but base-producing fruits and vegetables (F + V) might yield more and better health outcomes, making the intervention cost-effective. DESIGN AND METHODS: In this post hoc analysis of a clinical trial we randomized 108 macroalbuminuric, nondiabetic CKD stage 3 participants with metabolic acidosis to receive F + V (n = 36) calculated to reduce dietary acid by half, oral NaHCO3 (HCO3-, n = 36) 0.3 mEq/kg body weight/day, or Usual Care (UC, n = 36) assessed annually for 5 years. We calculated a mean overall health score for the groups as follows: 1 for improved, 0 for no change, and -1 for worsened at 5 years for plasma total CO2, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, change in medication dose (reduction = 1, increased = -1, no change = 0), and 1 for met goal and 0 for not meeting goal for estimated glomerular filtration rate (>30 mL/min/1.73 m2) and systolic blood pressure (<130 mm Hg). We also assessed the number of participants with cardiovascular disease events (myocardial infarctions + strokes) and group medication and hospitalization costs. RESULTS: Net plasma total CO2 increase at 5 years was no different between HCO3- and F + V. Average health scores at 5 years differed among groups (P < .01) with F + V (7.4 [mean] ± 1.6 [standard deviation]) being descriptively larger than HCO3- and UC (2.9 ± 1.6 and 1.2 ± 1.6, respectively). The number of participants suffering cardiovascular disease events differed among groups (P = .009) with none (0) in F + V, 6 in UC, and 2 in HCO3-. Total 5-year household cost per beneficial health outcome differed among groups (P = .005) with UC being highest and that for HCO3- and F + V being comparable. CONCLUSIONS: Metabolic acidosis improved comparably with F + V or standard oral NaHCO3, but F + V yielded ancillary beneficial health outcomes, fewer participants with adverse cardiovascular events, and per-household cost that was comparable to NaHCO3.

Resuscitation with macromolecular superoxide dismutase/catalase mimetic polynitroxylated PEGylated hemoglobin offers neuroprotection in guinea pigs after traumatic brain injury combined with hemorrhage shock.

BACKGROUND: Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as a carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a polytrauma model of traumatic brain injury (TBI) plus hemorrhagic shock. Guinea pigs were used because, like humans, they do not synthesize their own ascorbic acid, which is important in reducing methemoglobin. RESULTS: TBI was produced by controlled cortical impact and was followed by 20 mL/kg hemorrhage to a mean arterial pressure (MAP) of 40 mmHg. At 90 min, animals were resuscitated with 20 mL/kg lactated Ringer's solution or 10 mL/kg PNPH. Resuscitation with PNPH significantly augmented the early recovery of MAP after hemorrhagic shock by 10-18 mmHg; whole blood methemoglobin was only 1% higher and carboxyhemoglobin was 2% higher. At 9 days of recovery, unbiased stereology analysis revealed that, compared to animals resuscitated with lactated Ringer's solution, those treated with PNPH had significantly more viable neurons in the hippocampus CA1 + 2 region (59 ± 10% versus 87 ± 18% of sham and naïve mean value) and in the dentate gyrus (70 ± 21% versus 96 ± 24%; n = 12 per group). CONCLUSION: PNPH may serve as a small-volume resuscitation fluid for polytrauma involving TBI and hemorrhagic shock. The neuroprotection afforded by PNPH seen in other species was sustained in a species without endogenous ascorbic acid synthesis, thereby supporting potential translatability for human use.

Urine citrate excretion identifies changes in acid retention as eGFR declines in patients with chronic kidney disease.

Previous studies have shown that acid (H+) retention in patients with chronic kidney disease (CKD) but without metabolic acidosis increases as the estimated glomerular filtration rate (eGFR) decreases over time. The present study examined whether changes in urine excretion of the pH-sensitive metabolite citrate predicted changes in H+ retention over time in similar patients with CKD that were followed for 10 yr. We randomized 120 CKD2 nondiabetic, hypertension-associated nephropathy patients with plasma total CO2 of >24 mM to receive 0.5 meq·kg body wt-1·day-1 NaHCO3 ([Formula: see text]; n = 40), 0.5 meq·kg body wt-1·day-1 NaCl (NaCl; n = 40), or usual care (UC; n = 40). We assessed eGFR (CKD-EPI) and H+ retention by comparing the observed with expected plasma total CO2 increase 2 h after an oral NaHCO3 bolus (0.5 meq/kg body wt). Although 10 yr versus baseline eGFR was lower for each group, 10-yr eGFR was higher (P < 0.01) in [Formula: see text] (59.6 ± 4.8 ml·min-1·1.73 m-2) than NaCl and UC (52.1 ± 5.9 and 52.3 ± 4.1 ml·min-1·1.73 m-2, respectively) groups. Less eGFR preservation was associated with higher 10-yr versus baseline H+ retention in the NaCl group (26.5 ± 13.1 vs. 18.2 ± 15.3 mmol, P < 0.01) and UC group (24.8 ± 11.3 vs. 17.7 ± 10.9 mmol, P < 0.01) and with lower 10-yr versus baseline 8-h urine citrate excretion (UcitrateV) for the NaCl group (162 ± 47 vs. 196 ± 52 mg, respectively, P < 0.01) and UC group (153 ± 41 vs. 186 ± 42 mg, respectively, P < 0.01). Conversely, better eGFR preservation in the [Formula: see text] group was associated with no differences in 10-yr versus baseline H+ retention (14.2 ±13.5 vs. 16.1 ± 15.1 mmol, P = 1.00) or UcitrateV (212 ± 45 vs. 203 ± 49 mg, respectively, P = 0.74). An overall generalized linear model for repeated measures showed that UcitrateV predicted H+ retention (P < 0.01). Less eGFR preservation in patients with CKD2 without metabolic acidosis was associated with increased H+ retention that was predicted by decreased UcitrateV.

Urine citrate excretion as a marker of acid retention in patients with chronic kidney disease without overt metabolic acidosis.

Acid (H+) retention appears to contribute to progressive decline in glomerular filtration rate (GFR) in patients with chronic kidney disease (CKD), including some patients without metabolic acidosis. Identification of patients with H+ retention but without metabolic acidosis could facilitate targeted alkali therapy; however, current methods to assess H+ retention are invasive and have little clinical utility. We tested the hypothesis that urine excretion of the pH-sensitive metabolite citrate can identify H+ retention in patients with reduced GFR but without overt metabolic acidosis. H+ retention was assessed based on the difference between observed and expected plasma total CO2 after an oral sodium bicarbonate load. The association between H+ retention and urine citrate excretion was evaluated in albuminuric CKD patients with eGFR 60-89 ml/min/1.73m2 (CKD 2, n=40) or >90 ml/min/1.73m2 (CKD 1, n = 26) before and after 30 days of base-producing fruits and vegetables. Baseline H+ retention was higher in CKD 2, while baseline urine citrate excretion was lower in CKD 2 compared to CKD 1. Base-producing fruits and vegetables decreased H+ retention in CKD 2 and increased urine citrate excretion in both groups. Thus, H+ retention is associated with lower urine citrate excretion, and reduction of H+ retention with a base-producing diet is associated with increased urine citrate excretion. These results support further exploration of the utility of urine citrate excretion to identify H+ retention in CKD patients with reduced eGFR but without metabolic acidosis, to determine their candidacy for kidney protection with dietary H+ reduction or alkali therapy.

Fruit and Vegetable Treatment of Chronic Kidney Disease-Related Metabolic Acidosis Reduces Cardiovascular Risk Better than Sodium Bicarbonate.

BACKGROUND: Current guidelines recommend treatment of metabolic acidosis in chronic kidney disease (CKD) with sodium-based alkali. We tested the hypothesis that treatment with base-producing fruits and vegetables (F + V) better improves cardiovascular disease (CVD) risk indicators than oral sodium bicarbonate (NaHCO3). METHODS: We randomized 108 macroalbuminuric, matched, nondiabetic CKD patients with metabolic acidosis to F + V (n = 36) in amounts to reduce dietary acid by half, oral NaHCO3 (HCO3, n = 36) 0.3 mEq/kg bw/day, or to Usual Care (UC, n = 36) to assess the 5-year effect of these interventions on estimated glomerular filtration rate (eGFR) course as the primary analysis and on indicators of CVD risk as the secondary analysis. RESULTS: Five-year plasma total CO2 was higher in HCO3 and F + V than UC but was not different between HCO3 and F + V (difference p value < 0.01). Five-year net eGFR decrease was less in HCO3 (mean -12.3, 95% CI -12.9 to -11.7 mL/min/1.73 m2) and F + V (-10.0, 95% CI -10.6 to -9.4 mL/min/1.73 m2) than UC (-18.8, 95% CI -19.5 to -18.2 mL/min/1.73 m2; p value < 0.01) but was not different between HCO3 and F + V. Five-year systolic blood pressure was lower in F + V than UC and HCO3 (p value < 0.01). Despite similar baseline values, F + V had lower low-density lipoprotein, Lp(a), and higher serum vitamin K1 (low serum K1 is associated with coronary artery calcification) than HCO3 and UC at 5 years. CONCLUSION: Metabolic acidosis improvement and eGFR preservation were comparable in CKD patients treated with F + V or oral NaHCO3 but F + V better improved CVD risk indicators, making it a potentially better treatment option for reducing CVD risk.

Acid retention in chronic kidney disease is inversely related to GFR.

Greater H+ retention in animal models of chronic kidney disease (CKD) mediates faster glomerular filtration rate (GFR) decline and dietary H+ reduction slows eGFR decline in CKD patients with reduced eGFR and H+ retention due to the high acid (H+) diets of developed societies. We examined if H+ retention in CKD is inversely associated with estimated GFR (eGFR) using cross-sectional and longitudinal analysis of individuals with CKD stage 1 (>90 ml·min- 1·1.73 m-2), CKD stage 2 (60-89 ml/min per 1.73 m2), and CKD stage 3 (30-59 ml·min- 1·1.73 m-2) eGFR. H+ retention was assessed using the difference between observed and expected plasma total CO2 2 h after 0.5 meq/kg body wt oral NaHCO3. H+ retention was higher in CKD 2 vs. CKD 1 ( P < 0.01) and in CKD 3 vs. CKD 2 ( P < 0.02) at baseline and 5 yr, and was higher in CKD 2 vs. CKD 1 ( P < 0.01) at 10 yr. All groups had lower eGFR at subsequent time points ( P < 0.01) but H+ retention was not different among the three time points for CKD 1. By contrast, eGFR decrease was associated with higher H+ retention in CKD 2 at 5 yr ( P = 0.04) and 10 yr ( P < 0.01) and with higher H+ retention in CKD 3 at 5 yr ( P < 0.01). Yearly eGFR decline rate was faster in CKD 2 vs. CKD 1 ( P < 0.01) and in CKD 3 vs. CKD 2 ( P < 0.01). The data show that H+ retention is inversely associated with eGFR, with faster eGFR decline, and support the need for greater dietary H+ reduction therapy for CKD individuals with lower eGFR.

Acid retention with reduced glomerular filtration rate increases urine biomarkers of kidney and bone injury.

Diets high in acid of developed societies that do not cause metabolic acidosis in patients with chronic kidney disease nevertheless appear to cause acid retention with associated morbidity, particularly in those with reduced glomerular filtration rate. Here we used a rat 2/3 nephrectomy model of chronic kidney disease to study induction and maintenance of acid retention and its consequences on indicators of kidney and bone injury. Dietary acid was increased in animals eating base-producing soy protein with acid-producing casein and in casein-eating animals with added ammonium chloride. Using microdialysis to measure the kidney cortical acid content, we found that nephrectomized animals had greater acid retention than sham-operated animals when both ate the soy diet. Each increment in dietary acid further increased acid retention more in nephrectomized than in sham rats. Nephrectomized and sham animals achieved similar steady-state daily urine net acid excretion in response to increments in dietary acid but nephrectomized animals took longer to do so, contributing to greater acid retention that was maintained until the increased dietary acid was stopped. Acid retention was associated with increased urine excretion of both N-acetyl-ß-D-glucosaminidase and deoxypyridinoline, greater in nephrectomized than control rats, consistent with kidney tubulointerstitial and bone matrix injury, respectively. Greater acid retention in nephrectomized than control animals was induced by a slower increase in urinary net acid excretion rate in response to the increment in dietary acid and also maintained until the dietary acid increment was stopped. Thus, acid retention increased biomarkers of kidney and bone injury in the urine, supporting untoward consequences to these two tissues.

Cigarette Smoking Attenuates Kidney Protection by Angiotensin-Converting Enzyme Inhibition in Nondiabetic Chronic Kidney Disease.

BACKGROUND: Cigarette smoking exacerbates the estimated glomerular filtration rate (eGFR) decline in nondiabetic chronic kidney disease (CKD) despite the kidney protection that is achieved by angiotensin converting enzyme inhibition (ACEI). Whether smoking cessation restores ACEI-related kidney protection is not known. METHODS: This 5-year, prospective, prevention trial recruited 108 smokers and 108 nonsmokers with stage-2 nondiabetic CKD with primary hypertension and urine albumin-to-creatinine ratio (Ualb) >200 mg/g. All smokers underwent smoking cessation intervention programs. Blood pressure was reduced in all participants toward achieving a goal of <130 mm Hg with regimens including ACEI. The primary outcome was eGFR change, and secondary outcomes included Ualb and urine levels of angiotensinogen (UATG), a surrogate for kidney angiotensin II (AII) levels, and isoprostane 8-isoprostaglandin F2α (U8-iso), an indicator of oxidative stress. RESULTS: One-year Ualb was lower than baseline in nonsmokers but not in either smoking group, supporting greater ACEI-related kidney protection in nonsmokers than smokers. Higher Ualb at 1 year in continued smokers was associated with higher UATG and higher U8-iso, consistent with smoking-induced AII and increased oxidative stress contributing to less ACEI-related kidney protection in smokers. Baseline eGFR was not different among groups (p = 0.92), but 5-year eGFR was higher in quitters than in continued smokers (62.0 ± 5.4 vs. 52.9 ± 5.6 mL/min/1.73 m2, p < 0.001); this value was lower in quitters than in nonsmokers (64.7 ± 5.6 mL/min/1.73 m2, p = 0.02). CONCLUSIONS: Smoking cessation compared with continued smoking ameliorates eGFR decline in nondiabetic CKD treated with ACEI, possibly by restoring kidney-protective effects of ACEI through reductions in kidney AII and oxidative stress.

Angiotensin II-mediated GFR decline in subtotal nephrectomy is due to acid retention associated with reduced GFR.

BACKGROUND: Angiotensin II (AII) mediates glomerular filtration rate (GFR) decline in animals with subtotal nephrectomy (Nx), but the mechanisms for increased AII activity are unknown. Because reduced GFR of Nx is associated with acid (H(+)) retention that increases kidney AII, AII-mediated GFR decline might be induced by H(+) retention. METHODS: We measured GFR and kidney microdialyzate H(+) and AII content in Sham and 2/3 Nx rats in response to amelioration of H(+) retention with dietary NaHCO3, to AII receptor antagonism and to both. RESULTS: GFR was lower in Nx than that in Sham. Nx but not Sham GFR was lower at Week 24 than that at Week 1. Despite no differences in plasma acid-base parameters or urine net acid excretion, kidney H(+) content was higher in Nx than that in Sham, consistent with H(+) retention. Plasma and kidney microdialyzate AII were higher in Nx than that in Sham and dietary NaHCO3 reduced each in Nx but not in Sham. AII receptor antagonism was associated with higher Week 24 GFR in Nx with H(+) retention but not in Sham or in Nx in which H(+) retention had been corrected with dietary NaHCO3. Week 24 GFR after dietary NaHCO3 was higher than after AII receptor antagonism. Week 24 GFR was not different after adding AII receptor antagonism to dietary NaHCO3. CONCLUSIONS: AII-mediated GFR decline in 2/3 Nx was induced by H(+) retention and its amelioration with dietary HCO3 conserved GFR better than AII receptor antagonism in this CKD model. H(+) retention might induce AII-mediated GFR decline in patients with reduced GFR, even without metabolic acidosis.

Treatment of metabolic acidosis in patients with stage 3 chronic kidney disease with fruits and vegetables or oral bicarbonate reduces urine angiotensinogen and preserves glomerular filtration rate.

Alkali therapy of metabolic acidosis in patients with chronic kidney disease (CKD) with plasma total CO2 (TCO2) below 22 mmol/l per KDOQI guidelines appears to preserve estimated glomerular filtration rate (eGFR). Since angiotensin II mediates GFR decline in partial nephrectomy models of CKD and even mild metabolic acidosis increases kidney angiotensin II in animals, alkali treatment of CKD-related metabolic acidosis in patients with plasma TCO2 over 22 mmol/l might preserve GFR through reduced kidney angiotensin II. To test this, we randomized 108 patients with stage 3 CKD and plasma TCO2 22-24 mmol/l to Usual Care or interventions designed to reduce dietary acid by 50% using sodium bicarbonate or base-producing fruits and vegetables. All were treated to achieve a systolic blood pressure below 130 mm Hg with regimens including angiotensin converting enzyme inhibition and followed for 3 years. Plasma TCO2 decreased in Usual Care but increased with bicarbonate or fruits and vegetables. By contrast, urine excretion of angiotensinogen, an index of kidney angiotensin II, increased in Usual Care but decreased with bicarbonate or fruits and vegetables. Creatinine-calculated and cystatin C-calculated eGFR decreased in all groups, but loss was less at 3 years with bicarbonate or fruits and vegetables than Usual Care. Thus, dietary alkali treatment of metabolic acidosis in CKD that is less severe than that for which KDOQI recommends therapy reduces kidney angiotensin II activity and preserves eGFR.

Artificial oxygen carrier with pharmacologic actions of adenosine-5'-triphosphate, adenosine, and reduced glutathione formulated to treat an array of medical conditions.

Effective artificial oxygen carriers may offer a solution to tackling current transfusion medicine challenges such as blood shortages, red blood cell storage lesions, and transmission of emerging pathogens. These products, could provide additional therapeutic benefits besides oxygen delivery for an array of medical conditions. To meet these needs, we developed a hemoglobin (Hb)-based oxygen carrier, HemoTech, which utilizes the concept of pharmacologic cross-linking. It consists of purified bovine Hb cross-linked intramolecularly with open ring adenosine-5'-triphosphate (ATP) and intermolecularly with open ring adenosine, and conjugated with reduced glutathione (GSH). In this composition, ATP prevents Hb dimerization, and adenosine promotes formation of Hb polymers as well as counteracts the vasoconstrictive and pro-inflammatory properties of Hb via stimulation of adenosine receptors. ATP also serves as a regulator of vascular tone through activation of purinergic receptors. GSH blocks Hb's extravasation and glomerular filtration by lowering the isoelectric point, as well as shields heme from nitric oxide and reactive oxygen species. HemoTech and its manufacturing technology have been broadly tested, including viral and prion clearance validation studies and various nonclinical pharmacology, toxicology, genotoxicity, and efficacy tests. The clinical proof-of-concept was carried out in sickle cell anemia subjects. The preclinical and clinical studies indicate that HemoTech works as a physiologic oxygen carrier and has efficacy in treating: (i) acute blood loss anemia by providing a temporary oxygen bridge while stimulating an endogenous erythropoietic response; (ii) sickle cell disease by counteracting vaso-occlusive/inflammatory episodes and anemia; and (iii) ischemic vascular diseases particularly thrombotic and restenotic events. The pharmacologic cross-linking of Hb with ATP, adenosine, and GSH showed usefulness in designing an artificial oxygen carrier for multiple therapeutic indications.

Kidney and Cardiovascular Protection Using Dietary Acid Reduction in Primary Hypertension: A Five-Year, Interventional, Randomized, Control Trial.

BACKGROUND: High fruit and vegetable diets are associated with reduced chronic kidney disease and cardiovascular disease but are infrequently used in hypertension treatment. Low acid diets are also associated with reduced chronic kidney disease and cardiovascular disease, and fruits and vegetables or oral sodium bicarbonate (NaHCO3) lowers dietary acid. METHODS: We randomized 153 hypertensive macroalbuminuric patients receiving pharmacologic chronic kidney disease and cardiovascular disease protection to get fruits and vegetables, oral NaHCO3, or Usual Care. We assessed the course of kidney disease progression and cardiovascular disease risk indices over five years. RESULTS: Chronic kidney disease progression was slower in participants receiving fruits and vegetables or oral NaHCO3 than Usual Care [mean (SE)] [-1.08 (0.06) and -1.17 (0.07) vs. -1.94 (0.11) mL/min/1.73m2/ year, respectively, P's< .001). Yet, systolic blood pressure was lower, and cardiovascular disease risk indices improved more in participants receiving fruits and vegetables than in those receiving NaHCO3 or Usual Care. These cardiovascular benefits of fruits and vegetables were achieved despite lower doses of pharmacologic chronic kidney disease and cardiovascular disease protection. CONCLUSION: The trial supports fruits and vegetables as foundational hypertension treatment to reduce chronic kidney disease progression and cardiovascular disease risk.

Polynitroxylated PEGylated hemoglobin protects pig brain neocortical gray and white matter after traumatic brain injury and hemorrhagic shock.

Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as an anti-inflammatory carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a porcine model of traumatic brain injury (TBI) with and without accompanying hemorrhagic shock (HS). TBI was produced by controlled cortical impact over the frontal lobe of anesthetized juvenile pigs. Hemorrhagic shock was induced starting 5 min after TBI by 30 ml/kg blood withdrawal. At 120 min after TBI, pigs were resuscitated with 60 ml/kg lactated Ringer's (LR) or 10 or 20 ml/kg PNPH. Mean arterial pressure recovered to approximately 100 mmHg in all groups. A significant amount of PNPH was retained in the plasma over the first day of recovery. At 4 days of recovery in the LR-resuscitated group, the volume of frontal lobe subcortical white matter ipsilateral to the injury was 26.2 ± 7.6% smaller than homotypic contralateral volume, whereas this white matter loss was only 8.6 ± 12.0% with 20-ml/kg PNPH resuscitation. Amyloid precursor protein punctate accumulation, a marker of axonopathy, increased in ipsilateral subcortical white matter by 132 ± 71% after LR resuscitation, whereas the changes after 10 ml/kg (36 ± 41%) and 20 ml/kg (26 ± 15%) PNPH resuscitation were not significantly different from controls. The number of cortical neuron long dendrites enriched in microtubules (length >50 microns) decreased in neocortex by 41 ± 24% after LR resuscitation but was not significantly changed after PNPH resuscitation. The perilesion microglia density increased by 45 ± 24% after LR resuscitation but was unchanged after 20 ml/kg PNPH resuscitation (4 ± 18%). Furthermore, the number with an activated morphology was attenuated by 30 ± 10%. In TBI pigs without HS followed 2 h later by infusion of 10 ml/kg LR or PNPH, PNPH remained neuroprotective. These results in a gyrencephalic brain show that resuscitation from TBI + HS with PNPH protects neocortical gray matter, including dendritic microstructure, and white matter axons and myelin. This neuroprotective effect persists with TBI alone, indicating brain-targeting benefits independent of blood pressure restoration.

The Fruit and Veggies for Kidney Health Study: A Prospective Randomized Trial.

Rationale & Objective: Providing fruits and vegetables (F&Vs) to health care system patients with elevated urine albumin-creatinine ratio (ACR) reduced ACR, slowed chronic kidney disease (CKD) progression and reduced cardiovascular disease (CVD) risk factors in previous studies. This study evaluated a community-based strategy in lower-income populations to identify African Americans with elevated ACR before health care system involvement and sustain them in a 6-month F&V protocol with (F&V + Cook) and without (F&V Only) cooking instructions, with the hypothesis that adjuvant cooking instructions with F&Vs would further reduce ACR. Study Design: Prospective, randomized, parallel 2-arm design. Setting & Participants: African American adults with ACR >10 mg/g creatinine randomized to 1 of 2 study arms. Interventions: Two cups/day of F&Vs with or without cooking instructions in participants followed 6 months. Outcomes: Participants sustaining the F&V protocol and between-group indicators of CVD risk, kidney injury, and dietary intake at 6 weeks and 6 months. Results: A total of 142 African American adults (mean age, 57.0 years; ACR, 27.4 mg/g; body mass index, 34.4; 24.9% CKD 1; 24.8% CKD 2; 50.4% CKD 3; 55% female) randomized to F&V Only (n=72) or F&V + Cook (n=70), and 71% were retained at 6 months. Participants received 90% of available F&V pick-ups over 6 weeks and 69% over 6 months. In the adjusted model, 6-month ACR was 31% lower for F&V + Cook than F&V Only (P = 0.02). Net 6-week F&V intake significantly increased and biometric variables improved for participants combined into a single group. Limitations: Small sample size, low-baseline ACR, and potential nonresponse bias for 24-hour dietary recall measure. Conclusions: These data support the feasibility of identifying community-dwelling African Americans with ACR indicating elevated CVD and CKD risk and sustaining a F&V protocol shown to improve kidney outcomes and CVD risk factors and provides preliminary evidence that cooking instructions adjuvant to F&Vs are needed to lower ACR. Funding: National Institute on Diabetes, Digestive, and Kidney Diseases grant "Reducing chronic kidney disease burden in an underserved population" (R21DK113440). Trial Registration: NCT03832166. Plain-Language Summary: African Americans, particularly those in low-income communities, have increased rates of chronic kidney disease (CKD) with worsening outcomes over time. Giving fruits and vegetables to individuals with CKD identified in health care systems was previously shown to reduce kidney damage, measured by urine protein albumin, and slow kidney function decline. We recruited African Americans in low-income communities with increased urine albumin levels. They received fruits and vegetables for 6 months, and we tested whether added cooking instructions further reduced urine albumin levels. Most participants continued to receive fruits and vegetables throughout the 6 months. Those given cooking instructions had lower urine albumin levels after 6 months, indicating decreased kidney damage. Providing cooking instructions with fruits and vegetables appears to lessen kidney damage more than just fruits and vegetables alone.

Angiotensin II receptors mediate increased distal nephron acidification caused by acid retention.

Patients with a moderately reduced glomerular filtration rate (GFR) typically have no metabolic acidosis and a urine net acid excretion comparable to those with normal GFR, supporting greater per nephron acidification with moderately reduced GFR. We modeled such patients using rats with a surgical reduction of 2/3 kidney mass, yielding animals with reduced GFR without metabolic acidosis. We then tested the hypothesis that reduction of nephron mass augments distal nephron acidification in remnant nephrons mediated by increased angiotensin II activity, and that the latter is induced by underlying acid retention. Nephron mass reduction yielded lower GFR than controls (sham operation), higher acid retention (measured by microdialysis of kidney cortex), higher distal nephron acidification, and higher plasma and kidney levels of angiotensin II, but plasma total CO(2) and urine net acid excretion were not different. Angiotensin II receptor antagonism reduced distal nephron acidification to levels similar to control. Dietary alkali that lowered acid retention to that of control also reduced plasma and kidney levels of angiotensin II and reduced distal nephron acidification to control. Angiotensin II receptor antagonism with dietary alkali had no significant added effect on distal nephron acidification. Thus, nephron reduction that moderately reduced GFR with no metabolic acidosis is characterized by increased angiotensin II activity. This mediates increased distal nephron acidification and is induced by acid retention.

Dietary acid reduction with fruits and vegetables or bicarbonate attenuates kidney injury in patients with a moderately reduced glomerular filtration rate due to hypertensive nephropathy.

The neutralization of dietary acid with sodium bicarbonate decreases kidney injury and slows the decline of the glomerular filtration rate (GFR) in animals and patients with chronic kidney disease. The sodium intake, however, could be problematic in patients with reduced GFR. As alkali-induced dietary protein decreased kidney injury in animals, we compared the efficacy of alkali-inducing fruits and vegetables with oral sodium bicarbonate to diminish kidney injury in patients with hypertensive nephropathy at stage 1 or 2 estimated GFR. All patients were evaluated 30 days after no intervention; daily oral sodium bicarbonate; or fruits and vegetables in amounts calculated to reduce dietary acid by half. All patients had 6 months of antihypertensive control by angiotensin-converting enzyme inhibition before and during these studies, and otherwise ate ad lib. Indices of kidney injury were not changed in the stage 1 group. By contrast, each treatment of stage 2 patients decreased urinary albumin, N-acetyl ß-D-glucosaminidase, and transforming growth factor ß from the controls to a similar extent. Thus, a reduction in dietary acid decreased kidney injury in patients with moderately reduced eGFR due to hypertensive nephropathy and that with fruits and vegetables was comparable to sodium bicarbonate. Fruits and vegetables appear to be an effective kidney protective adjunct to blood pressure reduction and angiotensin-converting enzyme inhibition in hypertensive and possibly other nephropathies.

Adenosine-5'-triphosphate-adenosine-glutathione cross-linked hemoglobin as erythropoiesis-stimulating agent.

An effective hemoglobin (Hb)-based blood substitute that acts as a physiological oxygen carrier and volume expander ought to stimulate erythropoiesis. A speedy replacement of blood loss with endogenous red blood cells should be an essential feature of any blood substitute product because of its relatively short circulatory retention time and high autoxidation rate. Erythropoiesis is a complex process controlled by oxygen and redox-regulated transcription factors and their target genes that can be affected by Hb physicochemical properties. Using an in vitro cellular model, we investigated the molecular mechanisms of erythropoietic action of unmodified tetrameric Hb (UHb) and Hb cross-linked with adenosine-5'-triphosphate (ATP), adenosine, and reduced glutathione (GSH). These effects were studied under normoxic and hypoxic conditions. Results indicate that these Hb solutions have different effects on stabilization and nuclear translocation of hypoxia-inducible factor (HIF)-1 alpha, induction of the erythropoietin (EPO) gene, activation of nuclear factor (NF)-kappa B, and expression of the anti-erythropoietic agents-tumor necrosis factor-alpha and transforming growth factor-beta 1. UHb suppresses erythropoiesis by increasing the cytoplasmic degradation of HIF-1 alpha and decreasing binding to the EPO gene while inducing NF-kappa B-dependent anti-erythropoietic genes. Cross-linked Hb accelerates erythropoiesis by downregulating NF-kappa B, stabilizing and facilitating HIF-1 alpha binding to the EPO gene, under both oxygen conditions. ATP and adenosine contribute to normoxic stabilization of HIF-1 and, with GSH, inhibit the NF-kappa B pathway that is involved in the suppression of erythroid-specific genes. Proper chemical/pharmacological modification is required to consider acellular Hb as an erythropoiesis-stimulating agent.