Albumin Excretion Research Articles

Causal association between urine albumin-to-creatinine ratio and risk of colorectal cancer: Atwo-sample Mendelian randomization study.

Previous observational studies have investigated the association between urinary albumin excretion and the risk of colorectal cancer (CRC), but the results have been inconsistent. This study aimed to explore the causal association between urine albumin-to-creatinine ratio (ACR) and CRC risk through a two-sample Mendelian randomization (MR) analysis. The genome-wide association study (GWAS) data of ACR (n=382,500) and CRC (CRC: 6,509 cases and 287,137 controls) were obtained from the IEU OpenGWAS project website and the FinnGen database, respectively. The TwoSampleMR and MR-PRESSO R packages were used to search for and analyze genetic variations that served as instrumental variables for ACR. The odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using the inverse-variance weighted method, MR-Egger, and weighted median. Genetically predicted ACR was not associated with CRC risk (all P > 0.05). Further analysis based on the site of onset (colon or rectum) also did not show a significant association (all P > 0.05). MR-PRESSO, MR-Egger regression and leave-one-out sensitivity analysis all indicated that the current results were robust and reliable. These findings suggest that ACR does not affect CRC risk and may not be used as a marker of CRC risk in clinical practice. However, relevant studies especially in ethnically diverse populations are still needed to confirm the current findings.

Improvement in albumin excretion in patients with type 2 diabetes mellitus labelled as metformin-intolerant after rechallenge with extender-release metformin

Improvement in albumin excretion in patients with type 2 diabetes mellitus labelled as metformin-intolerant after rechallenge with extender-release metformin

Preventive effects of vasohibin-2-targeting peptide vaccine for diabetic nephropathy.

Diabetic nephropathy remains the leading cause of end-stage kidney disease in many countries, and additional therapeutic targets are needed to prevent its development and progression. Some angiogenic factors are involved in the pathogenesis of diabetic nephropathy. Vasohibin-2 (VASH2) is a novel proangiogenic factor, and our previous study showed that glomerular damage is inhibited in diabetic Vash2 homozygous knockout mice. Therefore, we established a VASH2-targeting peptide vaccine as a tool for anti-VASH2 therapy in diabetic nephropathy. In this study, the preventive effects of the VASH2-targeting peptide vaccine against glomerular injury were examined in a streptozotocin (STZ)-induced diabetic mouse model. The mice were subcutaneously injected with the vaccine at two doses 2 wk apart and then intraperitoneally injected with 50 mg/kg STZ for 5 consecutive days. Glomerular injury was evaluated 20 wk after the first vaccination. Treatment with the VASH2-targeting peptide vaccine successfully induced circulating anti-VASH2 antibody without inflammation in major organs. Although the vaccination did not affect blood glucose levels, it significantly prevented hyperglycemia-induced increases in urinary albumin excretion and glomerular volume. The vaccination did not affect increased VASH2 expression but significantly inhibited renal angiopoietin-2 (Angpt2) expression in the diabetic mice. Furthermore, it significantly prevented glomerular macrophage infiltration. The preventive effects of vaccination on glomerular injury were also confirmed in db/db mice. Taken together, the results of this study suggest that the VASH2-targeting peptide vaccine may prevent diabetic glomerular injury in mice by inhibiting Angpt2-mediated microinflammation.NEW & NOTEWORTHY This study demonstrated preventive effects of VASH2-targeting peptide vaccine therapy on albuminuria and glomerular microinflammation in STZ-induced diabetic mouse model by inhibiting renal Angpt2 expression. The vaccination was also effective in db/db mice. The results highlight the importance of VASH2 in the pathogenesis of early-stage diabetic nephropathy and the practicability of anti-VASH2 strategy as a vaccine therapy.

The Role of Interleukin-6 in Regulating Glomerular Filtration Rate during Angiotensin II and High Salt Conditions

Previous results demonstrate that interleukin-6 (IL-6) increases mean arterial pressure (MAP) during Angiotensin II (Ang II)-salt hypertension. In addition, albumin excretion was lower in IL-6 knockout mice (KO) when compared to wild-type (WT). This study investigated the role of IL-6 on regulating glomerular filtration rate (GFR), renal plasma flow (RPF) and MAP in WT and IL-6 KO mice treated with a slow pressor dose of Ang II (200 ng/kg/min) +/- 6% high-salt (HS) diet. Male C57BL6 and IL-6 KO mice (12 weeks old) were treated with Ang II +/- 6% HS diet for 12 – 14 days. Mean arterial pressure, RPF and GFR were measured in anesthetized mice. MAP was 130 ± 7 mmHg and 91 ± 4 mmHg in control WT and IL-6 KO mice, respectively. Ang II treatment increased MAP in WT (153 ± 5 mmHg) and no change in IL-6 KO (83 ± 4 mmHg) mice. Ang II + 6% HS increased MAP to 150 ± 11 mmHg in WT and 93 ± 4 mmHg in IL-6 KO mice. Baseline RPF was 1822 ± 229 and 1912 ± 402 ml/min/g in control WT and IL-6 KO mice, respectively. Ang II treatment increased RPF in WT (3156 ± 753 ml/min/g), while lowering RPF in IL-6 KO (1648 ± 422 ml/min/g) mice. RPF during Ang II + 6% HS treatment was decreased in WT (1095 ± 305 ml/min/g) and IL-6 KO (1133 ml/min/g) mice. GFR was 756 ± ml/min/g and 788 ± ml/min/g in control WT and IL-6 KO mice, respectively. Ang II increased GFR in WT (1009 ± 63 ml/min/g) and no change in IL-6 KO (756 ± 23 ml/min/g). Ang II + 6% HS increased GFR in WT (1095 ± 146 ml/min/g) and no change in GFR of IL-6 KO (540 ± 207 ml/min/g) mice. Our results suggest that IL-6 reduces MAP during baseline, Ang II, and Ang II + 6% HS. The absence of IL-6 prevented increases in MAP, RPF and GFR during Ang II treatment. Our data also suggest that IL-6 contributes to increased MAP and GFR during Ang II + 6% HS treatment. K01HL092593-05. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Salt-Sensitive Hypertension and Renal Damage are Amplified by Adoptive Transfer of CD4+ and Not CD8+ T Cells: A Role of NADPH Oxidase 2 (NOX2)

Infiltration of T cells into the kidney of the Dahl Salt Sensitive (SS) rat model of human disease accompanies the development of salt-sensitive hypertension and renal damage. Deletion of T cells on the Dahl SS genetic background (SSCD247−/−) attenuates hypertension and renal damage, and subsequent replacement of T cells by splenocyte transfer restores the disease phenotype. The present studies were performed to assess if the T cell subtype (CD4+ vs CD8+) is important in Dahl SS hypertension and interrogate the mechanism of action. In initial experiments, adoptive transfer of purified CD4+ or CD8+ T cells (~5 million) from splenocytes isolated from the Dahl SS (CD4+ SS→CD247 or CD8+ SS→CD247) or PBS vehicle (PBS→CD247) into the SSCD247−/− rat was performed on postnatal day 5. Animals were recovered and instrumented with radiotelemeters at 7 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 21, the rats were euthanized, and their kidneys were perfused and collected for immune cell analysis by flow cytometry. During the LS baseline period there was no significant difference observed in mean arterial pressure (MAP) between the CD4+ SS→CD247, CD8+ SS→CD247, or PBS→CD247 groups (113±1, 115±1, 114±1 mmHg; n=21, 10, 24, respectively). After three weeks of HS, rats receiving CD4+ T cells from the SS (CD4+ SS→CD247) demonstrated an amplified salt-sensitive hypertension (154±3 mmHg) and albuminuria (184±22 mg/day) compared to blood pressure (140±3 and 145±3 mmHg) and albuminuria (82±9 and 85±8 mg/day) in the CD8+ SS→CD247 or PBS→CD247 groups, respectively. Since the deletion of the p67phox subunit of NADPH oxidase 2 (NOX2) in adoptively transferred splenocytes attenuated Dahl SS hypertension and kidney damage, parallel studies examined the influence of deletion of p67phox in CD4+ (n=14) and CD8+ T cells (n=8). The adoptive transfer of either CD4+ or CD8+ T cells lacking functional NOX2 did not alter the LS or HS blood pressure or albuminuria when compared to the PBS control group. Finally, a flow cytometric analysis at the end of the experiment confirmed the absence of CD3+ T cells in the PBS→CD247 and documented the reconstitution of equal numbers of CD4+ cells in the blood and kidney of the rats receiving CD4+ cells with intact or deficient NOX2. Interestingly, no difference was observed in CD8+ T cells in the kidney and blood of rats receiving CD8+ cells with intact or deficient NOX2, but a significant number of CD4+ T cells were also observed in the SSCD247−/− rats receiving the purified CD8+ T cells. In summary, these studies demonstrate that CD4+ T cells amplify salt-sensitive hypertension and renal damage in the Dahl SS via a NOX2-dependent mechanism while CD8+ T cells do not alter disease phenotypes when compared to PBS control. GRANTS: HL161231, HL166458, Georgia Research Alliance. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Female Dahl SS rats exhibit greater increases in blood pressure on a high-fat, high-salt diet than males

Epidemiological data suggest that blood pressure (BP) in females is more sensitive to a high-fat (HF) diet than in males. To better understand the mechanisms driving greater increases in BP with HF in females, appropriate animal models are needed. Female Dahl rats have been shown to exhibit similar or greater increases in BP on a chronic HF diet compared to males. The goal of the current study was to test the hypothesis that HF loading will also exacerbate increases in BP in females vs. males when animals are placed on a HF/high-salt (HS) diet. To address this hypothesis, 6-wk-old male and female Dahl rats were implanted with telemetry devices to continuously measure mean arterial BP; n=4-7/group. Rats were allowed to recover for one week followed by one week of baseline BP recording. Rats were then placed on a HF diet (60% kcal from lard; Bio-Serv) for 2 weeks followed by a HF/HS diet (4% salt; Bio-Serv) for an additional 4 weeks. Controls were maintained on a normal-fat/normal-salt (NF/NS) diet throughout the study. Rats were weighed weekly and placed in metabolic cages at the end of the study to collect a 24-hr urine sample to measure protein and albumin excretion. Baseline BP values were comparable between males (119±2 mmHg) and females (122±3 mmHg). 2 wks of HFD alone increased BP in both sexes (males: 126±2 mmHg, females: 133±4 mmHg; Pdiet= 0.002, Psex=0.1, Pint=0.47). Switching rats to a HF/HS diet resulted in pronounced increases in BP, and females had a higher BP than males at the end of the study vs. HFD alone (156±6 vs. 173±10 mmHg; Pdiet<0.0001, Psex=0.024, Pint=0.2). Consistent with enhanced sensitivity of BP in females to HF/HS compared to males, protein excretion increased 78±3% when going from HF alone to HF/HS in females vs. 68±2% in males (p=0.040). Albumin excretion increased to comparable levels in both sexes when going from HFD to HF/HS (87±1 vs. 85±2 %; p=0.68). Importantly, HF/HS resulted in increased body weight in males but not females vs. control diet more (males: 370±9 vs. 398±7 g, females: 244±6 vs. 239±4 g), however, males weighed more than females (Pint=0.046, Pdiet=0.17, Psex<0.0001). Males on HF/HS also exhibited greater perirenal fat vs. control (17±1 vs. 13±2 g) with no differences in females (6±0.6 vs. 6±0.9 g; Pdiet=0.1368, Psex=<0.0001, Pint=0.1365). Neither sex nor diet impacted gonadal fat (males: 17±1 vs. 13±1 g; females: 18±4 vs. 20±1 g; Pdiet=0.4982, Psex=0.1106, Pint=0.1106). Total cholesterol was comparable between the sexes on HF/HS (2476±540 vs. 2216 ± 293 nM; p=0.65) and triglycerides were greater in males (0.46±0.06 vs. 0.22±0.02 mg/dL; p=0.012). In conclusion, HF increases BP sensitivity in female Dahl rats independently of overt increases in body weight or adiposity, establishing a model to study the mechanisms that cause females to lose their cardiovascular protection with HFD. Supported by 1U54HL169191. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanisms of Iron-Mediated Renal Injury in a Mouse Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is a hematologic disorder characterized by polymerization of the mutated hemoglobin S and sickling of red blood cells, leading to hemolysis, release of free heme/iron, and subsequent tissue iron accumulation. The kidney is one of the most frequently affected organs by iron overload, and chronic hemolysis is associated with chronic kidney disease severity in SCD. The potential role of iron overload in causing the observed renal injury in SCD has never been established. The goal of our study is to identify the impact of iron accumulation on the renal phenotype in SCD. We hypothesized that renal iron accumulation causes injury by oxidative stress dependent ferroptosis and/or mitochondrial dysfunction and subsequent kidney damage, that is worse in males than in females. Methods: We used 20-week-old male and female humanized SCD mice (HbSS) and age-matched genetic controls (HbAA). Mice were treated with either vehicle or the iron chelator Deferiprone (DFP, 100 mg/kg/day) for 8 weeks. Urinary excretion of protein and albumin as markers of renal injury were measured. Glomerular filtration rate (GFR) was measured transdermally after injection of FITC-sinistrin. Thiobarbituric acid reactive substance (TBARS) assay was performed for assessment of lipid peroxidation. Mitochondrial bioenergetics was assessed by Oroboros O2k Fluorometer of permeabilized renal cortices. Results: We previously demonstrated comparable iron accumulation in the kidney of male and female mice. However, HbSS males developed a more severe renal injury than females at 20 weeks of age, evidenced by proteinuria (2.52 ± 0.16 vs. 0.55 ± 0.16 mg/24hrs, p=0.0008) and albuminuria (76.44 ± 6.85 vs. 33.9 ± 8.81 μg/24hrs, p=0.0005). There was higher lipid peroxidation, a hallmark of ferroptosis, in the renal cortex of female HbSS than HbAA mice (1.31 ± 0.07 vs. 1.07 ± μM/mg, p=0.04). Bioenergetics studies revealed significantly lower state 3 oxygen consumption rate (108.2 ± 18.1 vs 164.8 ± 9.3 pmol/(s*mg), p=0.05), signifying lower oxidative phosphorylation capacity in HbSS males. In addition, there was significant reduction in state 4◦ respiration (36.4 ± 3.4 vs. 52.8 ± 3.4 pmol/(s*mg), p=0.03) in male HbSS mice. I also detected a decrease in maximum complex IV activity in male HbSS mice (325.5 ± 63.8 vs. 639.5 ± 51.3 pmol/(s*mg), p=0.02). Treatment with DFP improved proteinuria (2.66 ± 0.09 vs. 1.3 ± 0.14 mg/24hrs, p=0.0011) and GFR (165.3 ± 9.4 vs. 236.6 ± 12.6 μl/min, p=0.0058) in male HbSS mice. Conclusion: Iron accumulation causes renal injury which may be attributed to ferroptosis and mitochondrial dysfunction in SCD mice. Funding: NHLBI—R00HL144817 and ASN Norman Siegel Research Grant to MK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-Based Differences in Proximal Tubule Endocytic Capacity in Mice

Women are more protected from acute kidney injury, and chronic kidney disease (CKD) in women progresses more slowly to end-stage renal disease compared to men. CKD involves changes in the integrity of the glomerular barrier function with consequent proteinuria. The kidney proximal tubule (PT) normally reclaims albumin and other filtered proteins via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. PT health, and ultimately kidney function, is impacted by the uptake of excess albumin and by nephrotoxic drugs that enter cells in a megalin/cubilin dependent manner. Recent transcriptomic studies in mice have shown differences in the expression of megalin and cubilin between males and females and across the S1, S2, and S3 sub-segments that comprise the PT. Additionally, reduced reabsorption of water and salt by the PT has been demonstrated in female rodents. These differences in PT receptor expression and functionality likely contribute to sex-based differences in PT endocytic capacity. To determine if there are sex-based differences in the recovery of filtered proteins by the PT, urine and kidney tissue were collected from five male and five female 13-week-old C57BL/6 mice. Urinary albumin was quantified by ELISA. Kidneys were processed for Western blotting and immunofluorescence staining. We used imaging-based approaches to quantify the expression and distribution of albumin, megalin, and cubilin in SGLT2-positive S1 and SGLT2-negative, OAT1-positive S2 segments. Under normal conditions, S1 had greater intensity of subapical albumin staining in both males and females. However, females had greater intensity of albumin staining in S2 normalized to S1 compared to males. Female mice had lower levels of urinary albumin excretion when normalized to creatinine than males. Female mice had greater expression of total megalin (1.5x) and cubilin (1.4x) and decreased expression of NHE3 (0.7x) in the kidney cortex. Male and female mice had similar levels of megalin expression and surface localization in S1, however female mice had greater expression and surface localization of megalin in S2. Males had greater colocalization of megalin with lysosomal marker, LAMP1. In both males and females, the colocalization of megalin with LAMP1 decreased from S1 to S2. Together, these data suggest that, at baseline in female mice, S2 has greater endocytic capacity and contributes more to the uptake of normally filtered albumin than in males. This increased capacity could explain the reduced urinary excretion of albumin observed in rodent injury models and in women with CKD. Current studies are focused on incorporating these sex-based differences into a multiscale mathematical model of protein uptake along the length of the PT. National Institutes of Health: T32 DK007052, F31 DK121394, R01 DK125049, R01 DK118726, R01 DK083785, S10 OD021627, S10 OD028596, U54 DK137329, ASN Foundation for Kidney Research Pre-Doctoral Fellowship Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal tubular (pro)renin receptor deletion does not protect against kidney injury in db/db mice

Background: The (pro)renin receptor (PRR) is a multifunctional protein implicated in blood pressure regulation and kidney fibrosis. Previous studies report enhanced PRR expression in non-diabetic and diabetic kidney disease. In this study, we investigated whether deletion of renal tubular PRR attenuates kidney injury in type 2 diabetes. Methods: Floxed PRR mice were bred with mice expressing Pax8 rtTA and LC1 transgenes and db/db mice (B6.BKS) to obtain renal tubular PRR knockout (KO)-db/db mice. Age matched non-diabetic floxed controls, db/db mice and PRRKO-db/db mice were studied at 16, 20, 26 and 30 weeks of age. To induce PRR deletion, PRRKO-db/db mice were treated with 2 mg/ml doxycycline for 12 days at 8-10 weeks of age. Only male mice were included in this study. Results: All mice survived until the end of the study (N=5-7 mice/group). Compared to controls, db/db mice and PRRKO-db/db mice had higher body weights despite similar food intake throughout the study (p<0.001 by one-way ANOVA). PRRKO-db/db mice had higher urine volume and water intake compared to controls and db/db mice (p <0.01 by ANOVA). Db/db mice had elevated non-fasting blood glucose levels (average: 472 mg/dl at 16 weeks, 292 mg/dl at 30 weeks) while PRR KO-db/db mice had modestly elevated blood glucose levels (average 255 mg/dl at 20 and 30 weeks) relative to controls (average: 150 mg/dl). Urinary albumin excretion was markedly elevated in PRRKO-db/db mice relative to db/db mice and controls throughout the study (950 ug/day vs db/db:145 ug/day, controls: 50 ug/day, p<0.001). At 30 weeks, kidney histology by PAS stain showed minimal tubular injury, glomerulosclerosis or interstitial inflammation among all 3 groups. Interestingly, podocin staining by immunofluorescence was reduced in db/db mice but not PRR-KO db/db mice. Compared to control mice, db/db mice had similar KIM-1, NGAL and collagen-1 mRNA expression in kidney cortex and inner medulla. PRR KO db/db mice had increased cortical KIM-1, NGAL and collagen-I expression compared to control and db/db mice (p<0.01 by ANOVA). Plasma sPRR levels were almost two-fold higher in diabetic mice relative to controls with no difference between db/db mice and PRR KO-db/db mice. Urinary sPRR levels were undetectable in controls and mildly elevated in db/db mice (7.6 ± 0.7 pg/day) and PRR KO-db/db mice (47.6 ± 22.3 pg/ml). No differences in plasma prorenin/renin concentration was noted among the 3 groups while urinary prorenin/renin excretion was higher in PRRKO-db/db mice. Conclusion: Renal tubular deletion of PRR does not protect against kidney injury in type 2 diabetes. It is possible that loss of PRR impairs baseline tubular function which is exacerbated by type 2 diabetes. American Diabetes Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Toll-like Receptor 4 Promotes Salt-Sensitivity in Dahl SS Rats: An Investigation into the Mechanism

Renal inflammation accompanies hypertension and renal damage in Dahl Salt-Sensitive (SS) rats fed a high salt diet (HS, Dyets AIN-76A 4.0% NaCl). Previous studies demonstrated that toll-like receptor 4 (TLR4), a component of innate immune activation, is upregulated in macrophages infiltrating the kidneys of Dahl SS rats fed high salt. Preliminary studies in our laboratory indicated that mean arterial pressure (MAP) (SS: 162.5 ± 4.9 vs. SSTLR4−/−: 147.1 ± 3.5 mmHg, p<0.0001, n=10/group) and albuminuria (321.3 ± 62.7 vs. 170.6 ± 27.1 mg/day, p<0.05, n=10/group) were significantly reduced in male Dahl SS TLR4 knockout rats (SSTLR4−/−) compared to male SS after three weeks of HS. Furthermore, peritoneal macrophages isolated from SSTLR4−/− produce significantly less IL-1β compared to SS macrophages upon stimulation with lipopolysaccharide (LPS) . Since TLR4 is widely expressed in many tissues, we examined the specific effects of TLR4 deletion in immune cells via a total body irradiation/bone marrow transfer approach. The present study tested the hypothesis that TLR4 deletion specifically in hematopoietic cells would attenuate male Dahl SS hypertension and renal damage. At 6 weeks of age, Dahl SS male recipients were irradiated (11Gy) then received either SS or SSTLR4−/− bone marrow transplantation via tail vein injection (~15-20 million cells). Following recovery, rats (maintained on a low salt chow (LS, Dyets AIN-76A 0.4% NaCl)) underwent telemeter implantation at approximately 8 weeks of age for continuous monitoring of blood pressure. Animals were switched to HS following recovery and baseline blood pressure recordings. Urine samples were collected once during the LS period and weekly throughout a 3-week HS challenge. Upon euthanasia, kidneys were flushed and harvested for analysis. MAP was not found to be different between groups during the LS baseline period (SS: 113.4 ± 1.6 vs. SSTLR4−/−: 116.5 ± 2.3 mmHg, n=5-6/group), and our preliminary data also revealed that MAP was not different between groups throughout the high salt challenge (HS day 20: 136.4 ± 10.5 vs 144.8 ± 9.2). Urinary albumin excretion was also not found to be different between rats receiving either SS or TLR4−/− bone marrow transfer (165.2 ± 38.5 vs. 247.9 ± 58.7 mg/day, n=7/group). Furthermore, assessment of BUN at the conclusion of the experiment revealed no differences (33.0 ± 6.1 vs. 29.3 ± 3.2 mg/dL, n=7/group). Immune cells were isolated from the blood and kidneys of these animals and characterized by flow cytometric analysis. There was no difference in the number of circulating leukocytes or lymphocytes (2.1x106 vs. 2.3x106 CD45+ cells per mL blood, n=6-7/group) indicating comparable reconstitution of immune cells in both groups. We also found no difference in renal infiltrating leukocytes or lymphocytes (3.1 x106 vs. 3.2x106 CD45+ cells per kidney, n=7/group). Together, these data contrast the effects of whole-body TLR4 knockout and suggest that TLR4 in hematopoietic cells may not be responsible for promoting Dahl SS hypertension and renal damage in males. Further studies are needed to assess the inflammatory status of immune cells from these animals as well as alternative TLR4-expressing cell types responsible for promoting disease. 23PRE1020121, HL161231, HL166458. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loop Diuretic Resistance in a Mouse Model of Cardiorenal Syndrome

Nearly 30% of patients with heart failure (HF) develop Cardiorenal syndrome type 2 (CRS2), characterized by renal injury and lower glomerular filtration rate (GFR) secondary to lower cardiac function. Patients with uncompensated heart failure develop edema and are usually treated with a loop diuretic, such as furosemide, that acts in thick ascending limb (TAL) to inhibit NKCC2. However, over 50% of HF patients develop resistance to loop diuretics. The molecular mechanisms underlying the development of diuretic resistance are not clear and may involve decreased NKCC2-mediated NaCl transport, enhanced distal tubule NaCl reabsorption, or decreased accessibility of the diuretic drug to the nephron lumen. Few studies have used the mouse as a model of cardiorenal syndrome after myocardial infarction (MI). We hypothesize that C57Bl6J mice would develop kidney damage several months after myocardial infarction (MI) and resistance to loop diuretics. Heart failure was induced in C57Bl6J mice through ligation of the left anterior descending coronary artery (LAD) and classified as MI group and compared to a sham surgery group (without ligation) as controls. Both groups were assessed for heart function by echocardiography and renal filtration function by measuring GFR (FITC inulin) in conscious mice. The response to diuretics was studied in metabolic cages, 7-8 months after MI. MI mice had a 50% reduction in ejection fraction (EF) 4 weeks after MI compared to sham group (MI: 29.94 ± 1.94, Sham: 67.02 ± 1.14, n= 8 each group, p<0.05). Decreased EF continued to be lower 6 and 12 months after MI. Six months after surgery, the MI group showed decreased GFR compared to Sham (MI: 0.50 ± 0.06, Sham: 0.75±0.05 μl/min, n= 5 MI, n=7 sham, p<0.05). Despite lower GFR, 24h-urinary albumin excretion was similar in both groups (n=8 each group). In metabolic cage studies, a saturating dose of the NKCC2 inhibitor bumetanide (20 mg/Kg), induced diuresis and natriuresis in sham mice (Delta UV: 1.96 ± 0.27 ml/4h, Delta UNa: 117.2 ± 11.02μmols/4h, UCl: 175.65 ± 13.01 μmols/4h, n= 6) but had a smaller effect in MI mice which exhibited an attenuated diuretic response (Delta UV: 0.88 ± 0.23 ml/4h, Delta UNa: 65.00 ± 4.53 μmols/4h, UCl: 102.89 ± 14.08 μmols/4h, n= 4, p<0.05 vs sham), indicative of loop diuretic resistance. To study if bumetanide resistance is secondary to enhanced distal convoluted tubule (DCT) NaCl reabsorption, we measured the diuretic response to the NCC inhibitor hydrochlorothiazide (HCTZ). HCTZ (100 mg/Kg) induced diuresis and natriuresis in sham mice (Delta UNa: 75.24±11.18μmols/4h, UCl: 39.44±10.15 μmols/4h, n= 10) and the diuretic response was slightly higher in the MI group (Delta UNa: 129.54±11.59μmols/4h, UCl: 94.61±11.56 μmols/4h, n= 12, p<0.05 vs sham). We conclude that chronic heart failure in C57Bl6J mice, decreases GFR and induces loop diuretic resistance. Loop diuretic resistance after MI may be secondary to enhanced DCT NaCl transport in mice. This model could be useful to study the mechanisms causing decreased renal function and loop diuretic resistance after MI and pursuing new therapeutic avenues. No funding was received by the authors for conducting this study. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Urine Exosomal MicroRNA Profiling in the Family Investigation of Nephropathy and Diabetes (FIND)

About 1 in 3 adults with either type 1 or type 2 diabetes (T2D) has some degree of renal injury or diabetic nephropathy (DN). There is a need to develop more sensitive and specific means of detecting and gauging DN progression so that therapies can be started as early as possible. We hypothesized that UE microRNAs (miRs), small, non-coding 21-25 nucleotide species, a relatively stable readout of renal metabolic health, could be utilized in this fashion. From the NIH, NIDDK Central Repository, we obtained 299 urine samples (300 ml) from African American subjects previously enrolled in the FIND study. The FIND study was initially designed to elucidate genetic determinants of DN in related patient populations using both T2D and non-diabetic control (CTRL), male (M) and female (F) subjects. Our samples included: 20 CTRL-M, 46 CTRL-F, 43 T2D-M, and 90 T2D-F). Other deidentified demographic and clinical data was obtained from the NIH. We measured urine albumin and creatinine in the samples and calculated ratios (mg albumin/g creatinine). Patients were categorized as normal (N, < 30, 120 subjects), microalbuminuric (MIC, 30-300, 33 subjects), or macroalbuminuric (MAC, >300, 46 subjects). Urine exosomes (UE) were isolated from each urine using the Total Exosome Isolation (from urine) kit (Thermofisher), which allows for precipitation of water-insoluble exosomes via centrifugation at medium speed (10,000g) for 1 hour. Small RNA was isolated followed by cDNA synthesis using commercially-available kits (Qiagen). Previously, we found miR-451a to be more highly expressed in UE from human subjects with chronic kidney disease, than in control subjects. We also found this miR to be a potential predictive biomarker for albuminuria in diabetic rats. Therefore, first we used conventional qRT-PCR to evaluate UE levels of miR-451a, as well as, the anti-sense product, miR-451b. MiR-127 was used as the internal standard, as it has been reported not to change with T2D. In MAC subjects, we found that the UE median miR-451a level increased with a fold change (FC) of 3.5 (relative to N). Similarly, miR-451b showed a 3.6-fold increase. FC in the MIC groups were 2.9 and 2.5 for miR-451a and b, respectively (relative to N). Next we conducted an unbiased screen of commonly excreted miRs in these subjects using a miR Array (miRCURY LNA miRNA Focus PCR panel, Qiagen) configured with 88 targets highly expressed in UE. We screened 19 subjects representing a range of conditions, i.e., albuminuric and normal, diabetic and non-diabetic, male and female. We found positive signals in 5 or more subjects for 16 miRs. Of those, 12, i.e., let-7b-5p, miR-16-5p, miR-23b-3p, miR-26a-5p, miR-27a-3p, miR-30a-5p, miR-30b-5p, miR-30c-5p, miR30e-5p, miR-141-3p, miR-203a, and miR-204-5p were expressed at mean levels 2-fold higher in MAC versus N subjects. Furthermore, 13 of these miRs were also over 2-fold higher when subjects were divided as T2D versus CTRL, i.e., the above list plus miR-200c-3p and miR-30d-5p, but not let-7b-5p in this case. When subjects were not divided by groups, miR-598-5p had the highest positive correlation with urinary albumin excretion, R = 0.53 and p = 0.035 (for slope ≠ 0). Using miRNet (McGill University), a freely available miR analysis software tool, we mined for common targets of miR-451(a & b) and miR-598. One putative target of both miR-451b and miR-598 was vascular endothelial growth factor A (VEGFA), which has been shown to play a role in DN. In sum, these miRs represent candidate targets to undergo additional scrutiny. We conclude UE miRs may hold clinical value in the non-invasive assessment of DN severity and predicting progression. NIH/NINDS U01-DK103225 (Feldman, PI); Ecelbarger Pilot. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neutrophil to lymphocyte ratio (NLR) as a surrogate marker of diabetic nephropathy

Background and objectives: Diabetic nephropathy and retinopathy are microvascular complications of diabetes. Nephropathy is clinically manifested as an increase in urine albumin excretion. Total white blood cell count is a crude but sensitive indicator of inflammation and studied in many cardiac and non-cardiac diseases as an inflammatory marker such as acute myocardial infarction, stroke, and heart failure. In this study, the association of neutrophil to lymphocyte ratio (NLR) with diabetic nephropathy and retinopathy. Methods: This was a prospective observational study performed on patients with diabetic nephropathy +/- retinopathy. A total of v104 subjects diagnosed with 3type 2 diabetes mellitus registered in this study. NLR was calculated by analyzing differential leukocyte count in complete blood picture. Spot Albuminuria was tested by immune turbidimetry method. Urine creatinine was tested by jaffe’s kinetic method by which spot urine microalbumin to creatinine ratio is calculated. ANOVA, Independent t-test, chi-square test, Karl Pearson correlation were applied to find significance. P<0.05 was considered as statistically significant. Results: Totally 104 diabetic subjects were registered, 52 subjects had DN and 52 had normal urine albumin. Mean±SD NLR for a normal group is 2.5±1.1 and in DN group is 3.4±1.4 which was highly significant (P =0.001). Conclusion: Our study showed that there was a significant relation between NLR and DN; and positive correlation between NLR and urine microalbumin to creatinine ratio. Therefore, NLR may be considered as a novel surrogate marker of DN.

Chronic kidney disease and risk of bloodstream infections and sepsis: a 17-year follow-up of the population-based Trøndelag Health Study in Norway

PurposeBloodstream infections (BSI) and sepsis are important causes of hospitalization, loss of health, and death globally. Targetable risk factors need to be identified to improve prevention and treatment. In this study, we aimed to evaluate the association of chronic kidney disease (CKD) and risk of and mortality from BSI and sepsis in the general population during a 22-year period.MethodsWe conducted a prospective cohort study among participants in the population-based Norwegian HUNT Study, where 68,438 participated. The median follow-up time was 17.4 years. The exposures were estimated glomerular filtration rate (eGFR) and albumin–creatinine ratio (ACR) in urine. The outcomes were hazard ratios (HR) of hospital admission or death due to BSI or sepsis. The associations were adjusted for age, sex, diabetes, obesity, systolic blood pressure, smoking status, and cardiovascular disease.ResultsParticipants with eGFR < 30 ml/min/1.732 had HR 3.35 for BSI (95% confidence intervals (CI) 2.12–5.3) and HR 2.94 for sepsis (95% CI 1.82–4.8) compared to normal eGFR (≥ 90 ml/min/1.732). HRs of death from BSI and sepsis were 4.2 (95% CI 1.71–10.4) and 4.1 (95% CI 1.88–8.9), respectively. Participants with severely increased albuminuria (ACR > 30 mg/mmol) had HR 3.60 for BSI (95% CI 2.30–5.6) and 3.14 for sepsis (95% CI 1.94–5.1) compared to normal albumin excretion (ACR < 3 mg/mmol). HRs of death were 2.67 (95% CI 0.82–8.7) and 2.16 (95% CI 0.78–6.0), respectively.ConclusionIn this large population-based cohort study, CKD was clearly associated with an increased risk of BSI and sepsis and related death.

Metabolic control in type 2 diabetic patients treated with empagliflozin: A case series.

Type 2 diabetes mellitus is a highly prevalent disease and is associated with increased morbidity and mortality. Due to the low percentage of adequate glycemic control, new strategies for the treatment of type 2 diabetes mellitus have been sought, including sodium-glucose cotransporter type 2 inhibitorss. To describe the evolution of patients with type 2 diabetes mellitus with insulin requirements treated with empagliflozin at the Peñaflor Hospital. The primary objective was to evaluate the efficacy of the medication regarding glycosylated hemoglobin A1c (HbA1c). The secondary objectives were: 1) achievement of HbA1c equal to or less than 7.5% according to survival analysis. 2) Change in glomerular filtration rate and urinary albumin excretion post treatment. Review of clinical records of all patients treated with empagliflozin from November 2019 to June 2023. Average follow-up of 19 (16.3 to 40) months. To compare HbA1c values according to follow-up ranges, the paired T test or Wilcoxon test was used. We included 58 patients, 15 men and 43 women (74.1%), with an average age of 58.5 ± 9.2 years, ranging from 35 to 75 years. Baseline HbA1c of 10.3 ± 1.6% and 8.98% ± 2.2 in a follow-up of 18 to 24 months post-treatment, resulted in a decrease of 1.27% (p = 0.002; confidence interval 95%: 0.5 to 2.03). The most common adverse effect was urinary tract infection. Patients with type 2 diabetes mellitus with insulin requirements treated with empagliflozin at the Peñaflor Hospital achieved better glycemic control with few adverse effects.

Complications during Adolescence Predict Mortality in Young Adults with Childhood Onset Type 1 Diabetes

Objective. Microvascular complications increase the risk of cardiovascular disease and premature death in adults with type 1 diabetes. We examined the association between microvascular complications during adolescence, including cardiac autonomic nerve dysfunction and subsequent mortality. Research Design and Methods. We undertook data linkage with the Australian National Death Index in a cohort of 409 adolescents (diagnosed between 1973 and 1993), 48% male, median age at final complications assessment 17.4 years (interquartile range: 16.0–18.9), followed longitudinally for median 22.3 years (21.0–23.4) from diagnosis. Generalized estimating equations (GEE) were used to examine associations between mortality and adolescent complications. Mortality risk was calculated as standardized mortality ratio (SMR). Results. At final adolescent visit, 20% had CAN abnormality, 30% abnormal pupillary response, 20% albuminuria, 40% early elevation of albumin excretion rate (AER) and 45% retinopathy. Data linkage 8–13 years later showed 14 were deceased (3% of cohort), 57% male, median age 28.3 years (24.8–32.9). Acute or chronic diabetes complications accounted for 25% of deaths. In multivariable GEE, elevated AER (OR 4.54, 1.23–16.80, p=0.030), pupillary abnormality (OR 4.27, 1.20–15.22, p=0.023), systolic blood pressure SDS (OR 2.17, 1.26–3.74, p=0.005) and CAN (OR 4.65, 1.03–21.0, p=0.045) predicted mortality. HbA1c was not significant. SMR was 2.5 (1.4–4.2) and was higher in females (SMR 3.5, 1.3–7.8) but not in males (SMR 2.1, 0.9–4.0). Conclusion. Mortality in young adults with type 1 diabetes is predicted by subclinical markers of autonomic neuropathy and elevated AER during adolescence, but not glycemia. Mortality was over twice that of the background population in females but not in males.

Association of kidney function with physical performance: the Maastricht study.

Kidney failure has been associated with decreased physical capacity, although evidence regarding the physical performance of individuals with earlier stages of chronic kidney disease (CKD)remains limited. Cross-sectional data were derived from the prospective, population-based Maastricht Study. Multivariate linear regression models were fitted to assess the association of estimated glomerular filtration rate (eGFR) and albuminuria categories with physical performance test outcomes. Overall, 7396 participants were included. Compared to eGFR 60-90ml/min/1.73m2, values < 60ml/min/1.73m2 were associated with significantly shorter 6-min walk distance (β: - 13.04m, 95% confidence intervals-CI - 19.95; - 6.13), worse timed chair rise stand test time (β: 0.91s, 95% CI 0.36; 1.47), lower maximal grip (β: - 0.83kg, 95% CI - 1.50; - 0.15) and elbow flexion (β: - 3.64 Nm, 95% CI - 7.11; - 0.16) strength. Additionally, eGFR > 90ml/min/1.73m2 was linked to significantly shorter 6-min walk distance (β: - 6.13m, 95% CI - 9.44; - 2.82). Urinary albumin excretion > 30mg/24h was associated with shorter 6-min walk distance (β: - 12.48m, 95% CI - 18.28; - 6.68), worse timed chair rise stand test time (β: 0.51s, 95% CI 0.11; 1.06), lower maximal grip (β: - 1.34kg, 95% CI - 1.91; - 0.76) and elbow flexion strength (β: - 3.31Nm, 95% CI - 5.80; - 0.82). Reduced eGFR and higher albuminuria levels were associated with worse physical performance, especially shorter 6-min walk distance and lower muscle strength. The relationship between eGFR and physical function was non-linear, with also high eGFR values being associated with worse performance, especially in the six-minute walk test.

A Comparative Analysis of Fractional Excretion of Sodium, Potassium and Magnesium among T2DM Patients with Increased Albuminuria and Normal Albuminuria in a Tertiary Care Hospital- An Observational Study

Background: T2DM often leads to diabetic kidney disease, a condition marked by change in GFR and urinary albumin excretion. The renal handling of key electrolytes like sodium, potassium, and magnesium plays a crucial role in the progression of renal tubulointerstitial damage in diabetic patients. Aim of the study: The aim of this study was to compare the fractional excretion of sodium, potassium and magnesium among T2DM patients with increased albuminuria and normal albuminuria. Methods: This observational study was conducted in Department of Nephrology, Chattogram, Bangladesh from February 2021 to January 2022. Total 125 patients with T2DM were included in this study. Result: Baseline characteristics showed no significant differences across the study groups. Biochemically, eGFR decreased non-significantly from 95.5 to 86.1 ml/min/1.73 m² across groups, while UACR significantly increased from 14.3 to 1056 mg/gm (P&lt;0.001). FBG levels were similar, but 2hPPBG showed a significant decrease from 8.2 to 7.4 mmol/l (P=0.0041). HbA1C levels varied slightly. FENa, FEK, and FEMg showed significant differences across groups, with FENa ranging from 0.59% to 0.56% (P=0.0016), FEK from 9.4% to 6.9% (P=0.0358), and FEMg from 2.87% to 5.58% (P=0.001). Correlations revealed significant negative associations of eGFR with FENa, FEK, and FEMg, and a significant positive correlation of FEMg with UACR. No significant correlations were found between FBG, 2hPPBG, HbA1C, and the electrolytes excretion. Conclusion: Significant variations in the fractional excretion of sodium, potassium, and magnesium were observed. Higher eGFR was associated with lower electrolyte excretion, and a significant positive correlation was found between magnesium excretion and UACR.

Cell Salvage Using the Autotransfusion Device CATSmart®: A Randomized Controlled Bicentric Trial Evaluating the Quality of Two New Flex Wash Programs

Background: The use of cell salvage and autologous blood transfusion is an important and widespread method of blood conservation during surgeries with expected high blood loss. The continuous autotransfusion device CATSmart^® (Fresenius Kabi, Germany) contains two new washing programs on the device called Flex wash 3 and Flex wash 5. To the best of our knowledge, there are no published clinical data regarding the performance of the two new washing programs. Methods: In total, 69 patients undergoing cardiac or orthopedic surgery were included in this randomized, controlled, bicentric trial to validate the red cell separation process and washout quality of Flex wash 3 compared to Flex wash 5. After washing, the primary quality target was to determine hematocrit value, recovery rate, albumin, and total protein elimination rate in the packed red cells (PRCs). The secondary objective was to assess the elimination of heparin by measuring the factor anti-Xa activity by a 1- and 2-stage assay in PRC after washing. Results: In the whole cohort of patients, hematocrit was 16.00% [9.15%; 21.30%] (median [Q1; Q3]) in the wound blood and 69.90% [51.10%; 80.90%] in the PRC resulting in a recovery rate of 63.92% [47.06%; 88.13%]. The albumin elimination rate was 98.77% [97.94%; 99.27%], and the total protein elimination rate was 98.85% [97.76%; 99.42%]. The heparin elimination rate was 99.95% [99.90%; 99.97%] in the 1-stage assay and 99.70% [99.41%; 99.87%] in the 2-stage assay. There was no difference between Flex wash 3 and Flex wash 5 washing procedure regarding the recovery rate 63.75% [46.64%; 78.65%] versus 67.89% [47.20%; 92.69%] (p = 0.85), albumin elimination rate 98.74% [97.67%; 99.27%] versus 98.78% [98.10%; 99.28%] (p = 0.97), protein elimination rate 98.79% [97.94%; 99.47%] versus 98.92% [97.58%; 99.42%] (p = 0.88), and anti-Xa elimination rate in the 1-stage assay 99.94% [99.79%; 99.97%] versus 99.95% [99.92%; 99.97%] (p = 0.24) and in 2-stage assay 99.66% [99.20%; 99.86%] versus 99.77% [99.47%; 99.90%] (p = 0.23). Conclusions: The two new washing procedures, Flex wash 3 and Flex wash 5, enable sufficient and comparable red cell separation and washout quality of albumin, total protein, as well as heparin.

Baicalein improves renal interstitial fibrosis by inhibiting the ferroptosis in vivo and in vitro

Evidence indicates that Baicalein can ameliorate renal interstitial fibrosis by inducing myofibroblast apoptosis and inhibit the RLS3-induced ferroptosis in melanocytes. However, the relationship between renal interstitial fibrosis and anti-ferroptosis affected by Baicalein remains unclear. In our study, the anti-fibrosis and anti-ferroptosis effects of Baicalein were assessed in a rat model induced by the UUO method in vivo, and the effects of Baicalein on Erastin-induced ferroptosis of renal MPC-5 cells were examined by Western blot of fibrosis-related and ferroptosis-related proteins in vitro. In the UUO-induced rat model, Baicalein decreased kidney weight loss, improved renal function assessed the biomarks of urinary albumin excretion, serum creatine, and BUN levels, and reduced renal tubular injury. Furthermore, Baicalein inhibited renal ferroptosis by reducing ROS and MDA levels and increasing SOD and GSH levels in the UUO rat model. In addition, Baicalein potently reduced the expression of fibrosis-related proteins such as TGF-β1, a-SMA, and Smad-2 to prevent renal interstitial fibrosis, and increased the expression of ferroptosis-related proteins such as SLC7A11, GPX4, and FTH to inhibit ferroptosis both in vitro and in vivo. Taken together, Baicalein exerts anti-fibrosis activity by reducing the ferroptosis response on the UUO-induced rat model and renal MPC5 cells. Therefore, Baicalein, as a novel therapeutic method on kidney diseases with strong activity in suppressing ferroptosis, could be a potential alternative treatment for renal interstitial fibrosis.