Renal Glomerular Filtration Research Articles

Evaluation of concordance and association with clinical outcomes across eGFR values calculated with eight different formulas in a large cohort of european patients with atrial fibrillation

Abstract Background In randomized clinical trials of Direct Oral Anticoagulants (DOACs), the assessment of renal function impairment glomerular filtration rate (eGFR) in patients with Atrial Fibrillation (AF) has predominantly utilized the Cockroft-Gault (CG) formula for estimating Creatinine clearance (CrCl). However, in clinical practice, several other formulas for estimating glomerular filtration rate (eGFR) are employed. Limited data exist regarding the evaluation and comparison of eGFR/CrCl in AF patients. Objectives This study aimed to assess the concordance in the categorisation of renal function in in 6 classes taking into account eight different formulas for eGFR/CrCl (CG, CKD-EPI, CG/body surface area [BSA], BIS1, LMK, EKFC, FAS, MDRD) and their associations with a composite outcome (Major Adverse Cardiovascular Events [MACE] and all-cause death) in AF patients. Methods A contemporary European AF patient registry was utilized, and eGFR/CrCl were calculated using CG, CKD-EPI, CG/BSA, BIS1, LMK, EKFC, FAS, and MDRD formulas. eGFR was categorized according to KDIGO classes, with CKD patients defined as having eGFR/CrCl <60 ml/min/m2. Concordance was estimated using weighted Cohen’s kappa. Associations were evaluated through adjusted Cox regression analyses for the composite outcome, and results were plotted as spline curves. Results: A total of 8516 patients were included in the analysis (Median [IQR] age: 70 [63-77] years; 3518 women, 41.3%). Among the formulas, EKFC exhibited the highest median values of eGFR and the lowest prevalence of CKD patients, while BIS1 had the lowest median eGFR values, and the FAS formula led to the highest prevalence of CKD patients [Figure 1]. In the concordance analysis, CKD-EPI demonstrated the highest concordance with all other formulas, even if Cohen’s kappa for measuring the degree of concordance ranged from 0.61 to 0.88 [Figure 1]. In adjusted Cox regression analyses, all formulas were associated with an increased risk for the composite outcome, progressively as eGFR decreased. When plotted as spline curves, the associations with the composite outcome visually showed a similar behaviour of risks across eGFR values in all formulas compared to the CG spline [Figure 2]. Conclusions Among AF patients, the CKD-EPI formula exhibited variable, but substantially good concordance with CG, CG/BSA, BIS1, LMK, EKFC, FAS, and MDRD formulas. All calculated eGFR values showed a strong association with the composite outcomes, similarly to the CG formula, with higher risk when renal function decreased.FIGURE 1FIGURE 2

225 Kidney function and nitrogen excretion in Brown Swiss and Holstein dairy cows

Abstract The agricultural sector contributes to nitrogen (N) emissions since farm animals excrete considerable N amounts via feces and urine. To reduce N emissions by dairy cattle, methods predicting accurately individual N emissions are of great interest. Recent data showed that Brown Swiss (BS) cows have greater urea concentrations in milk and blood than Holstein (HO) cows. However, it is still unknown if greater BUN concentrations in a particular breed are congruently reflected by concomitantly increased contents of nitrogenous compounds in other media (e.g., urine and saliva). Thus, the present study simultaneously assessed biomarkers for kidney function like urea, and creatinine and symmetric dimethylarginine (SDMA) concentrations in several body fluids (i.e., blood, urine, milk, and saliva) as well as nitrogen excretion in feces in BS and HO cows under identical feeding conditions. Blood, saliva, urine, and feces were sampled in 31 multiparous BS and 46 HO cows kept under identical feeding and management conditions. Samples were collected at different lactational stages after the monthly DHIA control test-day. Concentrations of urea and creatinine were measured in serum, urine, and saliva. Symmetric dimethylarginine was determined in serum, which is an established indicator for glomerular filtration rate (GFR). Feces were analyzed for dry matter content and nitrogen concentrations. Data on milk urea and protein concentrations, and daily milk yield were obtained from the monthly DHIA test-day records. The effects of breed, time, and parity number on blood, saliva, urine, feces, and milk parameters were evaluated with the GLM procedure with breed, time, and parity number as fixed effects. Differences between BS and HO were assessed by the Tukey-corrected t-test at P < 0.05. Dry matter intake and body weight were similar in BS and HO cows (P > 0.05). No differences between BS and HO were observed for milk yield, fecal DM and nitrogen content. BS cows showed greater urea concentration (25.8 ± 0.7 vs 21.8 ± 0.7 mg/dL), and protein content in milk (3.70 ± 0.08 vs 3.45 ± 0.07%) than HO cows (P < 0.01). Concentrations of urea, creatinine, and SDMA in serum, were greater in BS than in HO cows (P < 0.01) with 5.46 ± 0.19 vs 4.72 ± 0.13 mmol/L for urea, 105.96 ± 2.23 vs 93.07 ± 1.50 mmol/l creatinine, and 16.78 ± 0.69 vs 13.39 ± 0.44 µg/dL SDMA, respectively. Urea and creatinine concentrations in urine and saliva did not differ among breeds. Despite greater urea and creatinine concentrations in blood, and milk urea content in BS compared with HO, excretion of these parameters did not differ between breeds in urine. Our results suggest a reduced renal glomerular filtration rate in BS compared with HO. The underlying mechanisms require further investigations.

Abstract P356: Sex-dependent Response of ATP-mediated Calcium in Renal Microvascular Smooth Muscle Cells in Ischemia-reperfusion Rats

Renal autoregulation is intrinsic to preglomerular microvascular smooth muscle cells (MVSMC) for maintaining stable renal blood flow and glomerular filtration rate during arterial pressure fluctuations. Our recent study suggests that renal ischemia-reperfusion (IR) leads to sex-dependent autoregulatory impairment of afferent arterioles. Renal autoregulation is linked to ATP sensitive-Ca 2+ signaling in MVSMC via activation of P2X purinoceptors (P2X). ATP vasoconstricts afferent arterioles by increasing intracellular calcium concentration ([Ca 2+ ] i ) in male rat MVSMC but female rats have not been studied. We postulate that female MVSMC exhibit highly sensitive ATP-mediated Ca 2+ responses which may protect females against IR. Blood-perfused juxtamedullary nephron experiments were performed on kidneys of Sprague-Dawley rats 24 hours after IR (60-min, bilateral renal artery clamping). ATP-induced changes in [Ca 2+ ] i were assessed in freshly isolated MVSMC using Fura-2. Superfusion of ATP (10 -8 –10 -4 M) elicited concentration-dependent vasoconstriction in sham rats of both sexes. The maximal vasoconstriction was 68±2% of the control diameters at 10 -4 mol/L in males and 72±9% in females (n=3/group). In contrast, ATP-induced vasoconstriction was nearly abolished in both sexes 24 h post-IR. The maximal vasoconstriction only reached 91±6% and 97±3% of control diameter in male and female IR rats (n=3/group, P <0.05 vs. shams), respectively, indicating impaired P2X signaling in afferent arterioles post-IR. ATP exposure increased [Ca 2+ ] i in control MVSMC of both sexes by 0.03±0.00 and 0.10±0.01 in males and 0.19±0.02 and 0.43±0.05 in females at 10 -5 and 10 -4 M ATP, respectively. The ATP-induced increase in [Ca 2+ ] i was 4-7 fold higher in female than in male controls, suggesting that MVSMC from normal female rats exhibit a highly sensitive response to ATP stimulation versus male’s. IR suppressed ATP-induced calcium responses in MVSMC of both sexes. [Ca 2+ ] i increased by 0.02±0.00 and 0.04±0.01 in IR males and 0.09±0.01 and 0.09±0.01 in IR females in response to 10 -5 and 10 -4 M ATP, respectively (P <0.05 vs control, n=60-150 cells/3-4 rats/group). Female IR MVSMC however, still possess higher reactivity to ATP than MVSMC from male shams. In conclusion, our studies suggest that MVSMC of male and female rats handle ATP-Ca 2+ signaling differently and the stronger ATP-mediated [Ca 2+ ] i response in female MVSMC may protect females against IR-induced kidney injury.

The efficacy of sacubitril/valsartan in patients receiving peritoneal dialysis with diabetes and heart failure with preserved ejection fraction.

To investigate the efficacy of sacubitril/valsartan in the treatment of peritoneal dialysis (PD) in patients with diabetes and heart failure with preserved ejection fraction (HFpEF). Patients with diabetes who underwent PD and had HFpEF (n = 64) were divided into two groups: the experimental group (n = 31), which was administered sacubitril/valsartan, and the control group (n = 33), administered valsartan alone. Data were collected before and after treatment to compare the inter-group changes in cardiac function indexes, residual renal function (RRF), and PD adequacy indexes. Compared with the control group, the N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were lower in the experimental group after treatment (Wilcoxon test, p < 0.05). The descent ranges of NT-proBNP, left ventricular end-systolic dimension, and left ventricular fraction shortening, as well as increases in the amplitude of left ventricular ejection fraction after treatment were better in the experimental group than in the control group (t-test, p < 0.05). The descent ranges of residual renal glomerular filtration rate, residual renal Kt/Vurea, and residual renal creatinine clearance, as well as increases in the amplitude of β2-microglobulin, were lower in the experimental group than in the control group (Wilcoxon test, p < 0.05). However, there were no significant differences between the two groups in the descent ranges of the PD adequacy indexes (Wilcoxon test, p > 0.05). Hyperkalemia occurred in 8 cases (25.81%) in the experimental group and 13 cases (39.39%) in the control group, while hypotension occurred in 2 cases (6.45%) and 1 case (3.03%), respectively. No other adverse effects were observed in either group. The findings suggest that sacubitril/valsartan can safely and effectively improve RRF and cardiac function in patients with diabetes combined with HFpEF receiving PD, but it has little effect on PD adequacy.

The physiological basis of renal nuclear medicine.

Renal physiology underpins renal nuclear medicine, both academic and clinical. Clearance, an important concept in renal physiology, comprises tissue uptake rate of tracer (tissue clearance), disappearance rate from plasma (plasma clearance), appearance rate in urine (urinary clearance) and disappearance rate from tissue. In clinical research, steady-state plasma clearances of para-amino-hippurate and inulin have been widely used to measure renal blood flow (RBF) and glomerular filtration rate (GFR), respectively. Routinely, GFR is measured at non-steady state as plasma clearance of a filtration agent, such as technetium-99m diethylenetriaminepentaacetic acid. Scaled to three-dimensional whole body metrics rather than body surface area, GFR in women is higher than in men but declines faster with age. Age-related decline is predominantly from nephron loss. Tubular function determines parenchymal transit time, which is important in renography, and the route of uptake of technetium-99m dimercaptosuccinic acid, which is via filtration. Resistance to flow is defined according to the pressure-flow relationship but in renography, only transit time can be measured, which, being equal to urine flow divided by collecting system volume, introduces further uncertainty because the volume is also unmeasurable. Tubuloglomerular feedback governs RBF and GFR, is regulated by the macula densa, mediated by adenosine and renin, and can be manipulated with proximal tubular sodium-glucose cotransporter-2 inhibitors. Other determinants of renal haemodynamics include prostaglandins, nitric oxide and dopamine, while protein meal and amino acid infusion are used to measure renal functional reserve. In conclusion, for measuring renal responses to exogenous agents, steady-state para-amino-hippurate and inulin clearances should be replaced with rubidium-82 and gallium-68 EDTA for measuring RBF and GFR.

Kidney function, but not nitrogen excretion, differs between Brown Swiss and Holstein dairy cows

Brown Swiss (BS) cows have greater urea concentrations in milk and blood compared with Holstein (HO) cows. We tested the hypothesis that BS and HO cows differ in kidney function and nitrogen excretion. Blood, saliva, urine, and feces were sampled in 31 multiparous BS and 46 HO cows kept under identical feeding and management conditions. Samples were collected at different lactational stages after the monthly DHIA control test-day. To test the glomerular filtration rate (GFR) and urea excretion, concentrations of creatinine and urea were measured in serum, urine, and saliva. As an additional marker to estimate GFR, we determined symmetric dimethylarginine (SDMA) in serum. Feces were analyzed for dry matter content and nitrogen concentration. Data on milk urea and protein concentrations, and daily milk yield were obtained from the monthly DHIA test-day records. The effects of breed, time, and parity number on blood, saliva, urine, feces, and milk parameters were evaluated with the GLM procedure with breed, time, and parity number as fixed effects. Differences between BS and HO were assessed by the Tukey-corrected t-test at P < 0.05. Concentrations of urea, creatinine, and SDMA in serum, were greater in BS than in HO cows (P < 0.01): 5.46 ± 0.19 vs 4.72 ± 0.13 mmol/L (urea), 105.96 ± 2.23 vs 93.07 ± 1.50 mmol/l (creatinine), and 16.78 ± 0.69 vs 13.39 ± 0.44 µg/dL (SDMA). We observed a greater urea concentration in BS cows (25.8 ± 0.7 vs 21.8 ± 0.7 mg/dL) and protein content in milk (3.70 ± 0.08 vs 3.45 ± 0.07%) than in HO cows (P < 0.01). Urea and creatinine concentrations in urine and saliva did not differ among breeds. No differences between BS and HO were observed for milk yield, fecal DM, and fecal nitrogen content. Dry matter intake and body weight were similar in BS and HO cows (P > 0.05). Despite greater urea, creatinine, and SDMA concentrations in blood as well as a higher milk urea content in BS compared with HO, respective concentrations in urine did not differ between breeds. In conclusion, our results demonstrate a lower renal GFR in BS compared with HO cows, thereby contributing to the greater plasma urea concentration in BS cows. However, estimation of nitrogen excretion via milk, urine, and feces does not entirely reflect nitrogen turnover within the animal.

Selection of a suitable animal model to evaluate secretion of drugs in the human milk: a systematic approach

Lack of data on drug secretion in human milk is a concern for safe use of drugs during postpartum. Clinical studies are often difficult to perform; despite substantial improvements in computational methodologies such as physiologically based pharmacokinetic modelling, there is limited clinical data to validate such models for many drugs. Various factors that are likely to impact milk to plasma ratio were identified. A literature search was performed to gather available data on milk composition, total volume of milk produced per day, milk pH, haematocrit, and renal blood flow and glomerular filtration rate in various animal models. BLAST nucleotide and protein tools were used to evaluate the similarities between humans and animals in the expression and predominance of selected drug transporters, metabolic enzymes, and blood proteins. A multistep analysis of all the potential variables affecting drug secretion was considered to identify most appropriate animal model. The practicality of using the animal in a lab setting was also considered. Donkeys and goats were identified as the most suitable animals for studying drug secretion in milk and future studies should be performed in goats and donkeys to validate the preliminary observations.

#246 Understanding the impact of antiangiogenic drug-induced proteinuria on kidney function in cancer patients

Abstract Background and Aims Proteinuria is a common adverse event associated with the use of vascular endothelial growth factor (VEGF) inhibitor drugs, which are widely employed in oncology for treating various tumors. The incidence of proteinuria of all degrees ranges from 10% to 90%, with proteinuria in the nephrotic range occurring in 1-5% of cases. VEGF plays a crucial role in maintaining the integrity of the glomerular capillary wall. Podocytes, constitutively express VEGF, and VEGF receptors are present on both the surface of glomerular capillary endothelial cells and podocytes. Any disruption in the podocyte-endothelial VEGF axis can lead to the loss of endothelial fenestrations in glomerular capillaries, proliferation of glomerular endothelial cells, loss of podocytes, and subsequent proteinuria. The most common pathological findings include focal segmental glomerulosclerosis and minimal change nephropathy, typically associated with tyrosine kinase inhibitors (TKIs), as well as thrombotic microangiopathy (TMA), more frequently observed in pts treated with anti VEGF. Proteinuria is a recognized risk factor for progressive renal damage and end-stage renal disease. However, it's worth noting that the average treatment duration for patients on anti-angiogenic therapy is considerably shorter than the time indicated by clinical studies as necessary to induce renal damage from proteinuria in healthy individuals. However, the long-term impact of drug-induced proteinuria on renal function remains uncertain. The aim of the study is to investigate the association between proteinuria induced by anti-VEGF/VEGFR drugs and its long-term effects on renal function in cancer patients. Method A retrospective study was conducted on 124 patients undergoing anti-VEGF/VEGFR therapy for ≥6 months. 45% of the patients enrolled in the study were on TKI monotherapy, 36% received TKI plus immune checkpoint inhibitors (ICIs), 10% received anti- VEGF treatment, 7% received TKI + chemotherapy (CT), and 2% received anti-VEGF + ICIs. Among patients, 51% were undergoing treatment for kidney cancer, 25% for thyroid cancer, 14% for colon cancer, 7% for liver cancer, and 5% for lung cancer. The study assessed the time course of estimated glomerular filtration rate (eGFR), the influence of baseline eGFR on subsequent variations, the association between proteinuria and renal function, and the identification of risk factors for proteinuria or renal failure. Proteinuria was quantified as proteinuria/24 h. Results Among the 124 patients, 53 developed proteinuria &amp;lt;1 g/24 h, 41 between 1-3 g/24 h, and 30 &amp;gt;3 g/24 h. Specific data are outlined in Table 1. A trend toward a reduction in eGFR of −5 ml/min/1.73 m2 was observed in all patients after 2 years of treatment. The high proteinuria group exhibited a greater percentage change in eGFR compared to the other groups (−1.5% vs −8.5% vs −12.3%) as depicted in Fig. 1A, along with a time-dependent decrease in eGFR. There was no significant difference in the development of eGFR &amp;lt;30 ml/min/1.73 m2 between groups, and no patients permanently discontinued treatment due to nephrotoxicity. No differences in eGFR reduction were noted among the three groups categorized by baseline eGFR (Fig. 1B). Importantly, renal function after the end of therapy was found to be reversible. Conclusion The study did not find a correlation between the degree of proteinuria secondary to anti-VEGF/VEGFR and significant reductions in renal function. Therefore, treatment can be continued irrespective of the degree of proteinuria. Clinical decisions regarding the continuation of therapy with these drugs should weigh the benefits in terms of survival against the potential risks of toxicity, including a modest reduction in renal glomerular filtration rate.

#621 The effect of intra-operative mean arterial pressure on early graft function in deceased donor kidney transplantation

Abstract Background and Aims The adequate perfusion pressure to the graft is essential for proper graft function in kidney transplantation, especially in deceased donor kidney transplantation. In particular, Mean arterial pressure (MAP) during operation can affect early graft function because MAP is associated with renal blood flow and glomerular filtration rate. Therefore, the analysis of intra-operative parameters are necessary to evaluate adequate graft perfusion. The aim of this study is to investigate the relationship between intra-operative parameters including MAP and early graft function in deceased donor kidney transplantation. Method We retrospectively analyzed 363 recipients who underwent deceased donor kidney transplantation from March 2010 to December 2020. Anesthetic monitoring data during intraoperative period was analyzed and basic clinical parameters were evaluated. Results In total 363 recipients, the mean recipient age was 48.9 ± 10.3 and mean donor age was 48.2 ± 15.2. Anesthetic time was mean 285.9 ± 63.8 (min) and operation time was 226.4 ± 48.7 (min). Median value of baseline MAP and MAP at reperfusion were 124 mmHg and 88 mmHg. After initial analysis, the recipients were divided into two groups, high MAP group (n = 185) and low MAP group (n = 178), according to median value of baseline MAP (124 mmHg) and these two groups were analyzed. High MAP group showed higher estimated glomerular filtration rate (eGFR) and urine output compared to low MAP group during immediate postoperative 1 week. At postoperative day 5, eGFRs were 43.4 ± 26.7 and 37.3 ± 24.2 mL/min/1.73 m2 respectively (p = 0.022) and urine outputs were 2942.6 ± 1368.6 and 2474.3 ± 1199.1 mL respectively (p = 0.001). The incidences of delayed graft function showed no significant difference between two groups (2.2% vs 6.2%, p = 0.066). We additionally analyzed the effect of MAP at reperfusion, and the result showed that there was no significant relationship between MAP at reperfusion and early graft function. Conclusion In this retrospective study, MAP at reperfusion was not significantly related to early graft function and incidence of delayed graft function. In baseline MAP analysis, high baseline MAP group showed early recovery of eGFR and more urine output than low baseline MAP group. As a result, the recipients with high baseline MAP were related to early recovery of graft function in deceased donor kidney transplantation.

#185 The role of angiotensin 2 type 1 receptor and type 2 receptor after postischemic acute kidney injury in spontaneously hypertensive rats

Abstract Background and Aims Renal ischemia/reperfusion injury is a common cause of acute kidney injury (AKI), and hypertension might contribute to the increased incidence of AKI. AKI has a multifactorial causality, but the mechanism of pathogenesis and the development of this disease are still incompletely defined. The purpose of this study was to compare the effects of AT1R blockade and AT2R simulation on AKI development in spontaneously hypertensive rats (SHR). Method We used male 24 weeks old SHR. Animals were divided into following experimental groups: sham operated rats (SHAM); rats with induced postischemic AKI (AKI); a group which received AT1R antagonist Losartan (10 mg/kg b.w.)(AKI+LOS) and group which received AT2R agonist novokinin (0.3 mg/kg b.w.)(AKI+NOVO) in the moment of kidney reperfusion. AKI was induced by surgical removal of right kidney followed by atraumatic clamp occlusion of the left renal artery for 40 minutes. Hemodynamic and biochemical measurements were done 24 hours after reperfusion. Results Conclusion Our results confirm the dominant role of AT1R on AKI development, but AT2R stimulation failed to buffer deleterious effects of AT1R hyperactivity on renal vasoconstriction and glomerular filtration. However, the role of AT2R during AKI development in hypertension will be in focus in our further studies.

#650 Predictive value of LKDPI on recipient's renal function one year after kidney transplantation: single-centre experience

Abstract Background and Aims The selection of the appropriate living kidney donor can be challenging, given the absence of a definitive method to determine the quality of the renal graft. This decision requires a delicate balance, weighing the recipient's benefits while prioritizing the safety of the donor. Recently, a tool known as the Living Kidney Donor Profile Index (LKDPI) was proposed to assess living donor kidneys, based on various donor characteristics, so as to predict the probability of recipient's graft loss; however, the validation of its effectiveness still needs to be proven. Method We evaluated retrospectively 147 stable living kidney donors, who donated between 01/2021 and 11/2023 and are currently attending the Living Donor Clinic of our Department. Our study aimed to assess the impact of the LKDPI on the recipient's estimated renal glomerular filtration rate (eGFR) (measured by the CKD-EPI) one-year posttransplant. Additionally, dialysis vintage, cause of CKD, comorbidities and medication were also determined in all recipients. Results The study comprised 147 stable kidney recipients (KTR), each with their respective living donor. Of these, 96 (65.3%) were males, with a mean age 44.02 ± 13.96 years and a mean dialysis vintage of 1.8 ± 2.3 years. In parallel, living kidney donors were predominantly females (70.7%), with a mean age of 59.08 ± 10.36 years and a mean eGFR prior to donation of 95 ± 13.11 ml/min/1.73 m². Furthermore, 20.1% of kidney transplantations were ABO incompatible and the mean LKDPI score was 34.6 ± 25.9%. Data analysis revealed a statistically significant negative correlation between LKDPI and the recipient's GFR one year after transplantation (p &amp;lt; 0.0001), suggesting that higher LKDPI scores were associated with lower renal function in recipients. In a post hoc analysis, KTRs who were on CKD stage 1, one-year post-transplantation, demonstrated no statistical difference in LKDPI values, compared to stage 2 (p = 0.35). Meanwhile recipients on stage 3 exhibited comparable LKDPI values to those on stage 4 (p = 0.821). The main statistical discrepancy was particularly evident between CKD stage 2 and 3, as recipients with a LKDPI surpassing 25.6, experienced a transition from stage 2 to stage 3 within the first year following transplantation (p &amp;lt; 0.0001). In addition, after multivariate analysis there were no correlations between KTR's eGFR and age difference between donor and recipient or recipient's smoking history or medication. Last, dialysis vintage or the number of previous transplantations did not affect recipient's kidney function. Conclusion LKDPI seems to be a promising tool for predicting the outcome of kidney transplantation, concerning the renal function of KTR particularly at the end of the first-year post-transplant However, it needs to be thoroughly evaluated before being considered as a decision-making factor in living kidney transplantations.

#2716 Selenoprotein-P1 (SEPP-1) in human proximal tubule cells after ischemic-reperfusion injury: an in-vitro model

Abstract Background and Aims Novel biomarkers, anticipating serum creatinine rise, are eagerly needed to have a speedy diagnosis of acute kidney injury (AKI) and thus to mitigate and to preserve kidney function with well-timed therapeutic plans. Recently, in a pilot study on patients undergoing elective major cardiac surgery with cardiopulmonary bypass (CPB), selenoprotein-p1 (SEPP-1) has emerged as a promising candidate plasmatic biomarker for an early AKI risk stratification. SEPP-1 increased particularly between 4th to 8th hour after CPB and with a strict relation with ischemia duration (Bolignano et Al. Rev. Cardiovasc. Med. 2022). SEPP1 is primarily secreted from liver and acts as a selenium transporter supplying tissues and organs with this trace mineral which elicits the activity of specific glutathione peroxidase selenoenzymes. Additionally, renal tubular cells are able to synthetize this protein but remain unknown if, during AKI, SEPP1 increases as defeat answer to renal cells damage or if it's hepatic SEPP1 that accumulates for a reduced renal glomerular filtration rate. The primary aim of this study was to investigate the release of SEPP-1 by renal tubular cells in an experimental model of AKI induced by ischemia-reperfusion and secondarily evaluate the effect of selenium supplying on renal cells injury. Method Human proximal tubular cells (PTC), HK2 cells and human embryonic kidney 293 cells (HEK293) were cultured in Keratinocyte-SFM a complete serum-free medium supplemented with human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE) (LGC Standards s.r.l., Milan, Italy) and in Eagle's Minimum Essential Medium (E-MEM) (LGC Standards s.r.l., Milan, Italy) containing 10% fetal bovine serum, respectively. The cells were pre-incubated with medium containing Sodium Selenite 100 nM for 24 h and then were treated for 24 hours with CoCl2 (Sigma Aldrich S.r.l., Milan, Italy) a chemical hypoxia inducer (500 µM for HK-2 and 250 µM for HEK-293). After that cell viability was evaluated by MTT and trypan bleu dye exclusion assay, while, protein expression of HIF (hypoxia marker) and SEPP1 was analyzed by western blot analysis. Results Ischemia-reperfusion was simulated by the exposure of HK-2 and HEK-293 to CoCl2 and confirmed by HIF expression. The expression of SEPP-1 increased significantly in HK-2 and HEK-293 after hypoxia stress (Fig. 1). Furthermore, the treatment with CoCl2 determined a decrease of cell viability (about 50% less) and Sodium selenite alone increased cell proliferation (about 15% more) in both cell lines. Interestingly cell growth after hypoxia augmented significantly if cell lines were pre-treated with sodium-selenite, highly in HK-2 cells (p &amp;lt; 0.001). Conclusion These preliminary data evidenced, in vitro, that AKI is able to induce renal tubular expression of SEPP-1 and probably SEPP-1 release in human after renal injury is not only a result of a reduced filtration of the hepatic released protein. Furthermore, SEPP-1 showed a possible protective role in AKI. In fact, AKI induced by ischemia-reperfusion, in our in vitro cellular model, was able to induce an increased expression of SEPP-1 in renal tubular cells suggesting a shielding mechanism against injury related to selenium transport. This hypothesis was supported by an exalted SEPP-1 expression after Sodium-Selenite adding to cells’ culture. Supplementary experiments, including functional molecular assays in vitro and in vivo studies will allow to better characterize the role of SEPP-1 in AKI.

#930 Elevated glucose in kidney organoids induces epithelial detachment driven by proinflammatory cytokines

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease affecting 40% of diabetics. The pathogenesis of DKD is linked with inflammation, altered metabolism, and hemodynamic change but the relationship between these factors is not well understood. Proteinuria and albuminuria are common features, suggesting that impairments in the renal glomerular filtration barrier may be among the primary disease initiators. Limited DKD models are available, which has hampered our disease understanding and the ability to develop new therapies. Therefore, creating an in-vitro model that harbors multiple renal cell types organized in their natural complex arrangement becomes an attractive option to fill in this gap. Here, we conducted a systematic investigation of the effects of high glucose on kidney organoids, with the goal of understanding whether these cultures can reflect specific features relevant to DKD. Method To evaluate the effect of high glucose, kidney organoids were differentiated until maturation (day 21) in standard glucose and then switched for six days to concentrations of 5.5, 11, 33 and 100 mM. Then, to test the effect of proinflammatory cytokines on the injury phenotypes we switched organoids, at day 21, in control glucose condition (11 mM) ± TNFα or high glucose (33 mM) ± inhibitors (TNFα). Morphology was analysed via immunostaining, LDH and live/dead assay were used for toxicity, qPCR and western blotting to investigate differences in gene and protein expression, and mannose was used as an osmotic control. scRNA-seq of organoid in 11 vs 33 mM was conducted to gain insight into the molecular mechanism(s) contributing to the high glucose. Results Organoids remain well preserved in glucose up to 11 mM whereas organoids cultured in 33 and 100 mM showed a ∼30% reduction in overall organoid number between days 3 and 6. Moreover, in high glucose (33 and 100 mM), we observed an increase in the number of small, single PODXL+ cells surrounding the main organoid body, suggesting detachment from the podocyte clusters that normally form in organoids (Fig. A). To quantify it, we divided the total PODXL+ area into two sub-regions, classified as intact area and detached area, and calculated their ratio (Fig. B). Results indicated a 2-fold reduction of the intact vs detached area between 11 and 33 mM (p = 0.0011), suggesting that organoids undergo a spreading process under the influence of high glucose (Fig. C). Comparison of equimolar glucose vs mannose, live/dead and LDH assays identify a non-osmotic and non-apoptotic cause of the injury. ScRNA-seq analysis identified TNF-α as an upregulated pathway, which was confirmed with qPCR (p = 0.0131). Comparison of organoids at 11mM ± TNFα indicated that TNFα addition sensitizes podocytes to detachment at lower glucose levels, specifically, we identified a 4-fold reduction of the PODXL+ intact vs detached area at 11 mM ± TNFα (p &amp;lt; 0.0001), Fig. D. To further validate these findings and investigate possible therapeutic strategies, organoids were cultured with 33 mM glucose with TNF-α inhibitor and we identified a protective effect of the inhibitor which prevented the podocyte spreading phenotype at 33 mM glucose (Fig. E). Conclusion Our experiments identify an injury phenotype under high glucose concentrations, in kidney organoids, characterized by podocyte detachment from the main organoid body. Moreover, no osmotic or toxicity effect was observed. Importantly, this phenotype resembles disease features identified in diabetic patients, where podocyte loss and detachment from the glomerular basement membrane is associated with pathophysiology. We identified a correlation between the injury phenotype, characterized by epithelial detachment, and proinflammatory cytokine upregulation, which is driven by high glucose, and we have shown it can be targeted therapeutically. Therefore, this new experimental model in-vitro offers up new insights into the pathophysiology of diabetes and its complications, and represents a new platform to screen for therapeutics.

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats

P2X7 receptors (P2X7) mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow (RBF) and glomerular filtration rate (GFR), suggesting that P2X7 receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. Here, we generated a novel P2X7 global knockout (KO) rat on the F344 background, hypothesising enhanced RBF and protection from chronic angiotensin II (ANG II)-induced injury. CRISPR/Cas9 technology introduced an early stop codon into exon 2 of P2rx7. In male and female KO and WT rats, renal P2X7 expression was measured by RTqPCR (n=4-5/group) and western blotting (n=3/group). Bone marrow-derived macrophages (BMDM) were used to assess P2X7 function by measuring interleukin (IL)-1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5-6/group). In vivo kidney function was assessed in anesthetized rats (thiopental, 50mg/kg, i.p.) at baseline and following serial arterial ligation to induce acute pressure natriuresis (n=9-10/group). Arterial blood pressure (BP) was measured directly, and urine was collected via tube cystotomy. RBF was measured using a Transonic Doppler flow probe placed around the main renal artery, and cortical and medullary flux by laser Doppler needle probes. GFR was determined by FITC-inulin clearance. Urinary sodium was measured by sodium-selective electrode analysis. In a separate experiment, BP was recorded over five consecutive days in conscious male KO and WT rats via implanted radiotelemetry devices (n=7-8/group). Finally, male KO and WT rats (n=9-10/group) were infused with ANG II (250ng/kg/min, s.c.) for 6 weeks. At end, one renal artery was isolated and vascular reactivity to phenylephrine, acetylcholine and sodium nitroprusside determined by wire myography. Bladder urine was collected to measure albumin/creatinine ratio (n=4-7/group), and kidneys were harvested for histological analyses (n=6/group). Data were analysed with unpaired t-test (two groups compared) or two-way ANOVA (two variables), with p&lt;0.05 statistically significant. P2rx7 mutation was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was partially decreased in kidney from male (-54%) and female (-25%) KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. In BMDM from WT rats, LPS+ATP stimulation induced IL-1β release; this response was markedly suppressed in KO BMDM (males: 2577±140 vs 413±175pg/mL, p&lt;0.0001; females: 1889±394 vs 265±121pg/mL, p&lt;0.0001). In vivo BP, renal haemodynamics and tubular sodium handling were not different between genotypes for either sex (P&gt;0.05). Radiotelemetry confirmed no difference in BP between KO and WT rats. Following chronic ANG II infusion, KO and WT rats had no renal artery dysfunction but showed similar increases in albuminuria (P=0.3587), renal perivascular fibrosis (Picrosirius Red, P=0.5393), cortical interstitium CD68-positive area (p=0.6546) and tubular casts (Periodic acid-Schiff, P=0.8803), compared with naïve controls. Contrary to our hypothesis, global genetic deletion of P2X7 did not affect renal hemodynamics and we found no significant role for P2X7 in the modulation of tubular sodium reabsorption. Our data do not support a major role of P2X7 in causing renal vascular and tubular injury. This work was supported by Diabetes UK grant 17/0005685, The British Heart Foundation(FS/15/60/31510; RE/18/5/34216), the Medical Research Council (MR/S01053X), and the University of Edinburgh College of Medicine &amp; Veterinary Medicine PhD Studentship. 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.

Alleviating renal injury and mitochondrial dysfunction following hemorrhagic shock

Resuscitation with polydatin (PD) has been shown to alleviate mitochondrial damage and ischemia reperfusion injury following hemorrhagic shock. However, whether PD has the same beneficial effect under prolonged hemorrhagic shock with delayed or limited resuscitation is unclear. Using a previously developed rat model of acute kidney injury (AKI) induced by prolonged hemorrhagic shock, we tested the hypothesis that PD preserves renal mitochondrial function, delays the onset of AKI, and decreases mortality following prolonged hemorrhagic shock without full resuscitation. Additionally, we determined whether polyethylene glycol-20k (PEG) can enhance the beneficial effects of PD via improved capillary perfusion and PD delivery into the ischemic tissues. Rats were randomized into 4 groups (n=8/group) based on treatment: saline (vehicle), PD, PEG, and PD+PEG. Trauma was induced in Inactin anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (mean arterial pressure (MAP) = 55 mmHg) for 45 minutes. Animals then received an intravenous injection of saline (vehicle), PD, PEG, or PD+PEG. MAP, blood gases, hematocrit, renal blood flow (RBF), and oxygen delivery were monitored for another 2 hours. Glomerular filtration rate (GFR), urinary sodium uptake, renal mitochondrial function, and mortality were recorded at the end of the experiment. The saline group exhibited AKI, renal mitochondrial dysfunction, and 100% mortality. PD alone showed no effect on mitochondrial function, renal injury, or mortality (100%). PD+PEG restored both complex II and IV activities, while PEG improved mitochondrial complex II function but showed no effect on complex IV. Nevertheless, PEG and PD+PEG similarly improved MAP, renal blood flow, renal oxygen delivery, GFR, sodium uptake, and mortality (PEG: 12.5%; PD+PEG: 0%). Our results suggest that polydatin requires suffcient tissue perfusion to exert its beneficial effects on the mitochondrial function of hemorrhagic shock animals. We used clinically meaningful endpoints to demonstrate the utility of PEG as a therapeutic tool for optimizing drug delivery to treat hemorrhagic shock. DoD disclaimer: The views expressed in this article are those of the author(s) and do not reflect the offcial policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. Funding: This study was funded by US Army Medical Research and Development Command. 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.

Use of Function Diagrams in Physiology Education: Nessie the Nephron and Navigating the Lochs

Function diagrams focus on physiological concepts rather than associated structures and can serve as elaboration tools and mnemonic aids. A function diagram prototype of the gastrointestinal system was recently described (Wilson &amp; Barrett, Adv Physiol Educ, 45:264–268, 2021). Currently, a functional diagram of the glomerulus, nephron, urinary system, and bladder is proposed. Colloquially named “Nessie the Nephron” to loosely capture the looping structure and illusive understanding of renal blood flow, glomerular filtration, and epithelial transport for the student, not to mention the nearly mythical countercurrent multiplier. “Navigating the Lochs” references Nessie’s possible habitat and more importantly the movement and storage of the modified ultrafiltrate from the nephron. The primary analogies that form the structure of this function diagram are: Rain Barrel, Soaker Hose, &amp; Hose Nozzle (afferent and efferent blood flow and filtration pressure); Water Purification System (glomerular filtration barrier forming multiple step filtration process involving the capillary, basement membrane, and podocytes); Mixed Recycling Machine (proximal tubule individual, co-, and bulk transport); Desiccator &amp; Briner (thin descending limb removal of water and thin/thick ascending limb movement of salt); Conveyor Belt Picker (distal nephron selective ion transport); and Concentrator (collecting duct water and urea transport). The primary analogies that elaborate “Navigating the Lochs” are Aqueduct &amp; Cistern System (fluid movement and collection); and Pressure Gauge, Syringe Bulb, and Two-valve Plumbing (bladder storage and micturition). Complimenting these analogies is the rich potential for inclusion of clinical and comparative applications and examples to link previous knowledge and strengthen memories for future retrieval. University of Kentucky College of Medicine. 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.

544 Interleukin-6 protects renal dysfunction in mouse models of hypertension and salt-sensitive hypertension

OBJECTIVES/GOALS: We are investigating the role of IL-6 in regulating renal function by measuring mean arterial pressure (MAP), renal plasma flow (RPF) and glomerular filtration rate (GFR) in wild type (WT) and IL-6-knockout (KO) mice in established mouse models of angiotensin II (AII)-dependent- hypertension and -salt-sensitive hypertension. METHODS/STUDY POPULATION: Twelve-week-old male WT and KO mice on the C57BL6 background strain were infused with vehicle (V; saline) or angiotensin II (AII; 200 ng/kg/min) for 12-14 days. Half of the AII-treatment groups were maintained on a high salt (HS; 6% NaCl) diet for the duration of the experiment, while the other half of the AII treatment groups and both vehicle groups were fed normal rat chow. MAP was continuously measured by a fluid filled catheter in conscious mice for the duration of the experiment. RPF and GFR were measured on days 12-14 in anesthetized mice by the para-aminohippurate, and fluorescein isothiocynate-Inulin techniques, respectively. All data were analyzed by 2-way ANOVA; *p&lt;0.05 vs. WT, same treatment; #p&lt;0.05 vs.V, same genotype; ^p&lt;0.05, AII vs. AII+HS, same genotype. RESULTS/ANTICIPATED RESULTS: MAP was 31% lower in KO vs WT mice. AII increased MAP (1.2-fold) in WT but not KO mice. HS diet magnified AII-induced increases in MAP in WT and moderately increased MAP in AII-KO mice: [MAP (mmHg): WT+V, 130±7.0; KO+V, 91.0+4.0*; WT+AII, 153±5.0#; KO+AII, 83.0±4.0*; WT+AII+HS, 150±11#; KO+AII+HS, 93.0±4.0#]. AII infusion reduced RPF in the KO but not WT mice. Addition of HS reduced RPF in WT and exacerbated AII-induced reductions in RPF in KO mice [RPF (ml/min/g): WT+V, 1.82±0.23; KO+V, 1.91+0.40; WT+AII, 3.16±0.75#; KO+AII, 1.65±0.42*; WT+AII+HS, 1.10±0.31#^; KO+AII+HS, 1.13±0.XX#^]. The HS diet reduced GFR in AII-infused KO but not WT mice [GFR (µl/min/g): WT+V, 756±XX; KO+V, 788±XX; WT+AII, 1010±63*#; KO+AII, 756±23*; WT+AII+HS, 1100±150#; KO+AII+HS: 540±210*#^]. DISCUSSION/SIGNIFICANCE: The absence of IL-6 in male mice attenuated AII- and/or AII+HS-induced increases in MAP; however, it exacerbated HS-induced reductions in RPF and GFR. These findings suggest inhibiting IL-6 has therapeutic potential as an antihypertensive but not as a renal protective agent in hypertension and salt-sensitive hypertension disease states.

Effect of Low-Frequency Renal Nerve Stimulation on Renal Glucose Release during Normoglycemia and a Hypoglycemic Clamp in Pigs.

Previously, we demonstrated that renal denervation in pigs reduces renal glucose release during a hypoglycemic episode. In this study we set out to examine changes in side-dependent renal net glucose release (SGN) through unilateral low-frequency stimulation (LFS) of the renal plexus with a pulse generator (2-5 Hz) during normoglycemia (60 min) and insulin-induced hypoglycemia ≤3.5 mmol/L (75 min) in seven pigs. The jugular vein, carotid artery, renal artery and vein, and both ureters were catheterized for measurement purposes, blood pressure management, and drug and fluid infusions. Para-aminohippurate (PAH) and inulin infusions were used to determine side-dependent renal plasma flow (SRP) and glomerular filtration rate (GFR). In a linear mixed model, LFS caused no change in SRP but decreased sodium excretion (p < 0.0001), as well as decreasing GFR during hypoglycemia (p = 0.0176). In a linear mixed model, only hypoglycemic conditions exerted significant effects on SGN (p = 0.001), whereas LFS did not. In a Wilcoxon signed rank exact test, LFS significantly increased SGN (p = 0.03125) and decreased sodium excretion (p = 0.0017) and urinary flow rate (p = 0.0129) when only considering the first instance LFS followed a preceding period of non-stimulation during normoglycemia. To conclude, this study represents, to our knowledge, the first description of an induction of renal gluconeogenesis by LFS.

Renal sympathetic denervation improves pressure-natriuresis relationship in cardiorenal syndrome: insight from studies with Ren-2 transgenic hypertensive rats with volume overload induced using aorto-caval fistula

The aim was to evaluate the effects of renal denervation (RDN) on autoregulation of renal hemodynamics and the pressure-natriuresis relationship in Ren-2 transgenic rats (TGR) with aorto-caval fistula (ACF)-induced heart failure (HF). RDN was performed one week after creation of ACF or sham-operation. Animals were prepared for evaluation of autoregulatory capacity of renal blood flow (RBF) and glomerular filtration rate (GFR), and of the pressure-natriuresis characteristics after stepwise changes in renal arterial pressure (RAP) induced by aortic clamping. Their basal values of blood pressure and renal function were significantly lower than with innervated sham-operated TGR (p < 0.05 in all cases): mean arterial pressure (MAP) (115 ± 2 vs. 160 ± 3 mmHg), RBF (6.91 ± 0.33 vs. 10.87 ± 0.38 ml.min–1.g–1), urine flow (UF) (11.3 ± 1.79 vs. 43.17 ± 3.24 µl.min–1.g–1) and absolute sodium excretion (UNaV) (1.08 ± 0.27 vs, 6.38 ± 0.76 µmol.min–1.g–1). After denervation ACF TGR showed improved autoregulation of RBF: at lowest RAP level (80 mmHg) the value was higher than in innervated ACF TGR (6.92 ± 0.26 vs. 4.54 ± 0.22 ml.min–1.g–1, p < 0.05). Also, the pressure-natriuresis relationship was markedly improved after RDN: at the RAP of 80 mmHg UF equaled 4.31 ± 0.99 vs. 0.26 ± 0.09 µl.min–1.g–1 recorded in innervated ACF TGR, UNaV was 0.31 ± 0.05 vs. 0.04 ± 0.01 µmol min–1.g–1 (p < 0.05 in all cases). In conclusion, in our model of hypertensive rat with ACF-induced HF, RDN improved autoregulatory capacity of RBF and the pressure-natriuresis relationship when measured at the stage of HF decompensation.

Clinical-grade human skin-derived ABCB5+ mesenchymal stromal cells exert anti-apoptotic and anti-inflammatory effects in vitro and modulate mRNA expression in a cisplatin-induced kidney injury murine model.

Acute kidney injury (AKI) is characterized by a rapid reduction in renal function and glomerular filtration rate (GFR). The broadly used anti-cancer chemotherapeutic agent cisplatin often induces AKI as an adverse drug side effect. Therapies targeted at the reversal of AKI and its potential progression to chronic kidney disease or end-stage renal disease are currently insufficiently effective. Mesenchymal stromal cells (MSCs) possess diverse immunomodulatory properties that confer upon them significant therapeutic potential for the treatment of diverse inflammatory disorders. Human dermal MSCs expressing ATP-Binding Cassette member B5 (ABCB5) have shown therapeutic efficacy in clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. In preclinical studies, ABCB5+ MSCs have also shown to reverse metabolic reprogramming in polycystic kidney cells, suggesting a capacity for this cell subset to improve also organ function in kidney diseases. Here, we aimed to explore the therapeutic capacity of ABCB5+ MSCs to improve renal function in a preclinical rat model of cisplatin-induced AKI. First, the anti-apoptotic and immunomodulatory capacity was compared against research-grade adipose stromal cells (ASCs). Then, cross-species immunomodulatory capacity was checked, testing first inhibition of mitogen-driven peripheral blood mononuclear cells and then modulation of macrophage function. Finally, therapeutic efficacy was evaluated in a cisplatin AKI model. First, ABCB5+ MSCs suppressed cisplatin-induced apoptosis of human conditionally-immortalized proximal tubular epithelial cells in vitro, most likely by reducing oxidative stress. Second, ABCB5+ MSCs inhibited the proliferation of either human or rat peripheral blood mononuclear cells, in the human system via the Indoleamine/kynurenine axis and in the murine context via nitric oxide/nitrite. Third, ABCB5+ MSCs decreased TNF-α secretion after lipopolysaccharide stimulation and modulated phagocytosis and in both human and rat macrophages, involving prostaglandin E2 and TGF-β1, respectively. Fourth, clinical-grade ABCB5+ MSCs grafted intravenously and intraperitoneally to a cisplatin-induced AKI murine model exerted modulatory effects on mRNA expression patterns toward an anti-inflammatory and pro-regenerative state despite an apparent lack of amelioration of renal damage at physiologic, metabolic, and histologic levels. Our results demonstrate anti-inflammatory and pro-regenerative effects of clinical grade ABCB5+ MSCs in vitro and in vivo and suggest potential therapeutic utility of this cell population for treatment or prevention of cisplatin chemotherapy-induced tissue toxicity.