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Diabetic kidney disease (DKD) is characterized by dysregulation of the renin-angiotensin system (RAS) and renal tubular injury. We investigated whether insulin treatment preserves renal homeostasis by modulating neprilysin (NEP), arginase-2 (Arg-2), and kidney injury molecule-1 (KIM-1) regulation in type 1 diabetic Akita mice. Diabetic Akita mice received three subcutaneous sustained-release insulin implants (0.1 U/day) for 16 weeks. Blood measurements and urine collections were performed weekly. Western blot, enzymatic activity assays, and ELISA were used to analyze renal and urinary NEP, KIM-1, and Arg-2. Full-length immunoreactive NEP (95 kDa) expression and activity were significantly reduced in Akita mice (p < 0.05 vs. wild type [WT] non-diabetic controls) in both kidney and urine. This decrease was found in both young (9-week-old) and older (27-week-old). Novel urinary immunoreactive NEP smaller fragments (70, 50, and 37 kDa) were detected in 27-week-old diabetic Akita mice but absent in non-diabetic controls mice (WT). Insulin treatment normalized hyperglycemia, reduced albuminuria, and decreased glomerular fibrosis. Furthermore, it restored renal and urinary full-length NEP expression (p < 0.05) and increased NEP activity, while reducing NEP fragment shedding. Notably, while Western blot and activity assays demonstrated reduced full-length NEP expression and activity in Akita mice, ELISA revealed a paradoxical increase in urinary NEP concentration, suggesting the detection of inactive smaller urinary NEP fragments in addition to the full-length. Urinary KIM-1 and renal Arg-2 were significantly increased in 27- weeks old diabetic Akita mice, effects that were significantly attenuated by insulin treatment ( < 0.05). Insulin therapy protects against diabetic nephropathy by: (i) augmenting renal NEP activity, (ii) reducing Arg-2-mediated injury, and (iii) attenuating tubular damage as evidenced by decreased urinary KIM-1 and NEP fragment shedding. The presence of low-molecular-weight NEP fragments in urine does warrant further investigation into their potential use as biomarkers for tracking the progression of DKD and monitoring the effectiveness of treatments.

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo . Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

Red blood cell (RBC) death could contribute to anemia in chronic kidney disease (CKD) patients. Recent observational research has suggested a relationship between RBC death (eryptosis) and hypoxemia in hemodialysis patients. Thus, we studied the isolated and joint effects of a uremic toxin (indoxyl sulfate; IS) and hypoxia on RBC biology.BACKGROUND/AIMSRed blood cell (RBC) death could contribute to anemia in chronic kidney disease (CKD) patients. Recent observational research has suggested a relationship between RBC death (eryptosis) and hypoxemia in hemodialysis patients. Thus, we studied the isolated and joint effects of a uremic toxin (indoxyl sulfate; IS) and hypoxia on RBC biology.We incubated RBC from healthy donors with IS at concentrations of 0.01mM, 0.09mM and 0.17mM under both normoxic (21% O2) and hypoxic (5% O2) conditions for 24 hours. Eryptosis was evaluated by RBC phosphatidylserine (PS) exposure, cell volume, and cytosolic calcium which were quantified by Annexin-V+, forward scatter, and Fluo-3AM+ binding, respectively. RBC redox balance was reported by reactive oxygen species (ROS) production and intracellular reduced glutathione (GSH). Analyses were performed by flow cytometry.METHODSWe incubated RBC from healthy donors with IS at concentrations of 0.01mM, 0.09mM and 0.17mM under both normoxic (21% O2) and hypoxic (5% O2) conditions for 24 hours. Eryptosis was evaluated by RBC phosphatidylserine (PS) exposure, cell volume, and cytosolic calcium which were quantified by Annexin-V+, forward scatter, and Fluo-3AM+ binding, respectively. RBC redox balance was reported by reactive oxygen species (ROS) production and intracellular reduced glutathione (GSH). Analyses were performed by flow cytometry.Hypoxia induced a 2-fold ROS production compared to normoxia. PS exposure and cytosolic calcium increased, while cell volume decreased by hypoxia and likewise by IS. IS increased ROS production in a dose-dependent manner under conditions of both normoxia and hypoxia. The same conditions promoted a GSH decrease with IS intensifying the hypoxia-induced effects.RESULTSHypoxia induced a 2-fold ROS production compared to normoxia. PS exposure and cytosolic calcium increased, while cell volume decreased by hypoxia and likewise by IS. IS increased ROS production in a dose-dependent manner under conditions of both normoxia and hypoxia. The same conditions promoted a GSH decrease with IS intensifying the hypoxia-induced effects.In summary, our results indicate that the concurrent presence of hypoxia and uremia augments RBC death and may therefore, contribute to the genesis of anemia in CKD.CONCLUSIONIn summary, our results indicate that the concurrent presence of hypoxia and uremia augments RBC death and may therefore, contribute to the genesis of anemia in CKD.

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.

Background Vascular calcification (VC) is a highly prevalent cardiovascular complication in maintenance hemodialysis (HD), affecting up to 80%–90% of subjects. It represents an actively regulated process of tissue biomineralization via the “bone-vascular axis”. VC contributes to morbidity and mortality in dialysis subjects causing arterial stiffening which may lead to cardiovascular events. This prospective observational pilot study assessed whether changes in osteoblastic turnover in aortic and cardiac VCs could be quantified by ¹⁸F-Sodium Fluoride Positron Emission Tomography / Computed Tomography ( 18 F-NaF PET/CT) in a serial manner. ¹⁸F-NaF PET provides a quantitative measure of ongoing vascular mineralization in calcifications of both the heart and aorta. Methods Seven subjects (three females, four males, age 65 ± 4.5 years) treated with HD were enrolled. Five subjects underwent baseline and follow-up 18 F-NaF PET/CT imaging at 9.9 ± 1.8 months. All subjects ( n  = 7) received coronary artery calcification (CAC) scoring with CT. Vascular mineralization rate (K i ) in 18 F-NaF PET was estimated using the Patlak method in regions of aortic and cardiac calcifications as well as thoracic spine. Results VCs were visually apparent in all subjects in both cardiac and aortic regions on both CT and PET. K i -Patlak in the aortic VCs increased in all subjects over time (13.0% ± 4.9%; p  = 0.005) indicating an increasing rate of aortic mineralization. Increased K i -Patlak was also seen in cardiac VCs in 4 of 5 subjects ( p  = 0.198). K i -Patlak in thoracic spine did not change during follow-up time ( p  = 0.953). The cardiac Agatston Calcification Score (ACS) ranged from moderate ( n  = 5) and severe ( n  = 1) to extensive ( n  = 1) showing the presence of atherosclerotic disease. The Agatston Calcification Score measured specifically in the aorta typically exceeded that in the heart and ranged from severe ( n  = 1) to extensive ( n  = 6). Changes in ACS were not significant during follow-up. Conclusion 18 F-NaF PET/CT showed the ability to quantify changes in cardiac and aortic vascular calcifications in subjects receiving HD and may present a method to monitor changes in 18 F-NaF uptake as a function of time.

Bisphenol A (BPA)-based materials are used in the manufacturing of hemodialyzers, including their polycarbonate (PC) housings and polysulfone (PS) membranes. As concerns for BPA’s adverse health effects rise, the regulation on BPA exposure is becoming more rigorous. Therefore, BPA alternatives, such as Bisphenol S (BPS), are increasingly used. It is important to understand the patient risk of BPA and BPS exposure through dialyzer use during hemodialysis. Here, we report the bisphenol levels in extractables and leachables obtained from eight dialyzers currently on the market, including high-flux and medium cut-off membranes. A targeted liquid chromatography–mass spectrometry strategy utilizing stable isotope-labeled internal standards provided reliable data for quantitation with the standard addition method. BPA ranging from 0.43 to 32.82 µg/device and BPS ranging from 0.02 to 2.51 µg/device were detected in dialyzers made with BPA- and BPS-containing materials, except for the novel FX CorAL 120 dialyzer. BPA and BPS were also not detected in bloodline controls and cellulose-based membranes. Based on the currently established tolerable intake (6 µg/kg/day), the resulting margin of safety indicates that adverse effects are unlikely to occur in hemodialysis patients exposed to BPA and BPS quantified herein. With increasing availability of new data and information about the toxicity of BPA and BPS, the patient safety limits of BPA and BPS in those dialyzers may need a re-evaluation in the future.

It has been estimated that in 2010, over two million patients with end-stage kidney disease may have faced premature death due to a lack of access to affordable renal replacement therapy, mostly dialysis. To address this shortfall in dialytic kidney replacement therapy, we propose a novel, cost-effective, and low-complexity hemodialysis method called allo-hemodialysis (alloHD). With alloHD, instead of conventional hemodialysis, the blood of a patient with kidney failure flows through the dialyzer’s dialysate compartment counter-currently to the blood of a healthy subject (referred to as a “buddy”) flowing through the blood compartment. Along the concentration and hydrostatic pressure gradients, uremic solutes and excess fluid are transferred from the patient to the buddy and subsequently excreted by the healthy kidneys of the buddy. We developed a mathematical model of alloHD to systematically explore dialysis adequacy in terms of weekly standard urea Kt/V. We showed that in the case of an anuric child (20 kg), four 4 h alloHD sessions are sufficient to attain a weekly standard Kt/V of >2.0. In the case of an anuric adult patient (70 kg), six 4 h alloHD sessions are necessary. As a next step, we designed and built an alloHD machine prototype that comprises off-the-shelf components. We then used this prototype to perform ex vivo experiments to investigate the transport of solutes, including urea, creatinine, and protein-bound uremic retention products, and to quantitate the accuracy and precision of the machine’s ultrafiltration control. These experiments showed that alloHD performed as expected, encouraging future in vivo studies in animals with and without kidney failure.

Activation of the renin angiotensin system plays a pivotal role in the regulation of blood pressure, which is mainly attributed to the formation of angiotensin-II (Ang II). The actions of Ang II are mediated through binding to the Ang-II type 1 receptor (AT1R) which leads to increased blood pressure, fluid retention, and aldosterone secretion. In addition, Ang II is also involved in cell injury, vascular remodeling, and inflammation. The actions of Ang II could be antagonized by its conversion to the vasodilator peptide Ang (1–7), partly generated by the action of angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Previous studies demonstrated increased urinary ACE2 shedding in the db / db mouse model of diabetic kidney disease. The aim of the study was to investigate whether renal and urinary ACE2 and NEP are altered in the 2K1C Goldblatt hypertensive mice. Since AT1R is highly expressed in the kidney, we also researched the effect of global deletion of AT1R on renal and urinary ACE2, NEP, and kidney injury marker (KIM-1). Hypertension and albuminuria were induced in AT1R knock out (AT1RKO) and WT mice by unilateral constriction of the renal artery of one kidney. The 24 h mean arterial blood pressure (MAP) was measured using radio-telemetry. Two weeks after 2K1C surgery, MAP and albuminuria were significantly increased in WT mice compared to AT1RKO mice. Results demonstrated a correlation between MAP and albuminuria. Unlike db / db diabetic mice, ACE2 and NEP expression and activities were significantly decreased in the clipped kidney of WT and AT1RKO compared with the contralateral kidney and sham control ( p < 0.05). There was no detectable urinary ACE2 and NEP expression and activity in 2K1C mice. KIM-1 was significantly increased in the clipped kidney of WT and AT1KO ( p < 0.05). Deletion of AT1R has no effect on the increased urinary KIM-1 excretion detected in 2K1C mice. In conclusion, renal injury in 2K1C Goldblatt mouse model is associated with loss of renal ACE2 and NEP expression and activity. Urinary KIM-1 could serve as an early indicator of acute kidney injury. Deletion of AT1R attenuates albuminuria and hypertension without affecting renal ACE2, NEP, and KIM-1 expression.

Monitoring intraperitoneal pressure (IPP) offers valuable insights into changes of intraperitoneal volume (IPV) during peritoneal dialysis (PD). This study aims to investigate the relationship between IPV and IPP during a PD dwell. Thirteen patients were studied during a 2‐h dwell using 2 L of dialysate containing 2.5% dextrose. IPP was measured using a pressure sensor integrated into an automated PD cycler. IPV was monitored concurrently by segmental bioimpedance (Hydra 4200). The density (ρ) of the PD dialysate was measured using a meter, and the creatinine and glucose concentrations in both dialysate (D) and serum (P) were measured pre‐ and post‐PD dwell. A physical model (IPP = ρ × g × h), was used to describe the relationship between IPP and IPV, where h is the apparent dialysate height and g is the gravitational acceleration. The change in IPP (ΔIPP, −21.2 ± 18%) was mainly determined by the change of h (Δh, −20.9 ± 18.5%), while the change ρ (Δρ, −0.34 ± 0.06%), was minor. The study demonstrated an association between ΔIPP and the ratio of D/P creatinine and D/D0 glucose, suggesting that ΔIPP may reflect membrane transport characteristics. Due to its noninvasive and seamless nature, the clinical utility of PD cycler‐based measurement of IPP warrants further exploration.

Erythropoietin deficiency is an extensively researched cause of renal anemia. The etiology and consequences of shortened red blood cell (RBC) life span in chronic kidney disease (CKD) are less well understood. Traversing capillaries requires RBC geometry changes, a process enabled by adaptions of the cytoskeleton. These changes are mediated by transient activation of the mechanosensory Piezo1 channel, resulting in calcium influx. Importantly, prolonged Piezo1 activation shortens RBC life span, presumably through activation of calcium‐dependent intracellular pathways triggering RBC death. Two Piezo1‐activating small molecules, Jedi1 and Jedi2, share remarkable structural similarities with 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid (CMPF), a uremic retention solute cleared by the healthy kidney. We hypothesize that in CKD the accumulation of CMPF leads to prolonged activation of Piezo1 (similar in effect to Jedi1 and Jedi2), thus reducing RBC life span. This hypothesis can be tested through bench experiments and, ultimately, by studying the effect of CMPF removal on renal anemia.