SGLT2 Inhibitors Decrease Overhydration and Proteasuria in Patients with Chronic Kidney Disease: A Longitudinal Observational Study.

INTRODUCTION: SGLT2 inhibitors are used to reduce the risk of progression of chronic kidney disease (CKD). In patients with type 2 diabetes, they have been found to reduce extracellular volume. Given the high prevalence of extracellular volume expansion and overhydration (OH) in CKD, we investigated whether SGLT2 inhibitors might correct these disturbances in CKD patients. METHODS: CKD patients who started treatment with an SGLT2 inhibitor were investigated in this prospective observational study for 6 months. Body composition and fluid status were measured by bioimpedance spectroscopy. In addition, spot urine samples were analyzed for albuminuria, glucosuria, and urinary aprotinin-sensitive serine protease activity. RESULTS: Forty-two patients (29% with diabetic/hypertensive CKD, 31% with IgA nephropathy; 88% dapagliflozin 10 mg, 10% dapagliflozin 5 mg, 2% empagliflozin 20 mg; median eGFR 46 mL/min/1.73 m2 and albuminuria 1,911 mg/g creatinine) participated in the study. Median glucosuria increased to 14 (10-19) g/g creatinine. At baseline, patients displayed OH with +0.4 (-0.2 to 2.2) L/1.73 m2, which decreased by 0.5 (0.1-1.2) L/1.73 m2 after 6 months. Decrease of OH correlated with higher OH at BL, decrease of albuminuria, glucosuria, and urinary aprotinin-sensitive protease activity. Adipose tissue mass was not significantly reduced after 6 months. CONCLUSION: SGLT2 inhibitors reduce OH in patients with CKD, which is pronounced in the presence of high albuminuria, glucosuria, and urinary aprotinin-sensitive protease activity.

Eryptosis: a driver of anemia in chronic kidney disease.

PURPOSE OF REVIEW: Anemia, characterized by a reduction in red blood cell (RBC) count or hemoglobin concentration, commonly accompanies chronic kidney disease (CKD), significantly impacting patients' quality of life. This review delves into the multifaceted nature of anemia in CKD, with a focus on novel mechanisms, particularly the dysregulation of eryptosis or programmed cell death of RBCs, leading to shortened RBC lifespan. RECENT FINDINGS: Recent studies in CKD patients and mouse models revealed that eryptosis, driven by factors such as uremic toxins, inflammation, and imbalances in calcium homeostasis, plays a pivotal role in the development of renal anemia. Dysregulated eryptosis results in premature RBC destruction, exacerbating the hypoproliferative character of anemia in CKD. SUMMARY: Recognizing the intricate relationship between eryptosis and anemia in CKD opens promising prospects for improving patient outcomes and enhancing our understanding of this complex condition. Future research and therapeutic development in this area hold the potential to improve anemia treatment of CKD patients.

Pathophysiology of Red Blood Cell Dysfunction in Diabetes and Its Complications.

Diabetes Mellitus (DM) is a complex metabolic disorder associated with multiple microvascular complications leading to nephropathy, retinopathy, and neuropathy. Mounting evidence suggests that red blood cell (RBC) alterations are both a cause and consequence of disturbances related to DM-associated complications. Importantly, a significant proportion of DM patients develop varying degrees of anemia of confounding etiology, leading to increased morbidity. In chronic hyperglycemia, RBCs display morphological, enzymatic, and biophysical changes, which in turn prime them for swift phagocytic clearance from circulation. A multitude of endogenous factors, such as oxidative and dicarbonyl stress, uremic toxins, extracellular hypertonicity, sorbitol accumulation, and deranged nitric oxide metabolism, have been implicated in pathological RBC changes in DM. This review collates clinical laboratory findings of changes in hematology indices in DM patients and discusses recent reports on the putative mechanisms underpinning shortened RBC survival and disturbed cell membrane architecture within the diabetic milieu. Specifically, RBC cell death signaling, RBC metabolism, procoagulant RBC phenotype, RBC-triggered endothelial cell dysfunction, and changes in RBC deformability and aggregation in the context of DM are discussed. Understanding the mechanisms of RBC alterations in DM provides valuable insights into the clinical significance of the crosstalk between RBCs and microangiopathy in DM.

Two cases of severe vitamin D3 intoxication treated with therapeutic plasma exchange and high cut-off hemodialysis.

We report on a 53-year-old female patient and a 33-year-old male patient presenting with life-threatening hypercalcemic crisis caused by self-induced vitamin-D intoxication. Both patients took high doses of vitamin D3 supplements, cumulatively up to 2,500,000-10,000,000 I.U. over several months. Accordingly, serum 25-OH-vitamin D concentrations were increased to 663 and 1289 nmol/L (reference 50-175 nmol/L), respectively. As forced diuresis and bisphosphonates failed to correct recurrent hypercalcemia, we hypothesized that add-on extracorporeal treatments might help overcome the refractory situation. Considering the binding of vitamin D3 metabolites to vitamin D-binding protein (VDBP, 59 kDa), we started extracorporeal treatments involving total plasma exchange with replacement by human albumin and by fresh frozen plasma, online hemodiafiltration and high cut-off hemodialysis. We found that in the former case, total plasma exchange with albumin and fresh frozen plasma and high cut-off hemodialysis lowered both 25-OH-vitamin D3 and 1,25-OH-vitamin D3, whereas in the latter case total plasma exchange with albumin was found to more effectively remove vitamin D metabolites compared to high cut-off hemodialysis. In contrast, the amount of total plasma calcium removed by high cut-off hemodialysis was higher compared to total plasma exchange with albumin. During follow up, patients 1 and 2 achieved almost normal total plasma calcium and vitamin D concentrations after 355 and 109 days, respectively. These two cases suggest that extracorporeal treatments with high cut-off hemodialysis and total plasma exchange with albumin may be considered as add-on treatment in refractory cases of vitamin D3-induced hypercalcemia to lower plasma 25-OH-vitamin D3 concentrations.

[Treatment strategies in diabetic nephropathy - Update 2022]. / Behandlungsstrategien bei diabetischer Nephropathie ­ Update 2022.

TREATMENT GOALS IN DIABETIC NEPHROPATHY: A new classification of patients with diabetes mellitus into so-called clusters can be used to assess the risk of developing diabetic nephropathy. Up to date treatment of patients with diabetic nephropathy includes not only glycemic control, but focuses on a holistic approach with prevention of (progression of) secondary diabetic complications and reduction of the patient's cardiovascular mortality. COMPONENTS OF TREATMENT OF DIABETIC NEPHROPATHY: To achieve this treatment goals, it requires comprehensive management of the patient to implement lifestyle interventions (regarding diet, physical activity, weight loss, smoking cessation) and selection of the medication with the greatest individual benefit. DIETARY RECOMMENDATIONS IN DIABETIC NEPHROPATHY: Part of lifestyle intervention is adherence to a dietary regimen that has again been shown in recent studies to be relevant in inhibiting progression of chronic kidney disease and reducing cardiovascular risk. In particular, this includes protein restriction with preference for plant sources of protein, and salt restriction. USE OF MEDICATION CLASSES WITH PROVEN CARDIORENAL BENEFITS: Adjustment of drug therapy is no longer based on antihyperglycemic effects alone, but takes into account inhibition of progression of nephropathy and cardiac risk, as well as support of body weight control. For this purpose, in addition to RAAS inhibitors from the field of antihypertensives, the antidiabetic classes of SGLT2 inhibitors and GLP-1 receptor agonists as well as the non-steroidal aldosterone antagonist finerenone are now available.

Thoracoscopic mesh implantation as a definitive treatment approach for peritoneal dialysis-associated hydrothorax.

Pleuroperitoneal leakage with the formation of hydrothorax is a rare complication of peritoneal dialysis, usually necessitating termination of peritoneal dialysis. We hypothesized that implantation of a polypropylene mesh on the diaphragm using video-assisted thoracoscopic surgery might induce permanent closure of pleuroperitoneal leakage. We report a case series of n = 12 peritoneal dialysis patients with pleuroperitoneal leakage and right-sided hydrothorax who underwent video-assisted thoracoscopy with mesh implantation from 2011 to 2020. Pleuroperitoneal leakage had been confirmed before surgery by intraperitoneal administration of toluidine blue, contrast-enhanced computer tomography or glucose determination from the pleural effusion. Median time from the start of peritoneal dialysis to manifestation of pleuroperitoneal leakage was 52 days. Video-assisted thoracoscopic surgery revealed multiple penetration points in the tendinous part of the diaphragm in all patients, which appeared as blebs. These were closed by covering the whole diaphragm with a polypropylene mesh. In all patients, peritoneal dialysis was paused for three months and bridged by hemodialysis. After restarting peritoneal dialysis and a median follow-up time of 1.9 years, none of the patients experienced a recurrence of pleuroperitoneal leakage. This case series demonstrates that pleuroperitoneal leakage in peritoneal dialysis patients can be permanently closed using thoracoscopic mesh implantation and allows peritoneal dialysis to be continued as renal replacement therapy.

EPCAM and TROP2 share a role in claudin stabilization and development of intestinal and extraintestinal epithelia in mice.

Epithelial cell adhesion molecule (EPCAM) is a transmembrane glycoprotein expressed on the surface of most epithelial and epithelium-derived tumor cells and reported to regulate stability of epithelial tight junction proteins, claudins. Despite its widespread expression, loss of EPCAM function has so far only been reported to prominently affect intestinal development, resulting in severe early onset enteropathy associated with impaired growth and decreased survival in both humans and mice. In this study, we show that the critical role of EPCAM is not limited to intestinal tissues and that it shares its essential function with its only known homolog, Trophoblast cell surface antigen 2 (TROP2). EPCAM-deficient mice show significant growth retardation and die within 4 weeks after birth. In addition to changes in small and large intestines, loss of EPCAM results in hyperkeratosis in the skin and forestomach, hair follicle atrophy leading to alopecia, nephron hypoplasia in the kidney, proteinuria, and altered production of digestive enzymes by the pancreas. Expression of TROP2 partially, but not completely, overlaps with EPCAM in a number developing epithelia. Although loss of TROP2 had no gross impact on mouse development and survival, TROP2 deficiency generally compounded developmental defects observed in EPCAM-deficient mice, led to an approximately 60% decrease in embryonic viability, and further shortened postnatal lifespan of born pups. Importantly, TROP2 was able to compensate for the loss of EPCAM in stabilizing claudin-7 expression and cell membrane localization in tissues that co-express both proteins. These findings identify overlapping functions of EPCAM and TROP2 as regulators of epithelial development in both intestinal and extraintestinal tissues.

Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

BACKGROUND: Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK). OBJECTIVE: We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC. METHODS: A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract. RESULTS: No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC. CONCLUSION: Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.

Rodent models to study sodium retention in experimental nephrotic syndrome.

Sodium retention and edema are hallmarks of nephrotic syndrome (NS). Different experimental rodent models have been established for simulating NS, however, not all of them feature sodium retention which requires proteinuria to exceed a certain threshold. In rats, puromycin aminonucleoside nephrosis (PAN) is a classic NS model introduced in 1955 that was adopted as doxorubicin-induced nephropathy (DIN) in 129S1/SvImJ mice. In recent years, mice with inducible podocin deletion (Nphs2Δipod ) or podocyte apoptosis (POD-ATTAC) have been developed. In these models, sodium retention is thought to be caused by activation of the epithelial sodium channel (ENaC) in the distal nephron through aberrantly filtered serine proteases or proteasuria. Strikingly, rodent NS models follow an identical chronological time course after the development of proteinuria featuring sodium retention within days and spontaneous reversal thereafter. In DIN and Nphs2Δipod mice, inhibition of ENaC by amiloride or urinary serine protease activity by aprotinin prevents sodium retention, opening up new and promising therapeutic approaches that could be translated into the treatment of nephrotic patients. However, the essential serine protease(s) responsible for ENaC activation is (are) still unknown. With the use of nephrotic rodent models, there is the possibility that this (these) will be identified in the future. This review summarizes the various rodent models used to study experimental nephrotic syndrome and the insights gained from these models with regard to the pathophysiology of sodium retention.

Sodium retention in nephrotic syndrome is independent of the activation of the membrane-anchored serine protease prostasin (CAP1/PRSS8) and its enzymatic activity.

Experimental nephrotic syndrome leads to activation of the epithelial sodium channel (ENaC) by proteolysis and promotes renal sodium retention. The membrane-anchored serine protease prostasin (CAP1/PRSS8) is expressed in the distal nephron and participates in proteolytic ENaC regulation by serving as a scaffold for other serine proteases. However, it is unknown whether prostasin is also involved in ENaC-mediated sodium retention of experimental nephrotic syndrome. In this study, we used genetically modified knock-in mice with Prss8 mutations abolishing its proteolytic activity (Prss8-S238A) or prostasin activation (Prss8-R44Q) to investigate the development of sodium retention in doxorubicin-induced nephrotic syndrome. Healthy Prss8-S238A and Prss8-R44Q mice had normal ENaC activity as reflected by the natriuretic response to the ENaC blocker triamterene. After doxorubicin injection, all genotypes developed similar proteinuria. In all genotypes, urinary prostasin excretion increased while renal expression was not altered. In nephrotic mice of all genotypes, triamterene response was similarly increased, consistent with ENaC activation. As a consequence, urinary sodium excretion dropped in all genotypes and mice similarly gained body weight by + 25 ± 3% in Prss8-wt, + 20 ± 2% in Prss8-S238A and + 28 ± 3% in Prss8-R44Q mice (p = 0.16). In Western blots, expression of fully cleaved α- and γ-ENaC was similarly increased in nephrotic mice of all genotypes. In conclusion, proteolytic ENaC activation and sodium retention in experimental nephrotic syndrome are independent of the activation of prostasin and its enzymatic activity and are consistent with the action of aberrantly filtered serine proteases or proteasuria.

Intraperitoneal extension of the peritoneal dialysis catheter-a new technique for catheter implantation in patients with obesity.

BACKGROUND: In patients with obesity and end-stage kidney disease, implantation of the peritoneal dialysis (PD) catheter may be complicated by increased abdominal circumference or skin folds. Relocation of the implantation site to the upper abdomen could solve this problem. However, this would require an extended catheter. METHODS: We developed an extended PD catheter based on a swan neck Missouri PD catheter with the help of two adaptors and a straight intraperitoneal extension segment. The extended catheter was assembled intraoperatively, and its length was adjusted individually to ensure correct positioning. After the operation, PD was commenced and handled as usual. RESULTS: In the period from 2011 to 2021, we implanted 31 extended PD catheters in 29 patients (38% men) with end-stage renal failure and obesity. Median age was 53 (range 28-77) years and body mass index was 35.5 (range 26.4-46.9) kg/m2. The postoperative course was unremarkable except for seroma formation in one patient and dialysate leakage in another. Continuous ambulatory peritoneal dialysis (CAPD) was initiated in 20 and APD in 9 patients. The achieved median Kt/V was 2.10 (range 1.50-3.10). During the follow-up period lasting up to 51 months, there was one case of intraperitoneal catheter disconnection due to an avoidable handling error. The peritonitis rate was 1:40 months. The 1- and 2-year catheter survival was 92% and 67%, respectively, and paralleled patient survival. CONCLUSIONS: When using a PD catheter with an intraperitoneal extension, PD catheter implantation can be relocated to the upper abdomen in patients with obesity, thus providing optimal position and easy surgical access.

Renal effects of the serine protease inhibitor aprotinin in healthy conscious mice.

Treatment with aprotinin, a broad-spectrum serine protease inhibitor with a molecular weight of 6512 Da, was associated with acute kidney injury, which was one of the reasons for withdrawal from the market in 2007. Inhibition of renal serine proteases regulating the epithelial sodium channel ENaC could be a possible mechanism. Herein, we studied the effect of aprotinin in wild-type 129S1/SvImJ mice on sodium handling, tubular function, and integrity under a control and low-salt diet. Mice were studied in metabolic cages, and aprotinin was delivered by subcutaneously implanted sustained release pellets (2 mg/day over 10 days). Mean urinary aprotinin concentration ranged between 642 ± 135 (day 2) and 127 ± 16 (day 8) µg/mL . Aprotinin caused impaired sodium preservation under a low-salt diet while stimulating excessive hyperaldosteronism and unexpectedly, proteolytic activation of ENaC. Aprotinin inhibited proximal tubular function leading to glucosuria and proteinuria. Plasma urea and cystatin C concentration increased significantly under aprotinin treatment. Kidney tissues from aprotinin-treated mice showed accumulation of intracellular aprotinin and expression of the kidney injury molecule 1 (KIM-1). In electron microscopy, electron-dense deposits were observed. There was no evidence for kidney injury in mice treated with a lower aprotinin dose (0.5 mg/day). In conclusion, high doses of aprotinin exert nephrotoxic effects by accumulation in the tubular system of healthy mice, leading to inhibition of proximal tubular function and counterregulatory stimulation of ENaC-mediated sodium transport.

Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice.

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2-/-) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2-/- mice. However, Habp2-/- mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2-/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

Role of mTOR Signaling for Tubular Function and Disease.

The mechanistic target of rapamycin (mTOR) forms two distinct intracellular multiprotein complexes that control a multitude of intracellular processes linked to metabolism, proliferation, actin cytoskeleton, and survival. Recent studies have identified the importance of these complexes for transport regulation of ions and nutrients along the entire nephron. First reports could link altered activity of these complexes to certain disease entities, i.e. diabetic nephropathy, acute kidney injury or hyperkalemia.

Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism.

Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on in vivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.

Experimental nephrotic syndrome leads to proteolytic activation of the epithelial Na+ channel in the mouse kidney.

Proteolytic activation of the renal epithelial Na+ channel (ENaC) involves cleavage events in its α- and γ-subunits and is thought to mediate Na+ retention in nephrotic syndrome (NS). However, the detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α-ENaC and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the NH2-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed Na+ retention and increased expression of fragments of α-ENaC and γ-ENaC cleaved at both the proximal cleavage site and, more prominently, the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented Na+ retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α-ENaC and γ-ENaC was similarly found in healthy mice treated with a low-salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and, more prominently, distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.NEW & NOTEWORTHY This study demonstrates that murine experimental nephrotic syndrome leads to aprotinin-sensitive proteolytic activation of the epithelial Na+ channel at both the α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.