Prospective long-term evaluation of incomplete distal renal tubular acidosis in idiopathic calcium nephrolithiasis diagnosed (treated) by low-dose NH4CL loading – gender prevalences and impact of alkali treatment

Purpose Prospective evaluation of the prevalence of incomplete distal renal tubular acidosis (idRTA) in idiopathic calcium stone formers (ICSF) diagnosed by half-dose ammonium chloride loading (NH4Cl, 0.05 g/kg body weight/day) and impact of alkali treatment of idRTA. Methods Evaluation of 386 consecutive idiopathic calcium stone formers (ICSF) (280 males, 106 females) for idRTA. If screening fasting urine pH was > 5.80, 1-day NH4Cl loading was performed without severe adverse effects. Normally, urine pH falls below 5.45. Results Sixty-four idiopathic calcium stone formers exhibited idRTA, one complete dRTA. Prevalence was higher in women (25.4%) than in men (13.6%). Thus, for more equilibrated comparisons, we formed pairs of 62 idiopathic calcium stone formers (ICSF) with and 62 without idRTA, matched for gender, age, BMI and serum creatinine. Idiopathic calcium stone formers with idRTA more often had hypercalciuria (p < 0.025) and urine citrate < 2 mmol/d (p < 0.05), formed calcium phosphate stones more frequently, exhibited higher numbers of stones/year (1.4 ± 1.5 vs. 0.9 ± 0.8, p = 0.034) and 2.5 times more intrarenal calcifications (4.6 ± 5.9 vs. 1.8 ± 3.6, p = 0.002). All idiopathic calcium stone formers with idRTA were recommended chronic alkali therapy. After 4–15 years of follow-up, stone events /years follow-up (stone passage or urologic intervention) were higher in patients non-adherent to alkali therapy (0.61 ± 0.92) than in patients adherent to treatment (0.11 ± 0.21, p = 0.006). Conclusion Incomplete distal renal tubular acidosis is 1.8-fold more prevalent among female idiopathic calcium stone formers, predicts more stone recurrences, predisposes to calcium phosphate stones and is associated with 2.5 times more intrarenal calcifications vs. non-idRTA patients. Chronic alkali treatment reduces clinical stone recurrences by 5.5 times. Graphical abstract

Physiologic Regulation of Systemic Klotho Levels by Renal CaSR Signaling in Response to CaSR Ligands and pHo

Background: The kidney is the source of sKlotho and kidney-specific loss of Klotho leads to a phenotype resembling the premature multi-organ failure phenotype in Klothohypomorphic mice (kl/kl mice). Klotho and the Ca-sensing receptor (CaSR) are highly expressed in the distal convoluted tubule (DCT). The physiologic mechanisms that regulate sKlotho levels are unknown. Methods: We measured sKlotho in WT and tubule-specific CaSR-/- (TS-CaSR-/-) mice treated with calcimimetics, alkali, or acid, and Klotho shed from minced mouse kidneys, as well as from HEK-293 cells expressing the CaSR and Klotho, in response to calcimimetics, calcilytics, alkalotic and acidic pH, and ADAM protease inhibitors. The CaSR, Klotho, and ADAM10 were imaged in mouse kidneys and cell expression systems using confocal microscopy. Results: The CaSR, Klotho, and ADAM10 co-localize on the baso-lateral membrane of the DCT. Calcimimetics and HCO3 increase serum sKlotho levels in WT but not in CaSR-/- mice and acid pH suppresses sKlotho levels in WT mice. In minced kidneys and cultured cells, CaSR activation with high Ca, calcimimetics, or alkali increase shed Klotho levels via ADAM10, as demonstrated using the ADAM10 inhibitor GI254023X and siRNA. In cultured cells the CaSR, Klotho, and ADAM10 form cell surface aggregates that disperse following CaSR activation. Conclusions: We identify a novel physiologic mechanism for regulation of sKlotho levels by the renal CaSR-ADAM10-Klotho pathway. We show that CaSR activators, including alkali, increase renal CaSR-stimulated Klotho shedding and predict that this mechanism is relevant to the effects of acidosis and alkali therapy on CKD progression.

FC 001ACIDOSIS AND ALKALI THERAPY ARE ASSOCIATED WITH TRANSCRIPTIONAL CHANGES AND ALTERED ABUNDANCE OF GENES INVOLVED IN CELL METABOLISM AND BICARBONATE TRANSPORT IN KIDNEY TRANSPLANT RECIPIENTS

Background and Aims Metabolic acidosis is a common event in kidney transplant recipients and has been associated to a higher risk of graft loss and mortality. In patients with CKD and acidosis, alkali therapy ameliorating acidosis appears to protect kidney function. However, it is still poorly understood how acidosis causes the detrimental effects to kidney graft function and how alkali therapy would interact with these mechanisms. Here we aim to identify transcriptomic alterations in kidney transplant recipients without metabolic acidosis in comparison to patients with metabolic acidosis with and without alkali therapy. Moreover, we examined immunolocalization of key proteins involved in acid-base base regulation in biopsies from these patients. Method We obtained 22 biopsies of patients 4-6 years after kidney transplantation. Among these patients, nine were not acidotic (serum [HCO3-] ≥ 22 mM), nine had acidosis ([HCO3-] &lt; 22 mM), and four had acidosis and received sodium bicarbonate (alkali therapy) fully correcting acidosis. Age, immunosuppressive drugs, time after transplantation, and eGFR were not statistically different between groups. RNA was extracted from biopsies and RNAseq was performed. Immunohistochemistry was performed for key proteins involved in the renal regulation of acid-base balance. Additionally, a control group of 6 non-transplanted healthy kidneys was included in the histology analysis. Results RNAseq analysis revealed 40 genes differentially expressed between acidosis and no acidosis groups. While most of the genes tended to be recovered by alkali therapy, only three fully recovered with bicarbonate supplementation (p-value &lt; 0.05 and log2(fold change) above 0.5). These genes were KCNJ15 (Kir4.2), SHMT1, and ACADSB. Renal localization of the genes was determined using single-cell RNA sequencing data (Ransick et al., Developmental Cell, 2019, doi.org/10.1016/j.devcel.2019.10.005). Most of the genes were expressed in the proximal tubule and were organized in the model shown in Figure 1A. Several of these genes participate in cell metabolism, such as beta-oxidation, and iron, folate, and methionine metabolism. Moreover, the K+-channel Kir4.2 regulates the activity of the electrogenic sodium bicarbonate cotransporter 1 (NBCe1, SLC4A4) and ammoniagenesis in renal proximal tubules. Immunofluorescence analysis showed that NBCe1 expression in proximal tubules was strongly reduced in patients who developed acidosis and was partially recovered in patients who received alkali therapy (Figure 1B). In type B intercalated cells, a similar pattern was observed for Pendrin (SLC26A4). No alteration in the expression of GDH (GLUD1), AE1 (SLC4A1), AQP2, CA2, RhCG (SLC42A3), and B1 subunit of the H+ATPase (ATP6V1B1) was observed in kidneys of treated or untreated patients with acidosis. Conclusion Kidney transplant recipients suffering from metabolic acidosis show distinct expression pattern of genes involved in cell metabolism and acid-base transport.

A Case of BRASH Syndrome, Refractory Acidosis Necessitating Multimodal Approach to Treat it

A 69-year-old female patient, with hypertension and diabetes mellitus, was brought to Emergency Department in unresponsive state. The initial evaluation revealed that the patient had wide complex junctional bradycardia owing to hyperkalemia; hypoglycaemia, metabolic acidosis. Treatment was started in an attempt to establish definitive airway; correct glucose levels, cardiac membrane stabilisation. Interim, patient had a cardiac arrest and the Return Of Spontaneous Circulation (ROSC) was achieved and then stabilised. Later, she was found to have viral hepatitis complicated with ischemic hepatitis due to urosepsis. Managing a coding patient with severe metabolic acidosis in a resource limited setting is always challenging. Numerous paradoxes including usage of alkali therapy, choice of inotropes, achievement of haemodynamic neutral intubation is extensively studied yet debated. The present case encompasses the difficulties and possible solutions in managing such patient with refractory acidosis, Bradycardia, Renal failure, AV nodal blockers, Shock, Hyperkalemia syndrome (BRASH).

P0004REDUCED EXPRESSION OF PROXIMAL ACID-BASE TRANSPORT PROTEINS IN KIDNEY TRANSPLANT PATIENTS WITH METABOLIC ACIDOSIS

Background and Aims Metabolic acidosis (MA) is a frequent complication of chronic kidney disease and an independent risk factor for kidney disease progression and mortality. MA is highly prevalent after kidney transplantation (12%-58%)(1). However, there are scarcely any data available on the underlying pathomechanisms and in particular molecular mechanisms involved in metabolic acidosis after kidney transplantation. Thus, we wanted to investigate the expression of key acid base transport proteins in kidney biopsies of kidney transplant recipients with and without metabolic acidosis. Method We evaluated 22 kidney transplant biopsies including 9 biopsies from kidney transplant recipients (KTR) with MA, nine biopsies from KTRs without MA (control) and four biopsies from KTRs with MA that were consequently subjected to alkali therapy (Alkali therapy). Immunofluorescence staining was used to identify key renal acid-base transport proteins. Additionally, six control kidneys were analyzed. Immunofluorescence staining was used to identify key renal acid-base transport proteins along the nephron. In addition, RNA extraction and full RNA sequencing analysis of all biopsies –where available- was performed. Results In the proximal tubule, we observed reduced immunostaining for the sodium bicarbonate cotransporter NBCe1 (SLC4A4) in the MA group compared to the control and alkali group, whereas the alkali group demonstrated the strongest staining of all three groups. In the distal nephron, expression of the chloride/bicarbonate exchanger Pendrin (SLC26A4) and the B1 subunit of the V-ATPase (ATP6V1B1) were markedly stronger in the alkali and control group compared to the MA group. Expression of other acid base proteins such as Renal ammonia transporter RhCG (SLC42A3), Carbonic Anhydrase II, Glutamate dehydrogenase, anion exchanger AE1 (SLC4A1) and the B2 subunit of the V-ATPase (ATP6V1B2) showed no difference among all groups. Interestingly, the B2 subunit was absent in the proximal tubule in transplant biopsies of all groups. In kidney biopsies of transplant recipients with metabolic acidosis RNA abundance of NBCe1, CAII and Pendrin was lower while RhCG and B1 RNA counts were not different when compared to recipients without metabolic acidosis. Conclusion Our data demonstrate altered protein and mRNA expression of several key acid base transporters in kidney biopsies of transplant recipients with metabolic acidosis. Treatment with alkali may have the potential to reverse or prevent these changes in renal allografts after transplantation.

Low Serum Bicarbonate and CKD Progression in Children

Background and objectivesStudies of adults have demonstrated an association between metabolic acidosis, as measured by low serum bicarbonate levels, and CKD progression. We evaluated this relationship in children using data from the Chronic Kidney Disease in Children study.Design, setting, participants, & measurementsThe relationship between serum bicarbonate and a composite end point, defined as 50% decline in eGFR or KRT, was described using parametric and semiparametric survival methods. Analyses were stratified by underlying nonglomerular and glomerular diagnoses, and adjusted for demographic characteristics, eGFR, proteinuria, anemia, phosphate, hypertension, and alkali therapy.ResultsSix hundred and three participants with nonglomerular disease contributed 2673 person-years of follow-up, and 255 with a glomerular diagnosis contributed 808 person-years of follow-up. At baseline, 39% (237 of 603) of participants with nonglomerular disease had a bicarbonate level of ≤22 meq/L and 36% (85 of 237) of those participants reported alkali therapy treatment. In participants with glomerular disease, 31% (79 of 255) had a bicarbonate of ≤22 meq/L, 18% (14 of 79) of those participants reported alkali therapy treatment. In adjusted longitudinal analyses, compared with participants with a bicarbonate level >22 meq/L, hazard ratios associated with a bicarbonate level of <18 meq/L and 19–22 meq/L were 1.28 [95% confidence interval (95% CI), 0.84 to 1.94] and 0.91 (95% CI, 0.65 to 1.26), respectively, in children with nonglomerular disease. In children with glomerular disease, adjusted hazard ratios associated with bicarbonate level ≤18 meq/L and bicarbonate 19–22 meq/L were 2.16 (95% CI, 1.05 to 4.44) and 1.74 (95% CI, 1.07 to 2.85), respectively. Resolution of low bicarbonate was associated with a lower risk of CKD progression compared with persistently low bicarbonate (≤22 meq/L).ConclusionsIn children with glomerular disease, low bicarbonate was linked to a higher risk of CKD progression. Resolution of low bicarbonate was associated with a lower risk of CKD progression. Fewer than one half of all children with low bicarbonate reported treatment with alkali therapy. Long-term studies of alkali therapy’s effect in patients with pediatric CKD are needed.