The Less Well-Known Little Brothers: The SLC9B/NHA Sodium Proton Exchanger Subfamily—Structure, Function, Regulation and Potential Drug-Target Approaches

The SLC9 gene family encodes Na(+)/H(+) exchangers (NHEs), a group of membrane transport proteins critically involved in the regulation of cytoplasmic and organellar pH, cell volume, as well as systemic acid-base and volume homeostasis. NHEs of the SLC9A subfamily (NHE 1–9) are well-known for their roles in human physiology and disease. Much less is known about the two members of the SLC9B subfamily, NHA1 and NHA2, which share higher similarity to prokaryotic NHEs than the SLC9A paralogs. NHA2 (also known as SLC9B2) is ubiquitously expressed and has recently been shown to participate in renal blood pressure and electrolyte regulation, insulin secretion and systemic glucose homeostasis. In addition, NHA2 has been proposed to contribute to the pathogenesis of polycystic kidney disease, the most common inherited kidney disease in humans. NHA1 (also known as SLC9B1) is mainly expressed in testis and is important for sperm motility and thus male fertility, but has not been associated with human disease thus far. In this review, we present a summary of the structure, function and regulation of expression of the SLC9B subfamily members, focusing primarily on the better-studied SLC9B paralog, NHA2. Furthermore, we will review the potential of the SLC9B subfamily as drug targets

The sodium/proton exchanger NHA2 regulates blood pressure through a WNK4-NCC dependent pathway in the kidney.

NHA2 is sodium/hydrogen exchanger that was associated with arterial hypertension in humans, but the role of NHA2 in kidney function and blood pressure homeostasis is currently unknown. Here we show that NHA2 localizes almost exclusively to distal convoluted tubules in the kidney. NHA2 knock-out mice displayed reduced blood pressure, normocalcemic hypocalciuria and an attenuated response to the thiazide diuretic hydrochlorothiazide. Phosphorylation of the thiazide-sensitive sodium/chloride cotransporter NCC and its upstream activating kinase Ste20/SPS1-related proline/alaninerich kinase (SPAK), as well as the abundance of with no lysine [K] kinase 4 (WNK4), were significantly reduced in kidneys of NHA2 knock-out mice. In vitro experiments recapitulated these findings and revealed increased WNK4 ubiquitylation and enhanced proteasomal WNK4 degradation upon loss of NHA2. The effect of NHA2 on WNK4 stability was Kelch-like 3 (KLHL3)-dependent. More specifically, loss of NHA2 selectively attenuated KLHL3 phosphorylation and the physiologically important PKAand PKC-mediated decrease of WNK4 degradation was blunted upon loss of NHA2. Phenotype analysis of NHA2/NCC double knock-out mice supported the notion that NHA2 affects blood pressure homeostasis by an intrarenal and NCC-dependent mechanism. In summary, our data reveal NHA2 as a critical component of the WNK4-NCC pathway and hence as a novel regulator of blood pressure homeostasis in the kidney.A readme.txt file with detailed re-use instructions has been uploaded. Uncropped blots can be visualized using Word (Version 2003 or higher), derived expression data can be visualized by Graph Pad Prism (Version 9 or higher). Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001711Award Number: 31003A_152829Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001711Award Number: NCCR TransCureDetailed description of methods can be found in the original, published manuscript (https://pubmed.ncbi.nlm.nih.gov/32956652/). 1. Uncropped immunoblots of all immunoblots published in the manuscript (incl. Supplemental Information). For details of SDS-PAGE and immunoblotting see Materials and Methods section of the published manuscript. Developed X-ray films were scanned. Unedited scans were safed as TIF files and compiled in a Word Document. 2. Basic data of normalized expression (immunoblots or real-time PCR, for details of quantification see Materials and Methods section of the published manuscript) were imported in Graph Pad Prism (Version 9) for statistical analysis and visualization

Prevalence and characteristics of genetic disease in adult kidney stone formers.

BACKGROUND Molecular mechanisms of kidney stone formation remain unknown in most patients. Previous studies showed high a heritability of nephrolithiasis, but data on prevalence and characteristics of genetic disease in unselected adults with nephrolithiasis are lacking. This study was conducted to fill this important knowledge gap. METHODS We performed whole exome sequencing in 787 participants of the Bern Kidney Stone Registry, an unselected cohort of adults with ≥ 1 past kidney stone episode (KSF), and 114 non-stone-forming individuals (NKSF). An exome-based panel of 34 established nephrolithiasis genes was analyzed and variants assessed according to ACMG criteria. Pathogenic (P) or likely pathogenic (LP) variants were considered diagnostic. RESULTS Mean age of KSF was 47±15 years, and 18% were first time KSF. A Mendelian kidney stone disease was present in 2.9% (23 of 787) of KSF. The most common genetic diagnoses were cystinuria (SLC3A1, SLC7A9; n=13), Vitamin D-24 hydroxylase deficiency (CYP24A1; n=5) and primary hyperoxaluria (AGXT, GRHPR, HOGA1; n=3). 8.1% (64 of 787) of KSF were monoallelic for LP/P variants predisposing to nephrolithiasis, most frequently in SLC34A1/A3 or SLC9A3R1 (n=37), CLDN16 (n=8) and CYP24A1 (n=8). KSF with Mendelian disease had a lower age at the first stone event (30±14 years vs. 36±14 years, p=0.003), were more likely to have cystine stones (23.4% vs. 1.4%) and less likely to have calcium oxalate monohydrates stones (31.9% vs. 52.5%) compared to KSF without genetic diagnosis. The phenotype of KSF with variants predisposing to nephrolithiasis was subtle and showed significant overlap with KSF without diagnostic variants. In NKSF, no Mendelian disease was detected, and LP/P variants were significantly less prevalent compared to KSF (1.8% vs. 8.1%). CONCLUSION Mendelian disease is uncommon in unselected adult KSF, yet variants predisposing to nephrolithiasis are significantly enriched in adult KSF

The role of urinary supersaturations for lithogenic salts in the progression of autosomal dominant polycystic kidney disease.

BACKGROUND Autosomal dominant polycystic kidney disease (ADPKD) is associated with significant risk of forming kidney stones, especially those made of calcium oxalate and uric acid, compared with the general population. Since crystals are able to activate the inflammasome and lead to cell injury, crystalluria might worsen ADPKD natural history, acting as a third hit. METHODS The Bern ADPKD registry is a prospective observational cohort study. Height-adjusted total kidney volume (ht-TKV) was measured at baseline and every 3 years. Twenty-four hour urinary solute excretions collected at baseline and eGFR measurements over time were included in this analysis. Twenty-four hour urinary supersaturations (SS) for calcium oxalate, calcium phosphate and uric acid were calculated using EQUIL-2. Linear regression models were used to assess linear and non-linear associations between slopes of ht-TKV and eGFR with SSs and 24 h urinary solute excretions. RESULTS Seventy-seven participants (mean age 45.0 [SD 12.9] years, eGFR 76.4 [28.3] mL/min/1.73 m2) were included, with a median follow-up of 4 years. The median slopes of ht-TKV and eGFR were 3.9 percent/year and 2.9 mL/min/1.73 m2/year, respectively. SS for uric acid showed a direct, linear association (p value for linearity 0.035) with ht-TKV slope. When analyzing individual components, urinary uric acid, ammonium, magnesium and sulfate were all directly associated with ht-TKV slope. Urinary sulfate was also directly associated with eGFR slope. CONCLUSIONS Uric acid supersaturation and several other urinary components are identified as predictors of cyst growth in patients with ADPKD. Future studies with a dedicated design are needed to investigate the pathophysiological mechanisms underlying these associations

Tolvaptan treatment is associated with altered mineral metabolism parameters and increased bone mineral density in ADPKD patients.

BACKGROUND Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by a unique bone and mineral phenotype. The impact of Tolvaptan treatment on mineral metabolism and bone mineral density is unknown. METHODS We conducted an analysis in the Bern ADPKD registry, a prospective observational cohort study. Mineral metabolism parameters were measured at baseline and every 12 months thereafter. Bone mineral density was determined by dual-energy X-ray absorptiometry at baseline and after 3 years. Multivariable mixed-effects regression models were applied to assess changes in mineral metabolism parameters and bone mineral density associated with Tolvaptan treatment. RESULTS A total of 189 participants (122 without and 67 with subsequent Tolvaptan treatment) were included in the analysis. During follow-up, Tolvaptan treatment was associated with increased bone mineral density at the femoral neck (β 0.092; 95% CI 0.001, 0.183; p = 0.047). In addition, Tolvaptan treatment was associated with higher plasma magnesium (β 0.019; 95% CI 0.001, 0.037; p = 0.037), bicarbonate (β 0.972; 95% CI 0.242, 1.702; p = 0.009) and urine pH (β 0.214; 95% CI 0.056, 0.372; p = 0.008), and lower parathyroid hormone (β -0.191; 95% CI -0.328, -0.053; p = 0.006), 1,25-(OH)2-Vitamin D3 (β -0.126; 95% CI -0.235, -0.164; p = 0.024) and fractional urinary magnesium excretion (β -0.473; 95% CI -0.622, -0.324; p < 0.001). CONCLUSIONS Chronic Tolvaptan treatment is associated with increased femoral bone mineral density and significant changes in both mineral metabolism and acid-base parameters in ADPKD patients

Urinary Lithogenic Risk Profile in ADPKD Patients Treated with Tolvaptan

Background and objectives: Nephrolithiasis is a common health problem in autosomal dominant polycystic kidney disease (ADPKD) and significantly contributes to patient morbidity. Recently, Tolvaptan has been introduced for the treatment of ADPKD, but whether it is associated with alterations of the urinary lithogenic risk profile remains unknown. Design, setting, participants, &amp; measurements: We conducted an analysis of participants enrolled in the Bern ADPKD registry, a prospective observational cohort study. Twenty-four-hour urine analyses were performed at baseline and then at yearly follow-ups. Relative supersaturation ratios for calcium oxalate, brushite, and uric acid were calculated with the program EQUIL2. Unadjusted and multivariable mixed-effects linear regression models, adjusted for age, sex, body mass index, eGFR, net acid excretion, and height-adjusted total kidney volume, were used to assess the association of Tolvaptan with urinary parameters relevant for kidney stone formation. The maximum individual follow-up time was 3 years, median follow-up time 1.9 years, and cumulative follow-up time 169 years. Results: In total, 125 participants (38 with and 87 without Tolvaptan treatment) were included in the analysis. In multivariable analysis, Tolvaptan treatment was associated [adjusted estimate of the difference between Tolvaptan and no Tolvaptan; 95% confidence interval (CI)] with lower urine relative supersaturation ratios for calcium oxalate (-0.56; 95% CI, -0.82 to -0.3; P&lt;0.001), brushite (-0.33; 95% CI, -0.54 to -0.11; P=0.004), and uric acid (-0.62; 95% CI, -0.88 to -0.37; P&lt;0.001), and with higher urine citrate in mmol/mmol creatinine per day (0.25; 95% CI, 0.05 to 0.46; P=0.02) and calcium in mmol/mmol creatinine per day (0.31; 95% CI, 0.09 to 0.53; P=0.006) excretion. In addition, Tolvaptan treatment was associated with lower net acid excretion in mEq/mmol creatinine per day (-0.54; 95% CI, -0.90 to -0.17; P=0.004) and higher net gastrointestinal alkali absorption in mEq/mmol creatinine per day (0.57; 95% CI, 0.26 to 0.88; P&lt;0.001). Conclusions: Tolvaptan treatment is associated with a significantly improved urinary lithogenic risk profile in patients with ADPKD

Prevalence and characteristics of monogenic disease in adult kidney stone formers

Molecular mechanisms of kidney stone formation remain unknown in most patients. Previous studies showed a high heritability of nephrolithiasis, but data on prevalence and characteristics of monogenic disease in unselected adults with nephrolithiasis are lacking. We performed whole exome sequencing in 787 participants of the Bern Kidney Stone Registry, an unselected cohort of adults with ≥ 1 past kidney stone episode (KSF), and 114 non-stone- forming individuals (NKSF). A panel of 34 established nephrolithiasis genes was analyzed and variants were assessed according to ACMG criteria. Pathogenic or likely pathogenic variants were considered diagnostic. Mean age of KSF was 47±15 years, and 18 % were first time KSF. A monogenic kidney stone disease was present in 8.4 % (66 of 787) of KSF and in 0.9 % (1 of 114) of NKSF. The most common genetic diagnoses were cystinuria (SLC3A1, SLC7A9; n=16), renal phosphate wasting (SLC34A1 or SLC34A3; n=20), Vitamin D-24 hydroxylase deficiency (CYP24A1; n=13) and renal magnesium wasting with hypercalciuria (CLDN16; n=8). KSF with monogenic disease had a lower mean age at the first stone event (30±14 years vs. 36±14 years, p=0.003), were more likely to have cystine stones (23.4 % vs. 1.4 %) and less likely to have calcium oxalate monohydrates stones (31.9 % vs. 52.5 %) compared to KSF without genetic diagnosis. All other clinical parameters were similar between the two groups. Thus, monogenic disease is prevalent in unselected adult KSF. The usefulness of clinical parameters for the prediction of monogenic disease is limited in adult KSF