Distal convoluted tubule-specific disruption of the COP9 signalosome but not its regulatory target cullin 3 causes tubular injury.

The disease familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome) is caused by aberrant accumulation of with-no-lysine kinase (WNK4) activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We used Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in the DCT and examined the mice after short- and long-term deletion. Short-term DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, pSPAKS373, and pNCCT53 abundance. However, neither model demonstrated changes in plasma K+, Cl-, or total CO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance and a higher abundance of kidney injury molecule-1, a marker of proximal tubule injury. No injury or reduction in NCC or parvalbumin was observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.NEW & NOTEWORTHY CUL3 degrades WNK4, which prevents activation of NCC in the DCT. CSN regulation of CUL3 is impaired in the disease FHHt, causing accumulation of WNK4. Short-term DCT-specific disruption of CUL3 or the CSN in mice resulted in activation of the WNK4-SPAK-NCC pathway but not hyperkalemic metabolic acidosis found in FHHt. Tubule injury was observed only after long-term CSN disruption. The data suggest that disruption of other cullins may be the cause for the injury.

Single-Nucleus RNA Sequencing Reveals Loss of Distal Convoluted Tubule 1 Renal Tubules in HIV Viral Protein R Transgenic Mice.

Although hyponatremia and salt wasting are common in patients with HIV/AIDS, the understanding of their contributing factors is limited. HIV viral protein R (Vpr) contributes to HIV-associated nephropathy. To investigate the effects of Vpr on the distal tubules and on the expression level of the Slc12a3 gene, encoding the sodium-chloride cotransporter (which is responsible for sodium reabsorption in distal nephron segments), single-nucleus RNA sequencing was performed on kidney cortices from three wild-type (WT) and three Vpr transgenic (Vpr Tg) mice. The percentage of distal convoluted tubule (DCT) cells was significantly lower in Vpr Tg mice compared with WT mice (P < 0.05); in Vpr Tg mice, Slc12a3 expression was not significantly different in DCT cells. The Pvalb+ DCT1 subcluster had fewer cells in Vpr Tg mice compared with those in WT mice (P < 0.01). Immunohistochemistry revealed fewer Slc12a3+Pvalb+ DCT1 segments in Vpr Tg mice. Differential gene expression analysis between Vpr Tg and WT samples in the DCT cluster showed down-regulation of the Ier3 gene, which is an inhibitor of apoptosis. The in vitro knockdown of Ier3 by siRNA transfection induced apoptosis in mouse DCT cells. These observations suggest that the salt-wasting effect of Vpr in Vpr Tg mice is likely mediated by Ier3 down-regulation in DCT1 cells and loss of Slc12a3+Pvalb+ DCT1 segments.

The IFNγ-PDL1 Pathway Enhances CD8T-DCT Interaction to Promote Hypertension.

BACKGROUND: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. METHODS: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8+ T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. RESULTS: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. CONCLUSIONS: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

Clinical and genetic approach to renal hypomagnesemia.

Magnesium (Mg2+) is an important intracellular cation and essential to maintain cell function including cell proliferation, immunity, cellular energy metabolism, protein and nucleic acid synthesis, and regulation of ion channels. Consequences of hypomagnesemia affecting multiple organs can be in overt or subtle presentations. Besides detailed history and complete physical examination, the assessment of urinary Mg2+ excretion is help to differentiate renal from extra-renal (gastrointestinal, tissue sequestration, and shifting) causes of hypomagnesemia. Renal hypomagnesemia can be caused by an increased glomerular filtration and impaired reabsorption in proximal tubular cells, thick ascending limb of the loop of Henle or distal convoluted tubules. A combination of renal Mg2+ wasting, familial history, age of onset, associated features, and exclusion of acquired etiologies point to inherited forms of renal hypomagnesemia. Based on clinical phenotypes, its definite genetic diagnosis can be simply grouped into specific, uncertain, and unknown gene mutations with a priority of genetic approach methods. An unequivocal molecular diagnosis could allow for prediction of clinical outcome, providing genetic counseling, avoiding unnecessary studies or interventions, and possibly uncovering the pathogenic mechanism. Given numerous identified genes responsible for Mg2+ transport in renal hypomagnesemia over the past two decades, several potential and specific molecular and cellular therapeutic strategies to correct hypomagnesemia are promising.

Molecular Basis, Diagnostic Challenges and Therapeutic Approaches of Bartter and Gitelman Syndromes: A Primer for Clinicians.

Gitelman and Bartter syndromes are rare inherited diseases that belong to the category of renal tubulopathies. The genes associated with these pathologies encode electrolyte transport proteins located in the nephron, particularly in the Distal Convoluted Tubule and Ascending Loop of Henle. Therefore, both syndromes are characterized by alterations in the secretion and reabsorption processes that occur in these regions. Patients suffer from deficiencies in the concentration of electrolytes in the blood and urine, which leads to different systemic consequences related to these salt-wasting processes. The main clinical features of both syndromes are hypokalemia, hypochloremia, metabolic alkalosis, hyperreninemia and hyperaldosteronism. Despite having a different molecular etiology, Gitelman and Bartter syndromes share a relevant number of clinical symptoms, and they have similar therapeutic approaches. The main basis of their treatment consists of electrolytes supplements accompanied by dietary changes. Specifically for Bartter syndrome, the use of non-steroidal anti-inflammatory drugs is also strongly supported. This review aims to address the latest diagnostic challenges and therapeutic approaches, as well as relevant recent research on the biology of the proteins involved in disease. Finally, we highlight several objectives to continue advancing in the characterization of both etiologies.

Distal Renal Tubular Acidosis in an Iranian Patient with Hereditary Spherocytosis

Background: Hereditary spherocytosis (HS) and hereditary hereditary distal renal tubular acidosis (dRTA) are associated with mutations in the SLC4A1 gene encoding the anion exchanger 1. In this study, some patients with clinical evidence of congenital HS and renal symptoms were investigated. Methods: Twelve patients with congenital HS and renal symptoms were recruited from Ali-Asghar Children's Hospital (Tehran, Iran). A patient suspected of having dRTA was examined using whole exome sequencing method, followed by Sanger sequencing. Results: One patient (HS03) showed severe failure to thrive, short stature, frequent urinary infection, and weakness. A homozygote (rs571376371 for c.2494C>T; p.Arg832Cys) and a heterozygote (rs377051298 for c.466C>T; p.Arg156Trp) missense variant were identified in the SLC4A1 and SPTA1 genes, respectively. The compound heterozygous mutations manifested as idRTA and severe HS in patient HS03. Conclusion: Our observations, for the first time, revealed clinical and genetic characteristics of idRTA and severe HS in an Iranian patient HS03.

Mapping the genetic architecture of human traits to cell types in the kidney identifies mechanisms of disease and potential treatments.

The functional interpretation of genome-wide association studies (GWAS) is challenging due to the cell-type-dependent influences of genetic variants. Here, we generated comprehensive maps of expression quantitative trait loci (eQTLs) for 659 microdissected human kidney samples and identified cell-type-eQTLs by mapping interactions between cell type abundances and genotypes. By partitioning heritability using stratified linkage disequilibrium score regression to integrate GWAS with single-cell RNA sequencing and single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing data, we prioritized proximal tubules for kidney function and endothelial cells and distal tubule segments for blood pressure pathogenesis. Bayesian colocalization analysis nominated more than 200 genes for kidney function and hypertension. Our study clarifies the mechanism of commonly used antihypertensive and renal-protective drugs and identifies drug repurposing opportunities for kidney disease.

Epidemiology of Distal Renal Tubular Acidosis: A Study Using Linked UK Primary Care and Hospital Data.

INTRODUCTION: Distal renal tubular acidosis (dRTA), or RTA type 1, a rare inherited or acquired disease, is a disorder of the distal tubule caused by impaired urinary acid secretion. Due to associated conditions and nonspecific symptoms, dRTA may go undetected. This analysis aims to estimate the prevalence of dRTA in the UK Clinical Practice Research Datalink (CPRD) databases and extrapolate it to European Union Five (EU5) populations. METHODS: A retrospective analysis was conducted using the CPRD GOLD database and linked Hospital Episode Statistics (HES) data to identify diagnosed and potentially undiagnosed or miscoded patients (suspected patients). Patients' records with at least one diagnosis code for dRTA, RTA, specific autoimmune diseases, or renal disorders recorded between January 1987 and November 2017 were obtained and analyzed. An algorithm was developed to detect potentially undiagnosed/miscoded dRTA, based on associated conditions and prescriptions. RESULTS: A total of 216 patients with diagnosis of RTA or dRTA were identified (with 98 linked to hospital data), and 447 patients were identified as having suspected dRTA. dRTA prevalence for 2017 was estimated between 0.46 (recorded cases, of which 22.1% were considered primary) and 1.60 when including suspected cases (7.6% primary) per 10,000 people. Prescription and clinical records of diagnosed patients revealed a wide range of comorbidities and a need for pharmacological treatment to manage associated symptoms. CONCLUSION: The study provides new estimates of dRTA prevalence in Europe and suggests that patients may often be unreported or miscoded, potentially confounding appropriate disease management.

Cathepsin K (Clone EPR19992) Demonstrates Uniformly Positive Immunoreactivity in Renal Oncocytoma, Chromophobe Renal Cell Carcinoma, and Distal Tubules.

Introduction. Cathepsin K is overexpressed in several tumors associated with microphthalmia transcription factor (MiTF) family or mechanistic target of rapamycin (mTOR) upregulation. Among renal neoplasms, MiTF translocation renal cell carcinoma (RCC), perivascular epithelioid cell neoplasms (PEComa), and eosinophilic solid and cystic RCC have demonstrated Cathepsin K immunoreactivity. In this study, we demonstrate a uniform Cathepsin K expression in oncocytoma, chromophobe RCC (CHRCC), and distal tubules. Design. We stained 13 oncocytomas, 13 CHRCC, 14 clear cell RCC (CCRCC), 9 papillary RCC (PRCC), 9 PEComas, and 5 MiTF RCC. Additionally, we assessed immunoreactivity for Cathepsin K in non-neoplastic renal parenchyma. Immunolabeling was performed on regularly charged slides from formalin-fixed paraffin-embedded tissue with monoclonal anti-rabbit antibodies to human Cathepsin K (clone EPR19992, Abcam). Results. All oncocytomas demonstrated diffuse strong cytoplasmic immunolabeling. CHRCC demonstrated uniform less intense immunolabeling in all cases with membranous accentuation. The assessment of the non-neoplastic renal parenchyma in all cases showed strong cytoplasmic immunoreaction in distal tubules and proximal tubules stained faintly. Mesangial cells were not immunoreactive. All MiTF RCC and PEComas were immunoreactive for Cathepsin K, whereas CCRCC and PRCC were negative in all cases. Conclusions. In this study, we expand the spectrum of renal neoplasms reactive with a particular clone of Cathepsin K (EPR19992). Distal tubules are strongly immunoreactive for Cathepsin K. Our conclusions need to be taken into consideration when differential diagnosis includes MiTF RCC or PEComa and this Cathepsin K clone is included in the immunohistochemical panel. This newer antibody clone was not tested in prior publications, potentially explaining the difference in conclusions.

Expression pattern of apoptosis-inducing factor in the kidneys of streptozotocin-induced diabetic rats.

BACKGROUND: It is believed that tubulo-interstitial fibrosis and atrophy in diabetic patients are directly associated with the progression of chronic kidney disease, CKD. AIF is one of the crucial factors responsible for mitochondrial apoptosis, however, it can also promote cell survival independently from its role in apoptosis, and therefore can be potentially used as a tool in prevention of the onset of CKD in diabetic patients. Our aim was to investigate the significance of AIF expression in the development of CKD by observing the expression of AIF in 2 weeks' and 2 months' kidneys of diabetic rats compared to their controls. METHODS: Male Sprague-Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). After 2 weeks and 2 months kidney samples were collected and analysed in different renal areas. RESULTS: Characteristic morphologic changes were found between the 2 months' control and 2 months' diabetic groups. Those changes, including fibrosis and possible replacement of podocytes with connective tissue were mainly present in the glomeruli. AIF expression was seen in the both cortex, and in the collecting ducts of the medulla. Strong intensity of AIF expression was seen in proximal and distal convoluted tubules in both diabetic groups. In the control groups the glomeruli showed no AIF staining but moderate staining was seen in both diabetic groups. Overall, the percentage of AIF positive cells in the glomeruli was the lowest. The greatest rise in cell positivity was displayed from the 2 weeks' control group to 2 weeks' diabetes group (38 %) in glomeruli. The cell positivity of the 2 weeks' diabetic group is significantly reduced to 18 % in the 2 months' diabetic group in glomeruli. A similar pattern was seen in the proximal tubular cells (92 % positivity 2 weeks diabetic groups; 89 % positivity 2 months diabetic groups), as well as in the distal tubules. The highest percentage of AIF positive cells was seen in the collecting ducts, more than 80 % in all groups. CONCLUSIONS: Our study provides insight into AIF expression pattern during short term diabetes model, confirming possible dual role of AIF, not only in apoptosis but also in cell function and homeostasis, and proving AIF as potential therapeutic target and marker of advancement of CKD.

SATB1 and PTEN expression patterns in biopsy proven kidney diseases.

BACKGROUND: In present study we investigated expression pattern of the special tissue markers. SATB1 and PTEN to evaluate possible influence in pathophysiology and development of various biopsy proven kidney diseases. METHODS: The 32 kidney biopsy samples were analysed using light, immunofluorescence and electron microscopy. There were 19 samples in proliferative and 13 samples in non- proliferative group of renal diseases. As control group, 9 specimens of healthy kidney tissue taken after surgery of kidney tumour were used. SATB1 and PTEN markers were used for immunofluorescence staining. Analysed tissue structures were glomeruli, proximal convoluted tubules (PCT) and distal convoluted tubules (DCT). The number of SATB1 and PTEN cells were calculated and the data compared between kidney structures, disease groups and control specimens. RESULTS: Both markers were positive in all investigated kidney structures, with expression generally, more prominent in tubular epithelial cells than in glomeruli, with the highest staining intensity rate as well as highest rate of both markers in DCT of proliferative diseases group (SATB1 64.5 %, PTEN 52 %). There was statistically significant difference in SATB1 expression in all tissue structures of interest in proliferative as well as non- proliferative group compared to control group (p < 0.01-p < 0.0001). PTEN expression were found significantly decreased in PCT of both disease groups in regard to control (PTEN 25.3 % and 23.8 % vs. 41.1 % (p < 0.01 and p < 0.001 respectively). CONCLUSION: SATB1 and PTEN could be considered as markers influenced in kidney disease development. SATB1/PTEN expression should be further investigated as useful markers of kidney disease activity as well as potential therapeutic target.

Inherited salt-losing tubulopathies are associated with immunodeficiency due to impaired IL-17 responses.

Increased extracellular sodium activates Th17 cells, which provide protection from bacterial and fungal infections. Whilst high salt diets have been shown to worsen autoimmune disease, the immunological consequences of clinical salt depletion are unknown. Here, we investigate immunity in patients with inherited salt-losing tubulopathies (SLT). Forty-seven genotyped SLT patients (with Bartter, Gitelman or EAST Syndromes) are recruited. Clinical features of dysregulated immunity are recorded with a standardised questionnaire and immunological investigations of IL-17 responsiveness undertaken. The effects of altering extracellular ionic concentrations on immune responses are then assessed. Patients are hypokalaemic and hypomagnesaemic, with reduced interstitial sodium stores determined by 23Na-magnetic resonance imaging. SLT patients report increased mucosal infections and allergic disease compared to age-matched controls. Aligned with their clinical phenotype, SLT patients have an increased ratio of Th2:Th17 cells. SLT Th17 and Tc17 polarisation is reduced in vitro, yet STAT1 and STAT3 phosphorylation and calcium flux following T cell activation are unaffected. In control cells, the addition of extracellular sodium (+40 mM), potassium (+2 mM), or magnesium (+1 mM) reduces Th2:Th17 ratio and augments Th17 polarisation. Our results thus show that the ionic environment typical in SLT impairs IL-17 immunity, but the intracellular pathways that mediate salt-driven Th17 polarisation are intact and in vitro IL-17 responses can be reinvigorated by increasing extracellular sodium concentration. Whether better correction of extracellular ions can rescue the immunophenotype in vivo in SLT patients remains unknown.

Mutation affecting the conserved acidic WNK1 motif causes inherited hyperkalemic hyperchloremic acidosis.

Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.

Short-chain fatty acid mitigates adenine-induced chronic kidney disease via FFA2 and FFA3 pathways.

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2-/- and FFA3-/- mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease.

The transcription factor Twist1 in the distal nephron but not in macrophages propagates aristolochic acid nephropathy.

Tubulointerstitial disease in the kidney culminates in renal fibrosis that portents organ failure. Twist1, a basic helix-loop-helix protein 38 transcription factor, regulates several essential biological functions, but inappropriate Twist1 activity in the kidney epithelium can trigger kidney fibrogenesis and chronic kidney disease. By contrast, Twist1 in circulating myeloid cells may constrain inflammatory injury by attenuating cytokine generation. To dissect the effects of Twist1 in kidney tubular versus immune cells on renal inflammation following toxin-induced renal injury, we subjected mice with selective deletion of Twist1 in renal epithelial cells or macrophages to aristolochic acid-induced chronic kidney disease. Ablation of Twist1 in the distal nephron attenuated kidney damage, interstitial fibrosis, and renal inflammation after aristolochic acid exposure. However, macrophage-specific deletion of Twist1 did not impact the development of aristolochic acid-induced nephropathy. In vitro studies confirmed that Twist1 in renal tubular cells underpins their susceptibility to apoptosis and propensity to generate pro-fibrotic mediators in response to aristolochic acid. Moreover, co-culture studies revealed that Twist1 in renal epithelia augmented the recruitment and activation of pro-inflammatory CD64+ macrophages. Thus, Twist1 in the distal nephron rather than in infiltrating macrophages propagates chronic inflammation and fibrogenesis during aristolochic acid-induced nephropathy.

FGF23 induced left ventricular hypertrophy mediated by FGFR4 signaling in the myocardium is attenuated by soluble Klotho in mice.

There is controversy regarding whether excess FGF23 causes left ventricular hypertrophy (LVH) directly through activation of fibroblast growth factor receptor 4 (FGFR4) in cardiomyocytes or indirectly through reductions in soluble Klotho (sK). We investigated the respective roles of myocardial FGFR4 and sKL in mediating FGF23-induced LVH using mouse genetic and pharmacological approaches. To investigate a direct role of myocardial FGFR4 in mediating the cardiotoxic effects of excess circulating FGF23, we administered rFGF23 to mice with cardiac-specific loss of FGFR4 (FGFR4 heart-cKO). We tested a model of sKL deficiency, hypertension and LVH created by the conditional deletion of FGFR1 in the renal distal tubule (FGFR1DT cKO mice). The cardioprotective effects of sKL in both mouse models was assessed by the systemic administration of recombinant sKL. We confirmed that FGF23 treatment activates PLCγ in the heart and induces LVH in the absence of membrane α-Klotho. Conditional deletion of FGFR4 in the myocardium prevented rFGF23-induced LVH in mice, establishing direct cardiotoxicity of FGF23 through activation of FGFR4. Recombinant sKL administration prevented LVH, but not HTN, in FGFR1DT cKO mice, consistent with direct cardioprotective effects. Co-administration of recombinant sKL with FGF23 in culture inhibited rFGF23-induced p-PLCγ signaling. Thus, FGF23 ability to include LVH represents a balance between FGF23 direct cardiac activation of FGFR4 and the modulating effects of circulating sKL to alter FGF23-dependent myocardial signaling pathways.

Bartter and Gitelman syndromes: Questions of class.

Bartter and Gitelman syndromes are rare inherited tubulopathies characterized by hypokalaemic, hypochloraemic metabolic alkalosis. They are caused by mutations in at least 7 genes involved in the reabsorption of sodium in the thick ascending limb (TAL) of the loop of Henle and/or the distal convoluted tubule (DCT). Different subtypes can be distinguished and various classifications have been proposed based on clinical symptoms and/or the underlying genetic cause. Yet, the clinical phenotype can show remarkable variability, leading to potential divergences between classifications. These problems mostly relate to uncertainties over the role of the basolateral chloride exit channel CLCNKB, expressed in both TAL and DCT and to what degree the closely related paralogue CLCNKA can compensate for the loss of CLCNKB function. Here, we review what is known about the physiology of the transport proteins involved in these disorders. We also review the various proposed classifications and explain why a gene-based classification constitutes a pragmatic solution.

Comparison of the renal cortex and the medulla for antibody mediated rejection.

OBJECTIVES: The presence of the peritubular capillaritis and its extent are important for diagnosis of the antibody-mediated rejection in kidneys. However, it is recommended that peritubular capillaritis should only be scored in the cortex. This study aims to focus on peritubular capillaritis scoring both in the cortex and the medulla to understand the value of the medulla in the diagnosis of antibody-mediated rejection. METHODS: Fifty-one allograft renal biopsy were re-evaluated for peritubular capillaritis, C4d and acute tubular injury, separately for the cortex and the medulla according to the Banff. RESULTS: Seventeen cases (33.3%) had peritubular capillaritis both in the cortex and the medulla and three (5.9%) cases had peritubular capillaritis only in the cortex while five (9.8%) cases had only in the medulla. Eighteen (35%) of the cases had C4d staining both in the cortex and the medulla and 14 (27.5%) cases had C4d positivity only in the cortex and 18 (35.3%) cases only in the medulla. Twenty-three (45%) cases had acute tubular injury both in the cortex and the medulla and 31 (60.7%) cases had acute tubular injury only in the cortex and 23 (45.1%) cases had only in the medulla. The sensitivity, specificity, positive and negative predictive values of medullar peritubular capillaritis predicting cortical peritubular capillaritis were 85.7%, 86.7%, 81.8% and 89.7%, respectively. CONCLUSION: In case of absence of the cortical tissue, medulla can be used as a reference for antibody-mediated rejection considering the morphological features, results of donor-specific antibody and renal function tests.

Loss of sodium chloride co-transporter impairs the outgrowth of the renal distal convoluted tubule during renal development.

BACKGROUND: Loss-of-function mutations in the sodium chloride (NaCl) co-transporter (NCC) of the renal distal convoluted tubule (DCT) cause Gitelman syndrome with hypokalemic alkalosis, hypomagnesemia and hypocalciuria. Since Gitelman patients are usually diagnosed around adolescence, we tested the idea that a progressive regression of the DCT explains the late clinical onset of the syndrome. METHODS: NCC wild-type and knockout (ko) mice were studied at Days 1, 4 and 10 and 6 weeks after birth using blood plasma analysis and morphological and biochemical methods. RESULTS: Plasma aldosterone levels and renal renin messenger RNA expression were elevated in NCC ko mice during the first days of life. In contrast, plasma ion levels did not differ between genotypes at age 10 days, but a significant hypomagnesemia was observed in NCC ko mice at 6 weeks. Immunofluorescent detection of parvalbumin (an early DCT marker) revealed that the fractional cortical volume of the early DCT is similar for mice of both genotypes at Day 4, but is significantly lower at Day 10 and is almost zero at 6 weeks in NCC ko mice. The DCT atrophy correlates with a marked reduction in the abundance of the DCT-specific Mg2+ channel TRPM6 (transient receptor potential cation channel subfamily M member 6) and an increased proteolytic activation of the epithelial Na+ channel (ENaC). CONCLUSION: After an initial outgrowth, DCT development lags behind in NCC ko mice. The impaired DCT development associates at Day 1 and Day 10 with elevated renal renin and plasma aldosterone levels and activation of ENaC, respectively, suggesting that Gitelman syndrome might be present much earlier in life than is usually expected. Despite an early downregulation of TRPM6, hypomagnesemia is a rather late symptom.