Gentamicin Inhibits Ca2+ Channel TPRV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway

Background Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal calcium wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. Methods We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase-reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient potential receptor vanilloid 5 (TPRV5), as determined by patch-clamp in HEK293 cells. Results Gentamicin increased urinary Ca2+ excretion in wild-type mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/-mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch-clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and that chronic gentamicin administration downregulated distal nephron Ca2+ transporters. Conclusions Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into calcium wasting in patients treated with gentamicin.

A Mathematical Model of Mitochondria in Proximal Tubule and Thick Ascending Limb Cells

Mitochondria are a key player in several kinds of tissue injury, and are even the ultimate cause of certain diseases. In this work we introduce new models of mitochondrial ATP generation in multiple tissues, including liver hepatocytes and the medullary thick ascending limb in the kidney. Using this model, we predict these tissues' responses to hypoxia, uncoupling, ischemia-reperfusion, and oxidative phosphorylation dysfunction. Our results suggest mechanisms explaining differences in robustness of mitochondrial function across tissues. The medullary thick ascending limb and proximal tubule in the kidney both experience a high metabolic demand, while having lower baseline activity of oxidative phosphorylation relative to the liver. These factors make these tissues susceptible to dysfunction of Complex III. A lower baseline oxygen tension observed in the thick ascending limb makes it susceptible to Complex IV. On the other hand, since the liver lacks these risk factors, and has higher baseline rates of glycolysis, it is less susceptible to all kinds of oxidative phosphorylation dysfunction.

Neonatal Bartter syndrome: A case report from Northern India

Bartter Syndrome is a rare genetic disorder affecting the renal tubular system causing a decreased absorption of sodium and chloride in the thick ascending limb of the Henle loop. Most children present in infancy with complaints of polyuria, polydipsia, vomiting, constipation and failure to thrive while older children present with recurrent episodes of dehydration, muscle weakness and cramps. The present study aimed to demonstrate a case of Bartter syndrome presenting as acute gastroenteritis.

Salt loading decreases urinary excretion and increases intracellular accumulation of uromodulin in stroke-prone spontaneously hypertensive rats.

Uromodulin (UMOD) is the most abundant renal protein secreted into urine by the thick ascending limb (TAL) epithelial cells of the loop of Henle. Genetic studies have demonstrated an association between UMOD risk variants and hypertension. We aimed to dissect the role of dietary salt in renal UMOD excretion in normotension and chronic hypertension. Normotensive Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) (n=8/sex/strain) were maintained on 1% NaCl for three weeks. A subset of salt-loaded SHRSP was treated with nifedipine. Salt-loading in SHRSP increased blood pressure (ΔSBP 35 ± 5 mmHg, p<0.0001) and kidney injury markers such as KIM-1 (fold change, FC 3.4; p=0.003), NGAL (FC, 2.0; p=0.012) and proteinuria. After salt-loading there was a reduction in urinary UMOD excretion in WKY and SHRSP by 26% and 55% respectively, compared to baseline. Nifedipine treatment reduced blood pressure in SHRSP, however, did not prevent salt-induced reduction in urinary UMOD excretion. In all experiments, changes in urinary UMOD excretion were dissociated from kidney UMOD protein and mRNA levels. Colocalization and ex-vivo studies showed that salt-loading increased intracellular UMOD retention in both WKY and SHRSP. Our study provides novel insights into the interplay between salt, UMOD, and blood pressure. The role of UMOD as a cardiovascular risk marker deserves mechanistic reappraisal and further investigations based on our findings.

Hypothetical Mechanism of Exercise-Induced Acute Kidney Injury Associated with Renal Hypouricemia

Renal hypouricemia (RHUC) is a hereditary disease that presents with increased renal urate clearance and hypouricemia due to genetic mutations in the urate transporter URAT1 or GLUT9 that reabsorbs urates in the renal proximal tubule. Exercise-induced acute kidney injury (EIAKI) is known to be a complication of renal hypouricemia. In the skeletal muscle of RHUC patients during exhaustive exercise, the decreased release of endothelial-derived hyperpolarization factor (EDHF) due to hypouricemia might cause the disturbance of exercise hyperemia, which might increase post-exercise urinary urate excretion. In the kidneys of RHUC patients after exhaustive exercise, an intraluminal high concentration of urates in the proximal straight tubule and/or thick ascending limb of Henle’s loop might stimulate the luminal Toll-like receptor 4–myeloid differentiation factor 88–phosphoinositide 3-kinase–mammalian target of rapamycin (luminal TLR4–MyD88–PI3K–mTOR) pathway to activate the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome and may release interleukin-1β (IL-1β), which might cause the symptoms of EIAKI.

SGLT2 Inhibition for Diabetic and Non-diabetic Kidney Disease

Chronic kidney disease (CKD) can be progressive, and its prognosis is worse because of increased mortality when it is associated with diabetes and cardiac disease. The outcomes of diabetic kidney disease (DKD) need to be improved, despite multifactorial interventions including glucose and blood pressure (BP) control, and the use of renin-angiotensin system (RAS) inhibitors, statins, and aspirin. Recent clinical trials suggest that sodium-glucose cotransporter-2 (SGLT2) inhibitors offer additional cardiorenal protection in DKD and non-diabetic CKD on top of RAS inhibition. The action of SGLT2 inhibitors is derived from the proximal tubule of the kidney, but their systemic effects beyond glucose-lowering involve hemodynamic and non-hemodynamic mechanisms. First, SGLT2 inhibitors restore tubuloglomerular feedback and relieve glomerular hypertension and albuminuria. Second, natriuresis and renal glycosuria lead to fluid and weight loss, resulting in BP lowering and prevention of heart failure. Third, SGLT2 inhibitors have anti-inflammatory and anti-oxidative actions that can reduce renal and cardiac inflammation and fibrosis, probably via adenosine monophosphate-activated protein kinase and sirtuin-1 activation. Finally, the proximal tubular workload is relieved, accompanied by increased erythropoiesis. Hypoxia-inducible factor 1 may be stimulated by renal outer medullary hypoxia when tubular sodium transport shifts from the proximal convoluted tubule to the proximal straight tubule and thick ascending limb, due to SGLT2 inhibition. These effects may also be beneficial in non-diabetic CKD, and we anticipate that SGLT2 inhibitors will prove effective for albuminuria reduction and preservation of kidney function in primary kidney diseases, including glomerulonephritis.

Mechanisms Underlying Calcium Nephrolithiasis

Nephrolithiasis is a worldwide problem with increasing prevalence, enormous costs, and significant morbidity. Calcium-containing kidney stones are by far the most common kidney stones encountered in clinical practice. Consequently, hypercalciuria is the greatest risk factor for kidney stone formation. Hypercalciuria can result from enhanced intestinal absorption, increased bone resorption, or altered renal tubular transport. Kidney stone formation is complex and driven by high concentrations of calcium-oxalate or calcium-phosphate in the urine. After discussing the mechanism mediating renal calcium salt precipitation, we review recent discoveries in renal tubular calcium transport from the proximal tubule, thick ascending limb, and distal convolution. Furthermore, we address how calcium is absorbed from the intestine and mobilized from bone. The effect of acidosis on bone calcium resorption and urinary calcium excretion is also considered. Although recent discoveries provide insight into these processes, much remains to be understood in order to provide improved therapies for hypercalciuria and prevent kidney stone formation. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

mTOR-Activating Mutations in RRAGD are Causative for Kidney Tubulopathy and Cardiomyopathy

Background Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all tubulopathy patients lack a genetic diagnosis. Methods We performed whole-exome and genome sequencings of a patient cohort with a novel inherited salt-losing tubulopathy, hypomagnesemia, and dilated cardiomyopathy. We also conducted subsequent functional analyses in vitro of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). Results In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD encoded by RRAGD plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. Conclusions Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

Integrative genomics uncover mechanisms of renal medullary carcinoma transformation, microenvironment landscape and therapeutic vulnerabilities.

Renal medullary carcinoma (RMC) is an aggressive desmoplastic tumour driven by bi-allelic loss of SMARCB1, however the cell-of-origin, the oncogenic mechanism and the features of its microenvironment remain poorly understood. Using single-cell and multi-region sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into at least three RMC cell states along an epithelial-mesenchymal gradient through a transcriptional switch involving loss of renal transcription factor TFCP2L1 and gain of a NFE2L2-associated ferroptosis resistance program. SMARCB1 re-expression in cultured RMC cells reactivates TFCP2L1 that relocates SWI/SNF from the promoters of the MYC-driven oncogenic program to the enhancers of TAL identity genes followed by ferroptotic cell death. We further show that RMC is associated with abundant M2-type macrophages and cancer-associated fibroblasts (CAFs) and we identify key regulatory cross-talks that shape this immunosuppressive microenvironment. Together our data describe the molecular events of RMC transformation and identify novel therapeutically targetable vulnerabilities.