Calcineurin inhibitor effects on kidney electrolyte handling and blood pressure: tacrolimus versus voclosporin.

BACKGROUND AND HYPOTHESIS: Calcineurin inhibitors affect kidney electrolyte handling and blood pressure through an effect on the distal tubule. The second generation calcineurin inhibitor voclosporin causes hypomagnesemia and hypercalciuria less often than tacrolimus. This suggests different effects on the distal tubule, but this has not yet been investigated experimentally. METHODS: Rats were treated with voclosporin, tacrolimus or vehicle for 28 days. Dosing was based on a pilot experiment to achieve clinically therapeutic concentrations. Drug effects were assessed by electrolyte handling at day 18 and 28, thiazide testing at day 20, telemetric blood pressure recordings, and analysis of mRNA and protein levels of distal tubular transporters at day 28. RESULTS: Compared to vehicle, tacrolimus but not voclosporin significantly increased the fractional excretions of calcium (>4-fold), magnesium and chloride (both 1.5-fold) and caused hypomagnesemia. Tacrolimus but not voclosporin significantly reduced distal tubular transporters at mRNA and/or protein level, including the sodium-chloride cotransporter, transient receptor melastatin 6, transient receptor potential vanilloid 5, cyclin M2, sodium-calcium exchanger and calbindin-D28K. Tacrolimus but not voclosporin reduced the mRNA level and urinary excretion of epidermal growth factor. The saluretic response to hydrochlorothiazide at day 20 was similar in the voclosporin and vehicle groups, whereas it was lower in the tacrolimus group. The phosphorylated form of the sodium-chloride cotransporter was significantly higher at day 28 in rats treated with voclosporin than in those treated with tacrolimus. Tacrolimus transiently increased blood pressure, whereas voclosporin caused a gradual but persistent increase in blood pressure which was further characterized by high renin, normal aldosterone, and low endothelin-1. CONCLUSIONS: In contrast to tacrolimus, voclosporin does not cause hypercalciuria and hypomagnesemia, but similarly causes hypertension. Our data reveal differences between the distal tubular effects of tacrolimus and voclosporin and provide a pathophysiological basis for the clinically observed differences between the two calcineurin inhibitors.

Inhibition of pannexin-1 does not restore electrolyte balance in precystic Pkd1 knockout mice.

Mutations in PKD1 and PKD2 cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid-filled cysts in the kidney. In a subset of ADPKD patients, reduced blood calcium (Ca2+) and magnesium (Mg2+) concentrations are observed. As cystic fluid contains increased ATP concentrations and purinergic signaling reduces electrolyte reabsorption, we hypothesized that inhibiting ATP release could normalize blood Ca2+ and Mg2+ levels in ADPKD. Inducible kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/-) exhibit hypocalcemia and hypomagnesemia in a precystic stage and show increased expression of the ATP-release channel pannexin-1. Therefore, we administered the pannexin-1 inhibitor brilliant blue-FCF (BB-FCF) every other day from Day 3 to 28 post-induction of Pkd1 gene inactivation. On Day 29, both serum Ca2+ and Mg2+ concentrations were reduced in iKsp-Pkd1-/- mice, while urinary Ca2+ and Mg2+ excretion was similar between the genotypes. However, serum and urinary levels of Ca2+ and Mg2+ were unaltered by BB-FCF treatment, regardless of genotype. BB-FCF did significantly decrease gene expression of the ion channels Trpm6 and Trpv5 in both control and iKsp-Pkd1-/- mice. Finally, no renoprotective effects of BB-FCF treatment were observed in iKsp-Pkd1-/- mice. Thus, administration of BB-FCF failed to normalize serum Ca2+ and Mg2+ levels.

Fluid shear stress stimulates ATP release without regulating purinergic gene expression in the renal inner medullary collecting duct.

In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1-/- IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1-/- IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1-/- IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release.

Extracellular vesicles contribute to early cyst development in autosomal dominant polycystic kidney disease by cell-to-cell communication.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1-/- mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1-/- mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1-/- iMCD3 cells to EVs derived from Pkd1-/- mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1-/- cells to mDCT15 Pkd1-/- EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth.

Differential parathyroid and kidney Ca2+-sensing receptor activation in autosomal dominant hypocalcemia 1.

BACKGROUND: Parathyroid Ca2+-sensing receptor (CaSR) activation inhibits parathyroid hormone (PTH) release, while activation of renal CaSRs attenuates Ca2+ transport and increases expression of the pore-blocking claudin-14. Patients with autosomal dominant hypocalcemia 1 (ADH1), due to activating CASR mutations, exhibit hypocalcemia but not always hypercalciuria (elevated Ca2+ in urine). The latter promotes nephrocalcinosis and renal insufficiency. Although CaSRs throughout the body including the kidney harbor activating CASR mutations, it is not understood why only some ADH1 patients display hypercalciuria. METHODS: Activation of the CaSR was studied in mouse models and a ADH1 patient. In vitro CaSR activation was studied in HEK293 cells. FINDINGS: Cldn14 showed blood Ca2+ concentration-dependent regulation, which was absent in mice with kidney-specific Casr deletion, indicating Cldn14 is a suitable marker for chronic CaSR activation in the kidney. Mice with a gain-of-function mutation in the Casr (Nuf) were hypocalcemic with low plasma PTH levels. However, renal CaSRs were not activated at baseline but only after normalizing blood Ca2+ levels. Similarly, significant hypercalciuria was not observed in a ADH1 patient until blood Ca2+ was normalized. In vitro experiments indicate that increased CaSR expression in the parathyroid relative to the kidney could contribute to tissue-specific CaSR activation thresholds. INTERPRETATION: Our findings suggest that parathyroid CaSR overactivity can reduce plasma Ca2+ to levels insufficient to activate renal CaSRs, even when an activating mutation is present. These findings identify a conceptually new mechanism of CaSR-dependent Ca2+ balance regulation that aid in explaining the spectrum of hypercalciuria in ADH1 patients. FUNDING: Erasmus+ 2018/E+/4458087, the Canadian Institutes for Health research, the Novo Nordisk Foundation, the Beckett Foundation, the Carlsberg Foundation and Independent Research Fund Denmark.

Gentamicin Inhibits Ca2+ Channel TRPV5 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 Ca2+ 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 receptor potential vanilloid 5 (TRPV5), as determined by patch clamp in HEK293 cells. RESULTS: Gentamicin increased urinary Ca2+ excretion in wild-type mice after 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 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 Ca2+ wasting in patients treated with gentamicin.

Renal claudin-14 expression is not required for regulating Mg2+ balance in mice.

The kidneys play a crucial role in maintaining Ca2+ and Mg2+ homeostasis by regulating these minerals' reabsorption. In the thick ascending limb of Henle's loop (TAL), Ca2+ and Mg2+ are reabsorbed through the tight junctions by a shared paracellular pathway formed by claudin-16 and claudin-19. Hypercalcemia activates the Ca2+-sensing receptor (CaSR) in the TAL, causing upregulation of pore-blocking claudin-14 (CLDN14), which reduces Ca2+ and Mg2+ reabsorption from this segment. In addition, a high-Mg2+ diet is known to increase both urinary Mg2+ and Ca2+ excretion. Since Mg2+ may also activate CaSR, we aimed to investigate whether CaSR-dependent increases in CLDN14 expression also regulate urinary Mg2+ excretion in response to hypermagnesemia. Here, we show that a Mg2+-enriched diet increased urinary Mg2+ and Ca2+ excretion in mice; however, this occurred without detectable changes in renal CLDN14 expression. The administration of a high-Mg2+ diet to Cldn14-/- mice did not cause more pronounced hypermagnesemia or significantly alter urinary Mg2+ excretion. Finally, in vitro evaluation of CaSR-driven Cldn14 promoter activity in response to increasing Mg2+ concentrations revealed that Cldn14 expression only increases at supraphysiological extracellular Mg2+ levels. Together, these results suggest that CLDN14 is not involved in regulating extracellular Mg2+ balance following high dietary Mg2+ intake.NEW & NOTEWORTHY Using transgenic models and in vitro assays, this study examined the effect of Mg2+ on regulating urinary excretion of Ca2+ and Mg2+ via activation of the Ca2+-sensing receptor-claudin 14 (CLDN14) pathway. The study suggests that CLDN14 is unlikely to play a significant role in the compensatory response to hypermagnesemia.

Renal sodium and magnesium reabsorption are not coupled in a mouse model of Gordon syndrome.

Active reabsorption of magnesium (Mg2+ ) in the distal convoluted tubule (DCT) of the kidney is crucial for maintaining Mg2+ homeostasis. Impaired activity of the Na+ -Cl- -cotransporter (NCC) has been associated with hypermagnesiuria and hypomagnesemia, while increased activity of NCC, as observed in patients with Gordon syndrome, is not associated with alterations in Mg2+ balance. To further elucidate the possible interrelationship between NCC activity and renal Mg2+ handling, plasma Mg2+ levels and urinary excretion of sodium (Na+ ) and Mg2+ were measured in a mouse model of Gordon syndrome. In this model, DCT1-specific expression of a constitutively active mutant form of the NCC-phosphorylating kinase, SPAK (CA-SPAK), increases NCC activity and hydrochlorothiazide (HCTZ)-sensitive Na+ reabsorption. These mice were normomagnesemic and HCTZ administration comparably reduced plasma Mg2+ levels in CA-SPAK mice and control littermates. As inferred by the initial response to HCTZ, CA-SPAK mice exhibited greater NCC-dependent Na+ reabsorption together with decreased Mg2+ reabsorption, compared to controls. Following prolonged HCTZ administration (4 days), CA-SPAK mice exhibited higher urinary Mg2+ excretion, while urinary Na+ excretion decreased to levels observed in control animals. Surprisingly, CA-SPAK mice had unaltered renal expression of Trpm6, encoding the Mg2+ -permeable channel TRPM6, or other magnesiotropic genes. In conclusion, CA-SPAK mice exhibit normomagnesemia, despite increased NCC activity and Na+ reabsorption. Thus, Mg2+ reabsorption is not coupled to increased thiazide-sensitive Na+ reabsorption, suggesting a similar process explains normomagnesemia in Gordon syndrome. Further research is required to unravel the molecular underpinnings of this phenomenon and the more pronounced Mg2+ excretion after prolonged HCTZ administration.