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Aim Methods Results Conclusion Calcineurin inhibitors (CNIs) are the backbone for immunosuppression after solid organ transplantation. Although successful in preventing kidney transplant rejection, their nephrotoxic side effects contribute to allograft injury. Renal parenchymal lesions occur for cyclosporine A (CsA) as well as for the currently favored tacrolimus (Tac). We aimed to study whether chronic CsA and Tac exposures, before reaching irreversible nephrotoxic damage, affect renal compartments differentially and whether related pathogenic mechanisms can be identified.CsA and Tac were administered chronically in wild type Wistar rats using osmotic minipumps over 4 weeks. Functional parameters were controlled. Electron microscopy, confocal, and 3D‐structured illumination microscopy were used for histopathology. Clinical translatability was tested in human renal biopsies. Standard biochemical, RNA‐seq, and proteomic technologies were applied to identify implicated molecular pathways.Both drugs caused significant albeit differential damage in vasculature and nephron. The glomerular filtration barrier was more affected by Tac than by CsA, showing prominent deteriorations in endothelium and podocytes along with impaired VEGF/VEGFR2 signaling and podocyte‐specific gene expression. By contrast, proximal tubule epithelia were more severely affected by CsA than by Tac, revealing lysosomal dysfunction, enhanced apoptosis, impaired proteostasis and oxidative stress. Lesion characteristics were confirmed in human renal biopsies.We conclude that pathogenetic alterations in the renal compartments are specific for either treatment. Considering translation to the clinical setting, CNI choice should reflect individual risk factors for renal vasculature and tubular epithelia. As a step in this direction, we share protein signatures identified from multiomics with potential pathognomonic relevance. [ABSTRACT FROM AUTHOR]

Aim: Perturbed calcium homeostasis limits life expectancy in familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC). This rare disease occurs by loss‐of‐function mutations in CLDN16 or CLDN19 genes, causing impaired paracellular reabsorption of divalent cations along the cortical thick ascending limb (cTAL). Only partial compensation takes place in the ensuing late distal convoluted tubule, connecting tubule, and collecting duct, where the luminal transient receptor potential channel V5 (TRPV5), as well as basolateral plasma membrane calcium ATPase (PMCA) and sodium‐potassium exchanger (NCX1) mediate transcellular Ca2+ reabsorption. The loop diuretic furosemide induces compensatory activation in these distal segments. Normally, furosemide enhances urinary calcium excretion via inhibition of the aforementioned cTAL. As Ca2+ reabsorption in the cTAL is already severely impaired in FHHNC patients, furosemide may alleviate hypercalciuria in this disease by activation of the distal transcellular Ca2+ transport proteins. Methods: Cldn16‐deficient mice (Cldn16−/−) served as a FHHNC model. Wild‐type (WT) and Cldn16−/− mice were treated with furosemide (7 days of 40 mg/kg bw) or vehicle. We assessed renal electrolyte handling (metabolic cages) and key divalent transport proteins. Results: Cldn16−/− mice show higher Ca2+ excretion than WT and compensatory stimulation of Cldn2, TRPV5, and NCX1 at baseline. Furosemide reduced hypercalciuria in Cldn16−/− mice and enhanced TRPV5 and PMCA levels in Cldn16−/− but not in WT mice. Conclusions: Furosemide significantly reduces hypercalciuria, likely via upregulation of luminal and basolateral Ca2+ transport systems in the distal nephron and collecting duct in this model for FHHNC. [ABSTRACT FROM AUTHOR]

Claudin‐10b is an important component of the tight junction in the thick ascending limb (TAL) of Henle's loop and allows paracellular sodium transport. In immunofluorescence stainings, claudin‐10b–positive cells exhibited extensive extra staining of basolateral, column‐like structures. The precise localization and function have so far remained elusive. In isolated cortical TAL segments from C57BL/6J mice, kidney‐specific claudin‐10 knockout mice (cKO), and respective litter mates (WT), we investigated the localization and protein expression and function by fluorescence microscopy and electrophysiological measurements. Ultrastructural analysis of TAL in kidney sections was performed by electron microscopy. Claudin‐10b colocalized with the basolateral Na+‐K+ ATPase and the Cl– channel subunit barttin, but the lack of claudin‐10b did not influence the localization or abundance of these proteins. However, the accessibility of the basolateral infolded extracellular space to ouabain or fluorescein was increased by basolateral Ca2+ removal and in the absence of claudin‐10b. Ultrastructural analysis by electron microscopy revealed a widening of basolateral membrane infoldings in cKO in comparison to WT. We hypothesize that claudin‐10b shapes neighboring membrane invaginations by trans interaction to stabilize and facilitate high‐flux salt transport in a water‐tight epithelium. [ABSTRACT FROM AUTHOR]

Aim: The use of calcineurin inhibitors such as cyclosporine A (CsA) for immunosuppression after solid organ transplantation is commonly limited by renal side effects. CsA‐induced deterioration of glomerular filtration rate and sodium retention may be related to juxtaglomerular dysregulation as a result of suppressed cyclooxygenase 2 (COX‐2) and stimulated renin biosynthesis. We tested whether CsA‐induced COX‐2 suppression is caused by hyperactive renin‐angiotensin system (RAS) and whether RAS inhibition may alleviate the related side effects. Methods: Rats received CsA, the RAS inhibitor candesartan, or the COX‐2 inhibitor celecoxib acutely (3 days) or chronically (3 weeks). Molecular pathways mediating effects of CsA and RAS on COX‐2 were studied in cultured macula densa cells. Results: Pharmacological or siRNA‐mediated calcineurin inhibition in cultured cells enhanced COX‐2 expression via p38 mitogen‐activated protein kinase and NF‐kB signalling, whereas angiotensin II abolished these effects. Acute and chronic CsA administration to rats led to RAS activation along with reduced cortical COX‐2 expression, creatinine clearance and fractional sodium excretion. Evaluation of major distal salt transporters, NKCC2 and NCC, showed increased levels of their activating phosphorylation upon CsA. Concomitant candesartan treatment blunted these effects acutely and completely normalized the COX‐2 expression and renal functional parameters at long term. Celecoxib prevented the candesartan‐induced improvements of creatinine clearance and sodium excretion. Conclusion: Suppression of juxtaglomerular COX‐2 upon CsA results from RAS activation, which overrides the cell‐autonomous, COX‐2‐stimulatory effects of calcineurin inhibition. Angiotensin II antagonism alleviates CsA nephrotoxicity via the COX‐2‐dependent normalization of creatinine clearance and sodium excretion. [ABSTRACT FROM AUTHOR]

R4514 --> 525.17 --> Calcineurin inhibitors (CNI) are widely in use for immunosuppression in transplant recipients, but their nephrotoxicity may cause or aggravate renal disease. CNI pathology includes vascular and tubulointerstitial alterations. We have compared the effects of cyclosporine A (CsA) and tacrolimus (Tac) upon chronic administration in a rat model. We have tested whether Tac caused less damage than CsA. To this end, functional as well as glomerular and tubulointerstitial parameters were compared. Adult Wistar rats received CsA (25 mg/kg/d), Tac (2 mg/kg/d), or vehicle via subcutaneously implanted minipumps. After four weeks, rats were sacrificed and organs removed for protein analysis or perfusion‐fixed and kidneys analyzed for histopathology. NRK cells were treated with graded concentrations of CNI for 24h. For evaluation, confocal immunohistochemistry, EM nanotomy, EM immunostaining, Western blot, and clinical chemistry methods were applied. In rats, CsA and Tac produced distinct alterations in the tubulointerstitium. With CsA, dysmorphic lysosomes with peripheral LAMP1 signal, autophagic and mitophagic vacuoles caused epithelial decay of the initial proximal tubule, while glomerular changes were less pronounced. With Tac, glomerulosclerosis and damage encroachment to proximal tubule were more prominent. Both drugs induced fibrotic upregulation of alpha‐smooth muscle actin in the interstitium. Alterations in unfolded protein response (UPR) parameters included rises in p‐eIF2α, p‐PERK, p‐IRE1, sXBP1, CHOP in CsA‐, but not in Tac‐treated rats. Epithelial TUNEL signal was stronger in the CsA than in the Tac groups. Results were verified in NRK cells using graded caspase‐3 and lysosome formation criteria. Data revealed distinct nephrotoxicity patterns in the two examined immunosuppressive medications. While CsA seems to be primarily tubulotoxic, a stronger glomerular component was detected with Tac. Results may serve to better adjust treatment combinations in transplant patients. [ABSTRACT FROM AUTHOR]

R3588 --> 651.6 --> Chronic calcineurin inhibitor (CNI) nephrotoxicity is a major drawback in current immunosuppressive regimens. In the chronic setting, arteriolar hyalinosis, decreased glomerular filtration rate, interstitial fibrosis and tubular dedifferentiation are the major adverse side effects. Since CsA is still widely used in transplant recipients, particularly in posttransplant diabetes, mechanistic insights into nephrotoxic mechanisms are required. To identify regulatory mechanisms in CsA nephrotoxicity we used quantitative transcriptomic, proteomic and phosphoproteomic methods. We tested the hypothesis that tubulointerstitial pathomechanisms play a predominant role in chronic CNI nephropathy. Whole transciptome RNA‐seq as well as global proteomic and phosphoproteomic methodologies were performed on kidney extracts from normal Wistar rats receiving CsA (25mg/kg b.w./day) or vehicle for 3 weeks. Differentially expressed genes and proteins as well as their phosphorylation status were obtained. Products of interest were tested using Western blot, qPCR, and immunohistochemistry. CsA treatment stimulated genome‐wide alterations in rat kidney according to RNA‐seq data. We identified 342 transcripts upregulated (padj<0.05) which included Ribosome (padj<0,0001) and Oxidative phosphorylation (padj<0,0001) pathways, whereas 331 transcripts were downregulated, with enrichment in genes critical for amino acid metabolism. Data were controlled by the established upregulation of renin and downregulation of calbindin (pdaj<0,05) in global proteomics. KEGG pathway and GO analysis from proteomics largely corresponded to the RNA‐seq results. Upregulated proteins were further related to ECM‐receptor interaction and focal adhesion pathways (padj<0,05). Phosphoproteomics demonstrated functional phosphorylation of components from unfolded protein response pathways, indicating an activation of the integrated stress response upon CsA. Products were verified at the tissue‐ and cell level. In sum, using integrated ‐omics analysis in CsA nephrotoxicity proved to be a powerful approach. Chronic CsA treatment was associated with enhanced energy metabolism and activation of the unfolded protein‐response pathways. A tubulointerstitial focus has been demonstrated. Potential biomarker candidates have been obtained. [ABSTRACT FROM AUTHOR]

R3571 --> 651.5 --> Calcineurin inhibitors (CNI), cyclosporine A (CsA) or tacrolimus (Tac), belong to the first‐line immunosuppressive strategies in organ‐transplanted patients. Both CNI may exert renal side effects, which are typically stronger in patients receiving CsA. Continued clinical demand for CsA requires improved understanding of its nephrotoxicity. Calcineurin inhibition by CsA occurs via complexes with cyclophilins, whereas Tac recruits FKBP12 instead. We hypothesized that impaired chaperone function of cyclophilins may aggravate CsA cytotoxicity due to induction of endoplasmic reticulum (ER) stress and pro‐apoptotic unfolded protein response (UPR). To address this hypothesis, effects of CsA vs. Tac (10 µM for 6 h) on the UPR signaling were evaluated in cultured human embryonic kidney cells (HEK293), human renal proximal tubular (PT) epithelial cells (HRPTEpC), and isolated rat PTs using quantitative PCR, immunoblotting, and immunofluorescence staining. An established ER stress inducer, thapsigargin (Tg) served as a positive control. CsA and Tg, but not Tac, induced ER stress and pro‐apoptotic UPR in HEK293 cells, which was reflected by increased levels of key UPR products (BiP, CHOP, spliced XBP1, and phosphorylated IRE1a) and cleaved caspase 3 (cCas3). Similar effects were observed in HRPTEpC cells and isolated rat PTs. Knockdown of cyclophilin A or B isoforms in HEK293 cells using siRNA augmented CHOP and cCas3 to an extent comparable with CsA treatment suggesting relevance of cyclophilin activity for intact proteostasis. Application of chemical chaperones, TUDCA or 4‐PBA, alleviated the CsA‐induced UPR suggesting that boosting the protein folding may have protective effects against CsA cytotoxicity. Along the same line, genetic suppression of UPR in HEK293 cells by CRISPR/Cas9‐mediated deletion of the key ER stress sensors, PERK or ATF6, blunted the pro‐apoptotic UPR in response to CsA. In summary, these results suggest that nephrotoxic effects of CsA are partially mediated by suppression of cyclophilins, ER stress, and pro‐apoptotic UPR. Pharmacological modulation of UPR bears the potential to alleviate CsA nephrotoxicity. [ABSTRACT FROM AUTHOR]

R3723 --> 614.1 --> The inhibition of calcineurin (PP2B) with tacrolimus (FK506) or cyclosporin A (CsA), two frequently used immunosuppressive drugs after organ transplantation, has been shown to stimulate cation‐coupled Cl‐ cotransporters such as NCC and NKCC2. Also, the stimulation of NCC induced by CsA or FK506 may be responsible for PP2B‐inhibition‐induced hypertension and hyperkalemia. The aim of the present study is to test whether acute application of FK506 or CsA also stimulates the basolateral K+ channels in the distal convoluted tubule (DCT) thereby activating NCC expression/activity. We first used patch‐clamp technique to examine the effect of FK506 or CsA on the basolateral Kir4.1/Kir5.1 activity in the DCT. Acutely addition of FK506 (5‐10 uM) stimulates the 40 pS K+ (Kir4.1/Kir5.1 heterotetramer) channel activity in the DCT and increases the NPo from 1.6 to 2.6. Moreover, acutely addition of FK506 (10 uM) increases the whole‐cell K+ currents from 1180 pA to 2050 pA in the DCT cells. As consequence of the stimulation of Kir4.1/Kir5.1, K‐currents (IK) reversal potential (an index of the membrane potential) becomes more negative in FK506 treated DCT than untreated (‐73 mV vs ‐62 mV). To examine whether FK506‐induced stimulation of Kir4.1/Kir5.1 in the DCT requires the FKBP12 (12 kDa FK506 binding protein), we then examined the effect of FK506 on the basolateral K+ channels in the DCT1 of kidney‐tubule‐specific (Ks)‐FKBP12 KO mice. The control and Ks‐FKBP12 KO mice were treated with FK506 (0.75 mg/Kg BW) by peritoneal injection 30 min before experiments. The stimulatory effect of FK506 on Kir4.1/Kir5.1 of the DCT is observed only in the control but is absent in Ks‐FKBP12 KO mice, suggesting the effect of FK506 is due to the inhibition of PP2B. Moreover, we have used the whole‐cell recording technique to examine the effect of CsA on the Ba2+‐sensitive K+ currents in the control and FKBP12‐deficient mice because CsA‐induced inhibition of PP2B requires binding to cyclophilin but not to FKBP12. We observed that CsA treatment (3 mg/Kg BW by peritoneal injection 30 min before experiments) significantly increases the whole‐cell K+ currents in both control and Ks‐FKBP12 KO mice. Fluorescence image shows that CsA treatment for 30 min increased the expression of Kir4.1 and pNCC (Serine 71) in the basolateral membrane and apical membrane, respectively. To determine whether the activation of the basolateral K+ channel activity in the DCT is essential for FK506‐induced stimulation of NCC expression, we next examined the effect of FK506 treatment on the expression of pNCC and tNCC in the control (Kcnj10flox/flox) and in kidney‐tubule‐specific (Ks‐Kir4.1) KO mice. Acute FK506 treatment (peritoneal injection of FK506 at 0.75 mg/Kg BW) robustly increases the expression of pNCC and tNCC. However, the stimulatory effect of FK506 on NCC is blunted in Ks‐Kir4.1 KO mice, suggesting the role of Kir4.1 in mediating PP2B‐inhibition induced stimulation of NCC. We conclude that acute inhibition of PP2B stimulates the basolateral Kir4.1/Kir5.1 activity of the DCT and NCC and that the acute effect of PP2B inhibition on NCC is partially achieves by stimulation of the basolateral Kir4.1/Kir5.1. [ABSTRACT FROM AUTHOR]