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Diabetic Kidney Disease (DKD) is a major microvascular complication for diabetic patients and is the most common cause of chronic kidney disease (CKD) and end-stage renal disease. Antidiabetic drugs, such as metformin and canagliflozin, have been shown to exert renoprotective effects. Additionally, quercetin recently showed promising results for the treatment of DKD. However, the molecular pathways through which these drugs exert their renoprotective effects remain partly unknown. The current study compares the renoprotective potential of metformin, canagliflozin, metformin + canagliflozin, and quercetin in a preclinical rat model of DKD. By combining streptozotocin (STZ) and nicotinamide (NAD) with daily oral N(ω)-Nitro-L-Arginine Methyl Ester (L-NAME) administration, DKD was induced in male Wistar Rats. After two weeks, rats were assigned to five treatment groups, receiving vehicle, metformin, canagliflozin, metformin + canagliflozin, or quercetin for a period of 12 weeks by daily oral gavage. Non-diabetic vehicle-treated control rats were also included in this study. All rats in which diabetes was induced developed hyperglycemia, hyperfiltration, proteinuria, hypertension, renal tubular injury and interstitial fibrosis, confirming DKD. Metformin and canagliflozin, alone or together, exerted similar renoprotective actions and similar reductions in tubular injury and collagen accumulation. Renoprotective actions of canagliflozin correlated with reduced hyperglycemia, while metformin was able to exert these effects even in the absence of proper glycemic control. Gene expression revealed that the renoprotective pathways may be traced back to the NF-κB pathway. No protective effect was seen with quercetin. In this experimental model of DKD, metformin and canagliflozin were able to protect the kidney against DKD progression, albeit in a non-synergistic way. These renoprotective effects may be attributable to the inhibition of the NF-κB pathway.

Chronic interstitial nephritis in agricultural communities is a devastating kidney disease with a globally increasing prevalence. Its cause is unknown. Two predominant etiologies are hypothesised: recurrent episodes of dehydration and exposure to environmental toxins, such as agrochemicals and metals. In this review, we summarise arguments on: 1) why heat stress/dehydration is an unlikely cause of this disease and 2) why chronic interstitial nephritis in agricultural communities is to be considered a toxin-induced nephropathy. Mechanistically, we provide arguments for a putative role of pesticides on the one hand, and the calcineurin pathway on the other hand, both of which require further investigation. Finally, we summarise several important perspectives for research on chronic interstitial nephritis in agricultural communities. (C) 2020 Published by Elsevier Masson SAS on behalf of Societe francophone de nephrologie, dialyse et transplantation.

Chronic interstitial nephritis in agricultural communities (CINAC), also named CKD of unknown origin (CKDu) or Mesoamerican nephropathy (MeN), is defined as a form of CKD affecting young men and—less often—women. Its etiology is unrelated to diabetes, GN, hypertension, or other known causes of CKD. Patients with CINAC live and work mainly in poor agricultural communities, often in hot tropical regions, and are exposed to potentially toxic agrochemicals through work by ingestion of contaminated food and water, and/or by inhalation (1,2). The epidemic dimension of CINAC was first observed in the 1990s in Sri Lanka and Central America and has since been found to be an important cause of CKD-related deaths in an increasing number of countries (3,4). Patients with CINAC have bilateral, small, irregularly contoured kidneys with mutual size discrepancy of

Almost 30 years after the detection of chronic interstitial nephritis in agricultural communities (CINAC) its etiology remains unknown. To help define this we examined 34 renal biopsies from Sri Lanka, El Salvador, India and France of patients with chronic kidney disease 2-3 and diagnosed with CINAC by light and electron microscopy. In addition to known histopathology, we identified a unique constellation of proximal tubular cell findings including large dysmorphic lysosomes with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound "aggregates". These aggregates associated with varying degrees of cellular/tubular atrophy, apparent cell fragment shedding and no-weak proximal tubular cell proliferative capacity. Identical lysosomal lesions, identifiable by electron microscopy, were observed in 9% of renal transplant implantation biopsies, but were more prevalent in six month (50%) and 12 month (67%) protocol biopsies and in indication biopsies (76%) of calcineurin inhibitor treated transplant patients. The phenotype was also found associated with nephrotoxic drugs (lomustine, clomiphene, lithium, cocaine) and in some patients with light chain tubulopathy, all conditions that can be directly or indirectly linked to calcineurin pathway inhibition or modulation. One hundred biopsies of normal kidneys, drug/toxin induced nephropathies, and overt proteinuric patients of different etiologies to some extent could demonstrate the light microscopic proximal tubular cell changes, but rarely the electron microscopic lysosomal features. Rats treated with the calcineurin inhibitor cyclosporine for four weeks developed similar proximal tubular cell lysosomal alterations, which were absent in a dehydration group. Overall, the finding of an identical proximal tubular cell (lysosomal) lesion in CINAC and calcineurin inhibitor nephrotoxicity in different geographic regions suggests a common paradigm where CINAC patients undergo a tubulotoxic mechanism similar to calcineurin inhibitor nephrotoxicity.

Current treatment strategies for chronic kidney disease (CKD) mainly focus on controlling risk factors. Metformin, a first-line drug for type-2 diabetes, exerts beneficial pleiotropic actions beyond its prescribed use and incipient data has revealed protective effects against the development of kidney impairment. This study evaluated the therapeutic efficacy of metformin and canagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor recently approved by the United States Food and Drug Administration to treat diabetic nephropathy, in slowing the progression of established non-diabetic CKD. Rats with adenine-induced CKD were assigned to different treatment groups to receive either 200 mg/kg metformin, four or five weeks after the start of the adenine diet (established mild-moderate CKD), or 25 mg/kg canagliflozin four weeks after the start of the diet, by daily oral gavage administered during four weeks. Each treatment group was compared to a vehicle group. Chronic adenine dosing resulted in severe CKD in vehicle-treated rats as indicated by a marked rise in serum creatinine levels, a marked decrease in creatinine clearance, and a disturbed mineral metabolism. Metformin, but not canagliflozin, halted functional kidney decline. Additionally, kidneys of metformin-treated animals showed less interstitial area and inflammation as compared to the vehicle group. Proteomic analyses revealed that metformin’s kidney-protective effect was associated with the activation of the Hippo signaling pathway, a highly conserved multiprotein kinase cascade that controls tissue development, organ size, cell proliferation, and apoptosis. Thus, metformin demonstrated therapeutic efficacy by halting the progression of established CKD in a rat model.

Background and Aims Acute kidney injury (AKI) is a global health concern with an incidence of 13.3 million patients per year, and increasing. AKI is recognized as an important risk factor for the development of chronic kidney disease (CKD). A crucial aspect for successful renal recovery after AKI is an efficient proliferative response of surviving tubular epithelial cells (TECs). Recently, we established a murine model in which the functional and histological recovery of a single kidney, injured by ischemia, is enhanced by removal of the unharmed contralateral kidney; a phenomenon termed nephrectomy-induced recovery. The renal epithelial reparative response in this unique physiological model has not been investigated, yet can provide new insights in unlocking the inherent regenerative potential of the renal epithelium. Method AKI was induced in R26RtdTomato and PAX2/Confetti mice by left unilateral ischemia/reperfusion (UIRI) for 21 min at 34°C, after which either right nephrectomy (Nx) or no Nx was performed 3 days later. Mice were euthanized 6 weeks and 28 days after UIRI, respectively. At week 6, kidneys were weighted and renal function was assessed by serum creatinine. At 28 days, renal tissue of Pax2/Confetti mice was collected to perform renal progenitor cell lineage tracing experiments by immunofluorescence and confocal microscopy. Results When nephrectomy was performed after UIRI, left kidney-to-body weight ratio did not change significantly over time, whereas, when no nephrectomy was performed, left kidney-to-body weight ratio gradually declined from 7,84 ± 0,48 mg/dl at day 3 till 3,26 ± 0,51 mg/dl at week 6, indicating severe atrophy in the injured left kidney. This loss of renal mass was associated with a significant increase in serum creatinine (1,76 ± 0,13 mg/dl) as compared to control (0,21 ± 0,12 mg/dl), whereas with nephrectomy, renal function fully restored. Clonal analysis in PAX2/Confetti mice revealed that nephrectomy after UIRI led to a significant increase in proliferating (i.e. clonogenic) Pax2+ progenitor cells, resulting in more multicellular clones as compared to un-nephrectomized controls. Conclusion Nephrectomy after UIRI overcomes chronic loss of renal mass and function within the investigated 6-week time frame. This study is the first to demonstrate that nephrectomy stimulates clonal expansion of renal progenitor cells in an injured kidney, beyond that observed for spontaneous repair after UIRI. Insight in the signaling mechanisms may reveal new therapeutic approaches to incite the inherent renal regeneration potential.

One of the most important risk factors for developing chronic kidney disease (CKD) is diabetes. To assess the safety and efficacy of potential drug candidates, reliable animal models that mimic human diseases are crucial. However, a suitable model of diabetic kidney disease (DKD) is currently not available. The aim of this study is to develop a rat model of DKD by combining streptozotocin and nicotinamide (STZ/NAD) with oral N(ω)-Nitro-L-Arginine Methyl Ester (L-NAME) administration. Diabetes was induced in male Wistar rats by intravenous injection of 65 mg/kg STZ, 15 min after intraperitoneal injection of 230 mg/kg NAD. Rats were assigned to different groups receiving L-NAME (100 mg/kg/day) (STZ/NAD/L-NAME) or vehicle (STZ/NAD) for a period of 9 or 12 weeks by daily oral gavage. All rats developed hyperglycemia. Hyperfiltration was observed at the start of the study, whereas increased serum creatinine, albumin-to-creatinine ratio, and evolving hypofiltration were detected at the end of the study. Daily L-NAME administration caused a rapid rise in blood pressure. Histopathological evaluation revealed heterogeneous renal injury patterns, which were most severe in the STZ/NAD/L-NAME rats. L-NAME-induced NO-deficiency in STZ/NAD-induced diabetic rats leads to multiple characteristic features of human DKD and may represent a novel rat model of DKD.

Clinical frailty in the elderly is defined by a composite measure of functional psychomotor decline. Herein, we develop the concept of haemodynamic frailty (HDF), a state of increased predisposition to disease prevalent in the elderly and characterised by impairment of the network of compensatory responses governing the defence of circulatory volume and adaptive haemodynamic function. We review the factors predisposing the elderly to HDF, with a focus on the impaired capacity to sustain total body water balance. As a component of HDF, dehydration generates vulnerability to diseases caused by tissue hypoperfusion, including acute kidney injury. We provide a detailed mechanistic explanation of how dehydration and depletion of the intravascular volume impacts on renal blood flow to become an important element of the heightened risk of acute kidney injury (AKI) in the elderly. We bring these mechanistic considerations into the clinical context with reference to examples of how pre-renal (haemodynamic) and intrinsic (involving renal parenchymal damage) AKI risk is elevated in the setting of dehydration. Finally, we present HDF as a state of opportunity to prevent disease, for which diagnostic and interventional standards need to be refined. Further prospective studies are warranted to help clarify the clinical utility of assessing and managing HDF with regard to the mitigation of AKI risk in the elderly.

Background and Aims Metformin, a biguanide drug, is the first-line oral antidiabetic agent used by 200 million patients suffering from type 2 diabetes mellitus. In the last decades, it has become clear that metformin exerts benign pleiotropic actions beyond its prescribed use and emerging data show protective effects against different age-related diseases. Additionally, recent preclinical and clinical data point towards a beneficial impact of metformin on the kidney. Current treatment strategies for chronic kidney disease (CKD) mainly focus on controlling important risk factors, such as high blood pressure and diabetes. To date, effective treatment directly targeting the kidney is lacking. Here, the ability of metformin to attenuate progression of already established CKD, independent from its effects on diabetes, was investigated. Method A rat model of adenine-induced CKD (n=64) was used. Metformin (200 mg/kg) or vehicle (1% carboxymethylcellulose) treatment, by daily oral gavage (7 days/week), was initiated after 4 and 5 weeks of adenine (0.25%) administration; i.e. after CKD had developed. Treatment was continued during 4 weeks until the end of the study (i.e. week 8 and 9, respectively). A constant dose volume of 10 mL/kg was used. The effect of metformin on renal function and histopathology was studied. Results Serum creatinine levels dramatically rose in vehicle-treated CKD rats: from 0.6 ± 0.1 mg/dL (week 0) to 1.3 ± 0.2 mg/dL (week 4) and 2.6 ± 1.2 mg/dL (week 5) and further to 5.7 ± 0.6 mg/dL (week 8) and 4.8 ± 1.1 mg/dL (week 9). Metformin treatment, which started after 4 and 5 weeks, prevented almost completely further increases in serum creatinine levels after 8 (2.0 ± 0.5 mg/dL) and 9 (2.9 ± 0.5 mg/dL) weeks compared to vehicle-treated CKD rats (p Conclusion Therapeutic metformin treatment is able to attenuate the progression of pre-existing adenine-induced CKD in rats.

Background and Aims In CINAC patients from Sri Lanka, we recently observed a sensitive constellation of proximal tubular cell findings including cellular/tubular atrophy, cell fragment shedding and, by electron microscopy (EM), the presence of an increased number of enlarged lysosomes. Here we focused on precisely defining the EM lysosomal phenotype and evaluating its presence in CINAC/MeN/CKDu cases and controls from other countries. Method Thirthy-two renal biopsies (18 Sri Lanka, 10 El Salvador, 1 India, 3 France) of patients with a diagnosis of CINAC (CKD 1-3A, 3B) were examined by electron microscopy (EM) in comparison to renal biopsies of normal kidneys at implantation, patients with calcineurin inhibitor (CNI) toxicity (n=17), proteinuric nephropathies (n=15), light chain disease (n=4), several cases on nephrotoxic drugs (lomustine, clomiphene, lithium, tenofovir, cisplatinum) and patients with reduced renal function of various causes (n=20). Results The aberrant lysosomal phenotypes can be defined as enlarged (>1.2µm) and dysmorphic with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound round to irregular shaped “aggregates”. In addition, indicative clusters of 4-6 smaller lysosomes with the same features could be observed. No cristae or other features of mitochondria, autophagic vacuoles, lipofuscin/ceroid droplets, peroxisomes (marginal plate), lysosomal myeloid bodies (aminoglycoside nephropathy) or electron dense laminated lysosomal inclusions (Fabry disease) were observed. Patients with calcineurin inhibitor nephrotoxicity and several cases on nephrotoxic drugs (lomustine, clomiphene, lithium) and a subset of patients with light chain disease, all conditions that can either directly or indirectly be linked to calcineurin inhibition, presented the same lesions. We present an image set demonstrating the phenotypical consistency of the diagnostic lysosomal lesion versus similar features that are non-diagnostic. Conclusion A rather sensitive constellation of lysosomal lesions in renal proximal tubular cells was detected associated with CINAC/CKDu/MeN and CNI nephrotoxicity in several countries, suggesting a common new pathomechanistic paradigm where CINAC patients are experiencing a tubulotoxic mechanism similar to CNI nephrotoxicity.

Background and Aims Calcineurin inhibitor therapy has changed the field of (renal) transplantation by considerably prolonging graft survival. Yet, all immunosuppressive calcineurin inhibitors are nephrotoxic that eventually contribute to complete scarring of the renal allograft. In renal biopsy analysis many histopathological features have been considered indicative of CNI nephrotoxicity, i.e. striped fibrosis, vascular hyalinosis, isometric tubular vacuolization, glomerulosclerosis, cellular infiltration and tubular atrophy, however, all are rather aspecific and can be secondary to many other causes. During the course of evaluating the specificity of a recently discovered proximal epithelial lysosomal lesion (i.e. multiple enlarged (>1,2µm) dysmorphic lysosomes containing dispersed electron dense non-membrane bound aggregates) in patients with Chronic Interstitial Nephropathy in Agricultural Communities (CINAC), we observed it to be present in renal transplant patients treated with cyclosporine or tacrolimus. Here, we test the hypothesis whether this lysosomal lesion is acquired during CNI therapy. Method A retrospective transmission electron microscopic analysis was performed to evaluate the presence of the typical lysosomal lesion on the following biopsies from renal transplant patients: 20 deceased donor implantation biopsies; 5 living donor implantation biopsies. For another 10 additional deceased donor renal allograft recipients, we evaluated implantation as well as protocol biopsies taken after 6 and 12 months of CNI treatment that started immediately after transplantation. Also included were 24 indication biopsies of CNI treated renal transplants. Results Of the total set of implantation biopsies (n=35), 2 (6%) were positive for the aberrant lysosomal phenotype on EM, whereas in the protocol and indication biopsies prevalence of the lesion was considerably higher ranging between 56% (protocol) and 80% (indication) of cases. Conclusion CNI therapy is associated with the fairly rapid appearance of a particular proximal tubular lysosomal phenotype observable on EM, that was not (or rarely) present at implantation. Whether this lesion is related to CNI toxicity and indicative for the outcome for the graft and/or patient survival after renal transplantation has to be investigated in a prospective trial.

Aims: Cytoglobin (CYGB) is a member of the mammalian globin family of respiratory proteins. Despite extensive research efforts, its physiological role remains largely unknown, but potential functions include reactive oxygen species (ROS) detoxification and signaling. Accumulating evidence suggests that ROS play a crucial role in podocyte detachment and apoptosis during diabetic kidney disease. This study aimed to explore the potential antioxidative renal role of CYGB both in vivo and in vitro. Results: Using a Cygb-deficient mouse model, we demonstrate a Cygb-dependent reduction in renal function, coinciding with a reduced number of podocytes. To specifically assess the putative antioxidative function of CYGB in podocytes, we first confirmed high endogenous CYGB expression levels in two human podocyte cell lines and subsequently generated short hairpin RNA-mediated stable CYGB knockdown podocyte models. CYGB-deficient podocytes displayed increased cell death and accumulation of ROS as assessed by 2 ' 7 '-dichlorodihydrofluorescein diacetate assays and the redox-sensitive probe roGFP2-Orp1. CYGB-deficient cells also exhibited an impaired cellular bioenergetic status. Consistently, analysis of the CYGB-dependent transcriptome identified dysregulation of multiple genes involved in redox balance, apoptosis, as well as in chronic kidney disease (CKD). Finally, genome-wide association studies and expression studies in nephropathy biopsies indicate an association of CYGB with CKD. Innovation: This study demonstrates a podocyte-related renal role of Cygb, confirms abundant CYGB expression in human podocyte cell lines, and describes for the first time an association between CYGB and CKD. Conclusion: Our results provide evidence for an antioxidative role of CYGB in podocytes.

Background and Aims CINAC patients present a newly discovered constellation of proximal tubular lysosomal lesions which is also observed in patients experiencing calcineurin inhibitor (CNI) nephrotoxicity, suggesting that CINAC is a toxin-induced nephropathy involving calcineurin inhibition. An alternate hypothesis advocates chronic heat stress/dehydration as the major etiological factor for CINAC. Here, we evaluated in rats to what extent heat stress/dehydration versus CNI exposure reflects proximal tubular CINAC histopathology. Method Wistar rats were divided in 3 groups. Group 1 (n=6) was given water ad libitum (control group). Group 2 (n=8) was water deprived for 10 hours per 24h, 5 days/week and were placed in an incubator (37°C) for 30 min/hr of water deprivation. Group 3 (n=8) underwent daily oral gavage with cyclosporine (50mg/kg body weight). Animals were weighed daily and urine was collected at day 3, 17 and 28. After 28 days, rats were sacrificed. Kidneys were collected for light (LM) and electron microscopic (EM) histopathological analysis as well as for cortical renal tissue proteomics. Results Cyclosporine rats developed focal cortical lesions mimicking those of CINAC patients: i.e. atrophic proximal tubuli with thickened basement membranes and associated tubulo-interstitial fibrosis, PASM staining demonstrating enlarged argyrophyllic granules in the affected proximal tubuli, LAMP1 immunofluorescent staining identifying a subset of these granules as lysosomes, and EM confirming the presence of enlarged lysosomes, some dysmorphic approaching CINAC lysosomes. In dehydrated rats, confirmed by urinary osmolality and fluctuating body weight associated with water deprivation, none of the cyclosporine features were observed. Proteomic analysis confirmed cellular toxicity by cyclosporine, whereas the dehydration group lacked any markers of such. Conclusion The histopathological analogy between CNI nephrotoxicity in rats and humans and CINAC suggests a toxicological etiology for CINAC. In rats, dehydration/heat stress alone does not lead to the constellation of proximal tubular lesions as observed in CINAC patients.

Background and Aims 30 years after the detection of CINAC, there is no consensus on its etiology. Heat stress/dehydration and toxic exposure are the two most likely etiologies. There are no diagnostic criteria that can directly identify CINAC patients. Method Renal biopsies (RB) (18 Sri Lanka, 10 El Salvador, 1 India, 3 France) of patients with CINAC (CKD2-3) were examined by light (LM) and electron microscopy (EM) in comparison to RB of normal kidneys at implantation and 6 and 12 months of calcineurin inhibitor (CNI) therapy, transplant patients on CNI with indication biopsies (n=24), proteinuric nephropathies (n=15), light chain disease (n=4), cases on nephrotoxic drugs (lomustine, clomiphene, lithium, tenofovir, cisplatinum) and CKD of various causes (n=20). A rat study was conducted comparing histopathology of heat stress/dehydration with cyclosporine nephrotoxicity. Results In addition to previously described histopathological changes, there was a unique constellation of proximal tubular cell (PTC) findings: cellular/tubular atrophy, cell fragment shedding, weak to non-proliferative capacity of the PTC and dysmorphic lysosomes increased in size and number with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound “aggregates”. Identical renal lesions were observed in 55-80% of renal transplant protocol biopsies taken after 6 and 12 months of calcineurin inhibitor (CNI) therapy and in indication biopsies, in implantation biopsies the prevalence was 6%. Several cases of nephrotoxic drugs (lomustine, clomiphene, lithium) and a subset of patients with light chain disease, all conditions that can be linked to CNI, presented the same lesion. Control RBs (n=66) of normal kidney, toxic nephropathies (tenofovir, cisplatinum), and overt proteinuric patients of different etiology to some extent could demonstrate the tubular cell changes observed by LM, but not or very rarely those that were observed by EM. Rats treated with cyclosporine for 4 weeks developed similar PTC lysosomal alterations, absent in a dehydration group. Conclusion A sensitive constellation of renal PTC lesions was detected associated with CINAC and CNI nephrotoxicity in several countries, suggesting a common new paradigm where CINAC patients are experiencing a tubulotoxic mechanism similar to CNI nephrotoxicity, the latter also being known as a direct or suggested indirect effect of pesticides.

Sclerosteosis is a rare autosomal recessive bone disorder marked by hyperostosis of the skull and tubular bones. Initially, we and others reported that sclerosteosis was caused by loss-of-function mutations in SOST, encoding sclerostin. More recently, we identified disease-causing mutations in LRP4, a binding partner of sclerostin, in three sclerosteosis patients. Upon binding to sclerostin, LRP4 can inhibit the canonical WNT signaling that is known to be an important pathway in the regulation of bone formation. To further investigate the role of LRP4 in the bone formation process, we generated an Lrp4 mutated sclerosteosis mouse model by introducing the p.Arg1170Gln mutation in the mouse genome. Extensive analysis of the bone phenotype of the Lrp4R1170Q/R1170Q knock-in (KI) mouse showed the presence of increased trabecular and cortical bone mass as a consequence of increased bone formation by the osteoblasts. In addition, three-point bending analysis also showed that the increased bone mass results in increased bone strength. In contrast to the human sclerosteosis phenotype, we could not observe syndactyly in the forelimbs or hindlimbs of the Lrp4 KI animals. Finally, we could not detect any significant changes in the bone formation and resorption markers in the serum of the mutant mice. However, the serum sclerostin levels were strongly increased and the level of sclerostin in the tibia was decreased in Lrp4R1170Q/R1170Q mice, confirming the role of LRP4 as an anchor for sclerostin in bone. In conclusion, the Lrp4R1170Q/R1170Q mouse is a good model for the human sclerosteosis phenotype caused by mutations in LRP4 and can be used in the future for further investigation of the mechanism whereby LRP4 regulates bone formation. © 2017 American Society for Bone and Mineral Research.

Human beings are exposed to various potentially toxic agents and conditions in their natural and occupational environments. The kidney, due to its concentrating ability and excretory function, is highly vulnerable to the effects of environmental toxins. Identifying the precise cause and mechanisms of environmentally induced renal injury remains a challenge for which various scientific disciplines need to be involved. Investigations in this field are confronted with the apparent infinite types of toxins, their mutual interaction, handling/metabolization by the body, ways of exposure, etc. Although interdisciplinary efforts and persistence are required to identify, mechanistically unravel and tackle environmental toxininduced pathologies, research eventually pays off in ameliorated working/living conditions and development of preventive/therapeutic strategies. This review was compiled to particularly emphasize the need for a maintained awareness of environmental threats in general and those targeting the kidney. Different mechanisms of renal toxicity are illustrated and discussed, thereby focusing on three types of environmental toxins, namely aristolochic acid, melamine and heavy metals.

Introduction Sucroferric oxyhydroxide (PA21) is an efficacious, well-tolerated iron-based phosphate binder and a promising alternative to existing compounds. We compared the effects of PA21 with those of a conventional phosphate binder on renal function, mineral homeostasis and vascular calcification in a chronic kidney disease–mineral and bone disorder (CKD-MBD) rat model. Methods To induce stable renal failure, rats were administered a 0.25% adenine diet for 8 weeks. Concomitantly, rats were treated with vehicle, 2.5 g/kg/day PA21, 5.0 g/kg/day PA21 or 3.0 g/kg/day calcium carbonate (CaCO3). Renal function and calcium/phosphorus/iron metabolism were evaluated during the study course. Renal fibrosis, inflammation, vascular calcifications and bone histomorphometry were quantified. Results Rats treated with 2.5 or 5.0 g/kg/day PA21 showed significantly lower serum creatinine and phosphorus and higher ionized calcium levels after 8 weeks of treatment compared with vehicle-treated rats. The better preserved renal function with PA21 went along with less severe anaemia, which was not observed with CaCO3. Both PA21 doses, in contrast to CaCO3, prevented a dramatic increase in fibroblast growth factor (FGF)-23 and significantly reduced the vascular calcium content while both compounds ameliorated CKD-related hyperparathyroid bone. Conclusions PA21 treatment prevented an increase in serum FGF-23 and had, aside from its phosphate-lowering capacity, a beneficial impact on renal function decline (as assessed by the renal creatinine clearance) and related disorders. The protective effect of this iron-based phosphate binder on the kidney in rats, together with its low pill burden in humans, led us to investigate its use in patients with impaired renal function not yet on dialysis.

Background: Current treatment options for chronic kidney disease (CKD) are limited and their focus is on slowing its progression by addressing comorbidities. Fibrosis, the common histopathological process in CKD, is a major therapeutic research target. In CKD, fibroblasts are terminally activated due to alterations in their DNA-methylation pattern, particularly hypermethylation. Preventing the copying of pathological DNA-methylation patterns in proliferating fibroblasts could be a new effective therapeutic strategy for treating CKD. Methods: To evaluate the therapeutic effect of short-term treatment with the DNA-methyltransferase (DNMT)-inhibitor decitabine on fibrosis (either developing or already established), male C57Bl/6 mice underwent warm unilateral ischemia-reperfusion injury. Respectively 3 days, 3 and 6 weeks after surgery, decitabine treatment (0.25 mg/kg) was initiated for 10 days after which animals were followed up to 12 weeks after ischemia. The efficacy of therapy on fibrosis was evaluated by collagen I and tgfβ gene expression and histological quantification of collagen I staining. In addition, the effect of decitabine treatment on tubular injury (Kim-1, Ngal), inflammation (TNFa, IL6), DNA-methyltransferases (Dnmt1, 3a, and 3b), and global methylation status was determined. Results: Following ischemia there was a significant increase in fibrotic, injury, and inflammatory markers as well as an increase of the various dnmts. Although decitabine treatment transiently increased renal injury and had a moderately decreasing effect on dnmt expression and on global DNA-methylation upon immediate treatment, none of the treatment regimens succeeded in preventing, attenuating, or diminishing fibrosis in the long run. Conclusion: Administration of untargeted nucleoside analogues seems unsuitable as a first-line treatment option in developing or established CKD.

The use of geo-engineering materials to manage phosphorus in lakes has increased in recent years with aluminium and lanthanum based materials being most commonly applied. Hence the potential impact of the use of these compounds on human health is receiving growing interest. This review seeks to understand, evaluate and compare potential unintended consequences on human health and ecotoxicological risks associated with the use of lanthanum- and aluminium-based materials to modify chemical and ecological conditions in water bodies. In addition to their therapeutic use for the reduction of intestinal phosphate absorption in patients with impaired renal function, the phosphate binding capacity of aluminium and lanthanum also led to the development of materials used for water treatment. Although lanthanum and aluminium share physicochemical similarities and have many common applications, their uptake and kinetics within the human body and living organisms importantly differ from each other which is reflected in a different toxicity profile. Whilst a causal role in the development of neurological pathologies, skeletal lesions, hematopoietic disorders and respiratory effects has unequivocally been demonstrated with increased exposure to aluminium, studies until now have failed to find such a clear association after exposure to lanthanum although caution is warranted. Our review indicates that lanthanum and aluminium have a distinctly different profile with respect to their potential effects on human health. Regular monitoring of both aluminium and lanthanum concentrations in lanthanum-/aluminium-treated water by the responsible authorities is recommended to avoid acute accidental or chronic low level accumulation.

Background Acute kidney injury (AKI) is an underestimated, yet important, risk factor for the development of chronic kidney disease (CKD). Persistence of inflammation after a renal ischemic injury has been observed, both in experimental models and patients, and is thought to be an important mechanisms underlying progression of acute-to-chronic renal injury. Temporary suppression of inflammation immediately after AKI might therefore be a good first-line therapeutic strategy towards a better long term outcome. Methods Male C57Bl/6 J mice (Charles River, 10–12 weeks of age) underwent warm (36 °C body temperature) unilateral ischemia-reperfusion of the kidney for 21 min, after which treatment with intraperitoneal injection of the corticosteroid dexamethasone (10 mg/kg) was initiated for 3 weeks. Both at that time point and after an additional 3 week post-treatment follow up period, fibrosis was quantified by collagen I gene expression and immunostaining, as well as gene expression analysis of fibrosis-related genes Tgfβ, Ccn2 (Ctgf), Pai-1 and Ccn3. Furthermore, inflammation was evaluated by Tnfα gene expression and protein expression of the F4/80 macrophage marker and the α-SMA fibroblast marker. Lastly, renal histopathology was quantified by a morphometric analysis of the tubulointerstitial area. Results Treatment with dexamethasone attenuated development of fibrosis, as evidenced by reduced collagen I gene expression and immunostaining, in combination with reduced gene expression of the pro-fibrotic Ccn2 and increased expression of the anti-fibrotic Ccn3. The effects of dexamethasone on renal fibrosis persisted during the 3 week follow up period, as evidenced by stagnation of collagen I deposition in the ischemic kidney, in contrast to vehicle-treatment, where progression of fibrosis was observed. However, expression levels of the pro-fibrotic genes re-approached those of vehicle-treated injured kidneys suggesting that the effects of dexamethasone on fibrosis beyond the treatment period are temporary. Conclusion A short term anti-inflammatory therapy with dexamethasone only transiently attenuates ischemia induced fibrosis. Prolonged or persistent anti-inflammatory treatment seems warranted to achieve long term benefit. Electronic supplementary material The online version of this article (10.1186/s12882-018-1151-7) contains supplementary material, which is available to authorized users.

Acute kidney injury (AKI), commonly caused by ischemia-reperfusion injury, has far-reaching health consequences. Despite the significant regenerative capacity of proximal tubular epithelium cells (PTCs), repair frequently fails, leading to the development of chronic kidney disease (CKD). In the last decade, it has been repeatedly demonstrated that dysregulation of the cell cycle can cause injured kidneys to progress to CKD. More precisely, severe AKI causes PTCs to arrest in the G1/S or G2/M phase of the cell cycle, leading to maladaptive repair and a fibrotic outcome. The mechanisms causing these arrests are far from known. The arrest might, at least partially, be attributed to DNA damage since activation of the DNA-damage response pathway leads to cell cycle arrest. Alternatively, cytokine signalling via nuclear factor kappa beta (NF-κβ) and p38-mitogen-activated protein kinase (p38-MAPK) pathways, and reactive oxygen species (ROS) can play a role independent of DNA damage. In addition, only a handful of cell cycle regulators (e.g., p53, p21) have been thoroughly studied during renal repair. Still, why and how PTCs decide to arrest their cell cycle and how this arrest can efficiently be overcome remain open and challenging questions. In this review we will discuss the evidence for cell cycle involvement during AKI and development of CKD together with putative therapeutic approaches.

Chronic kidney disease (CKD) causes dysregulation of mineral metabolism, vascular calcification and renal osteodystrophy, an entity called CKDMineral and Bone Disorder (CKD-MBD). Here we determine whether metformin, an anti-diabetic drug, exerts favorable effects on progressive, severe CKD and concomitant mineral metabolism disturbances. Rats with CKD-MBD, induced by a 0.25% adenine diet for eight weeks, were treated with 200 mg/kg/day metformin or vehicle from one week after CKD induction onward. Severe, stable CKD along with marked hyperphosphatemia and hypocalcemia developed in these rats which led to arterial calcification and high bone turnover disease. Metformin protected from development toward severe CKD. Metformin-treated rats did not develop hyperphosphatemia or hypocalcemia and this prevented the development of vascular calcification and inhibited the progression toward high bone turnover disease. Kidneys of the metformin group showed significantly less cellular infiltration, fibrosis and inflammation. To study a possible direct effect of metformin on the development of vascular calcification, independent of its effect on renal function, metformin (200 mg/kg/day) or vehicle was dosed for ten weeks to rats with warfarin-induced vascular calcification. The drug did not reduce aorta or small vessel calcification in this animal model. Thus, metformin protected against the development of severe CKD and preserved calcium phosphorus homeostasis. As a result of its beneficial impact on renal function, associated comorbidities such as vascular calcification and high bone turnover disease were also prevented.

Vascular calcification, albeit heterogeneous in terms of biological and physicochemical properties, has been associated with ageing, lifestyle, diabetes, and chronic kidney disease (CKD). It is unknown whether or not moderately impaired renal function (CKD stages 24) affects the physiochemical composition and/or the formation of magnesium-containing tricalcium phosphate ([Ca,Mg]3[PO4]2, whitlockite) in arterial microcalcification. Therefore, a high-resolution scanning X-ray diffraction analysis (European Synchrotron Radiation Facility, Grenoble, France) utilizing histological sections of paraffin-embedded arterial specimens derived from atherosclerotic patients with normal renal function (n = 15) and CKD (stages 24, n = 13) was performed. This approach allowed us to spatially assess the contribution of calcium phosphate (apatite) and whitlockite to arterial microcalcification. Per group, the number of samples (13 vs. 12) with sufficient signal intensity and total lengths of regions (201 vs. 232 μm) giving rise to diffractograms (informative regions) were comparable. Summarizing all informative regions per group into one composite sample revealed calcium phosphate/apatite as the leading mineral phase in CKD patients, whereas in patients with normal renal function the relative contribution of whitlockite and calcium phosphate/apatite was on the same order of magnitude (CKD, calcium phosphate/apatite 157 μm, whitlockite 38.7 μm; non-CKD, calcium phosphate/apatite 79.0 μm, whitlockite 94.1 μm; each p < 0.05). Our results, although based on a limited number of samples, indicate that chronic impairment of renal function affects local magnesium homeostasis and thus contributes to the physicochemical composition of microcalcification in atherosclerotic patients.

Over the past decades metformin has been the optimal first-line treatment for type 2 diabetes mellitus (T2DM). Only in the last few years, it has become increasingly clear that metformin exerts benign pleiotropic actions beyond its prescribed use and ongoing investigations focus on a putative beneficial impact of metformin on the kidney. Both acute kidney injury (AKI) and chronic kidney disease (CKD), two major renal health issues, often result in the need for renal replacement therapy (dialysis or transplantation) with a high socio-economic impact for the patients. Unfortunately, to date, effective treatment directly targeting the kidney is lacking. Metformin has been shown to exert beneficial effects on the kidney in various clinical trials and experimental studies performed in divergent rodent models representing different types of renal diseases going from AKI to CKD. Despite growing evidence on metformin as a candidate drug for renal diseases, in-depth research is imperative to unravel the molecular signaling pathways responsible for metformins renoprotective actions. This review will discuss the current state-of-the-art literature on clinical and preclinical data, and put forward potential cellular mechanisms and molecular pathways by which metformin ameliorates AKI/CKD. View Full-Text Keywords: metformin; type 2 diabetes mellitus; acute kidney injury; chronic kidney disease; lactic acidosis; renoprotection; AMP-activated protein kinase pathway

A dozen conditions are regularly associated with nephrocalcinosis, and two dozen more less frequently. Mechanisms of intratubular and interstitial crystal formation, and propagation or removal, as well as inhibitors of crystal formation, are described.

Environmental and occupational exposure to heavy metals should be considered an important health hazard, even more so since evidence has been provided in recent literature for toxic effects occurring even at low levels. The kidney is highly vulnerable to metal toxicity and the extent of renal damage depends on the nature, dose, route, and duration of exposure. Both acute and chronic intoxication have been demonstrated to cause kidney injury, with various levels of severity, ranging from tubular dysfunctions to severe chronic kidney disease. The epidemiology of heavy metals poisoning, their renal handling, pathophysiological mechanisms of renal toxicity, diagnosis, and monitoring, as well as therapeutic aspects are dealt with. The metals discussed include lead, cadmium, mercury, uranium, arsenic, and chromium.

Preconditioning of the distal tubular epithelium of the human kidney precedes nephrocalcinosis. Background Preterm neonates and renal transplant patients frequently develop nephrocalcinosis. Experimental studies revealed that crystal retention in the distal nephron, a process that may lead to nephrocalcinosis, is limited to proliferating/regenerating tubular cells expressing hyaluronan and osteopontin at their luminal surface. Fetal and transplant kidneys contain proliferating and/or regenerating cells since nephrogenesis is not completed until 36 weeks of gestation, while ischemia and nephrotoxic immunosuppressants may lead to injury and repair in renal transplants. This prompted us to investigate the expression of hyaluronan and osteopontin and to correlate this to the appearance of tubular calcifications both in fetal/preterm and transplanted kidneys. Methods Sections of fetal/preterm kidneys and protocol biopsies of transplanted kidneys (12 and 24 weeks posttransplantation from the same patients) were stained for osteopontin, hyaluronan, and calcifications (von Kossa). Results Hyaluronan and osteopontin were expressed at the luminal surface of the epithelial cells lining the distal tubules of all fetal kidneys at birth and in all kidney graft protocol biopsies 12 and 24 weeks posttransplantation. In 7 out of 18 surviving (at least 4 days) preterm neonates crystal retention developed. In renal allografts a striking increase (from 2/10 to 6/10) in tubular crystal retention between 12 and 24 weeks posttransplantation was observed. In addition, crystals were selectively retained in distal renal tubules containing cells with hyaluronan and osteopontin at their luminal surface. Conclusion The results of this study show that luminal expression of hyaluronan and osteopontin preceded renal distal tubular retention of crystals in preterm neonates and renal transplant patients. We propose that the presence of this crystal binding phenotype may play a general role in renal calcification processes.

Prior to colonoscopy, bowel cleansing is performed for which frequently oral sodium phosphate (OSP) is used. OSP results in significant hyperphosphatemia and cases of acute kidney injury (AKI) referred to as acute phosphate nephropathy (APN; characterized by nephrocalcinosis) are reported after OSP use, which led to a US-FDA warning. To improve the safety profile of OSP, it was evaluated whether the side-effects of OSP could be prevented with intestinal phosphate binders. Hereto a Wistar rat model of APN was developed. OSP administration (2 times 1.2 g phosphate by gavage) with a 12h time interval induced bowel cleansing (severe diarrhea) and significant hyperphosphatemia (21.79 ± 5.07 mg/dl 6h after the second OSP dose versus 8.44 ± 0.97 mg/dl at baseline). Concomitantly, serum PTH levels increased fivefold and FGF-23 levels showed a threefold increase, while serum calcium levels significantly decreased from 11.29 ± 0.53 mg/dl at baseline to 8.68 ± 0.79 mg/dl after OSP. OSP administration induced weaker NaPi-2a staining along the apical proximal tubular membrane. APN was induced: serum creatinine increased (1.5 times baseline) and nephrocalcinosis developed (increased renal calcium and phosphate content and calcium phosphate deposits on Von Kossa stained kidney sections). Intestinal phosphate binding (lanthanum carbonate or aluminum hydroxide) was not able to attenuate the OSP induced side-effects. In conclusion, a clinically relevant rat model of APN was developed. Animals showed increased serum phosphate levels similar to those reported in humans and developed APN. No evidence was found for an improved safety profile of OSP by using intestinal phosphate binders.

Purpose: Increased intestinal oxalate absorption leads to increased urinary oxalate excretion (secondary hyperoxaluria) and calcium oxalate crystal formation, contributing to nephrocalcinosis/lithiasis. Lanthanum carbonate is an intestinal phosphate binder that is orally administered to patients on dialysis to treat hyperphosphatemia. It is hypothesized that lanthanum can also bind oxalate, in addition to phosphate. We evaluated this in vitro and in vivo. Materials and Methods: In vitro oxalate binding was evaluated by oxalate precipitation from a solution by lanthanum. In vivo oxalate absorption kinetics and the effect of lanthanum carbonate on nephrocalcinosis development were assessed in male Sprague-Dawley (R) rats that received 1) 1,000 mg lanthanum carbonate and oxalate, 2) carboxymethylcellulose and oxalate or 3) carboxymethylcellulose by gavage for up to 12 hours (kinetics) or 7 days (nephrocalcinosis). Plasma and urinary oxalate concentrations were measured at several time points after gavage. The degree of nephrocalcinosis was assessed histomorphometrically on von Kossa stained sections and by measuring total calcium content in renal tissue. Results: In vitro lanthanum bound oxalate in a pH range comparable to the range of the intestine. In vivo oxalate administration in untreated animals resulted in a biphasic pattern of increased plasma oxalate levels, which was almost abolished in lanthanum treated rats. In the urine of treated rats oxaluria and calcium oxalate crystalluria were blunted. Moreover, significantly decreased nephrocalcinosis was observed compared with that in untreated rats. Conclusions: Lanthanum carbonate is a promising agent for the future prevention/treatment of secondary hyperoxaluria.

Hyperoxaluria leads to urinary calcium oxalate (CaOx) supersaturation, resulting in the formation and retention of CaOx crystals in renal tissue. CaOx crystals may contribute to the formation of diffuse renal calcifications (nephrocalcinosis) or stones (nephrolithiasis). When the innate renal defense mechanisms are suppressed, injury and progressive inflammation caused by these CaOx crystals, together with secondary complications such as tubular obstruction, may lead to decreased renal function and in severe cases to end-stage renal failure. For decades, research on nephrocalcinosis and nephrolithiasis mainly focused on both the physicochemistry of crystal formation and the cell biology of crystal retention. Although both have been characterized quite well, the mechanisms involved in establishing urinary supersaturation in vivo are insufficiently understood, particularly with respect to oxalate. Therefore, current therapeutic strategies often fail in their compliance or effectiveness, and CaOx stone recurrence is still common. As the etiology of hyperoxaluria is diverse, a good understanding of how oxalate is absorbed and transported throughout the body, together with a better insight in the regulatory mechanisms, is crucial in the setting of future treatment strategies of this disorder. In this review, the currently known mechanisms of oxalate handling in relevant organs will be discussed in relation to the different etiologies of hyperoxaluria. Furthermore, future directions in the treatment of hyperoxaluria will be covered.

Intratubular nephrocalcinosis is defined as the histological observation of calcium oxalate and/or calcium phosphate deposits retained within the lumen of the renal tubules. As the tubular epithelium is the primary interaction partner of crystals formed in the tubular fluid, the role of the epithelial cells in nephrocalcinosis has been investigated intensively. This review summarizes our current understanding on how the tubular epithelium mechanistically appears to be involved both in the initiation and in the course of nephrocalcinosis, with emphasis on in vivo observations.

Background. The fixed particle theory states that, besides crystal formation in the tubular fluid, crystal adhesion to the tubular epithelium is a prerequisite for the development of intratubular nephrocalcinosis. It has been hypothesized that the tubular epithelium, in order to bind crystals, needs to be phenotypically altered. Whereas most evidence hereto is provided by in vitro experiments, we set out to illustrate this theory in vivo. Methods. We simultaneously investigated the temporal changes of nephrocalcinosis-associated parameters during and shortly after a 4-day ethylene glycol (EG)-administration period in rats. We measured oxaluria, crystal formation, crystalluria, apoptosis, epithelial injury/ regeneration and luminal membrane expression of several crystal-binding molecules [hyaluronan (HA), osteopontin (OPN) and for the first time in vivo, annexin-2 (ANX2) and nucleolin-related-protein (NRP) and one of their receptors (CD44, HA/OPN-receptor]. Clinically, renal biopsies of preterm infants, transplant patients and acute phosphate nephropathy patients were stained for ANX2, NRP, HA and OPN. Results. In the presence of a rather constant and persistent intratubular crystal formation, crystal retention gradually increased during EG-administration and markedly increased after arrest thereof, indicating that the development of crystal adhesion requires more than just the presence of crystals in the tubular fluid. All luminal membrane markers and a regenerating/dedifferentiated epithelium, unlike apoptosis, to various extents were upregulated concurrently and in association with crystal adhesion. However, both in humans and rats, expression of luminal molecules was not confined to crystal-containing tubules. Conclusions. Altogether, these findings allow better insight into the mechanisms underlying the fixed particle theory in vivo and indicate that an altered epithelial phenotype with crystal-binding properties precedes crystal adhesion, thereby corroborating the requirement of tubular epithelial phenotypical changes in the development of intratubular nephrocalcinosis.

The most common form of renal stone disease, calcium nephrolithiasis , is defined as the presentation of a macroscopic concrement of inorganic (calcium phosphate and/or calcium phosphate) and organic material in the renal calyces and/or pelvis, either adhered to the papillae or pelvic urothelium or not. In search of the mechanism underlying calcium nephrolithiasis, in vitro and in vivo studies and observations in human biopsies have shown the presence of two distinct types of renal microscopical crystal deposition processes; one taking place within the tubular lumen ( intratubular nephrocalcinosis ), and the other in the interstitium ( interstitial nephrocalcinosis ). Recent observations, however, strongly suggest that nephrocalcinosis and calcium nephrolithiasis are to be considered two independent pathologies and that nephrocalcinosis may cause calcium nephrolithiasis only in particular conditions. In this review, we discuss our current understanding of the mechanisms involved in both types of nephrocalcinosis (intratubular and interstitial), their possible consequences and their relation to calcium nephrolithiasis.

The kidney has several defense mechanisms to avert nephrocalcinosis by preventing intratubular crystal formation and adherence. Little is known about the fate of luminally adhered crystals. In order to study post-crystal adhesion defense mechanisms we quantified the number and morphology of crystal-containing tubules in rats at various time points following ethylene glycol administration as well as in renal biopsies of patients diagnosed with nephrocalcinosis of different etiology. In rats, nephrocalcinosis was completely cleared by epithelial overgrowth of adherent crystals, which were then translocated to the interstitium and subsequently disintegrated. These processes correlated with a low to moderate infiltration of inflammatory cells. Patients with nephrocalcinosis due either to acute phosphate nephropathy, primary hyperoxaluria, preterm birth, or transplantation also showed epithelial crystal overgrowth independent of the underlying disorder or the nature of the crystals. Our study found a quantitative association between changes in tubular and crystalline morphology and crystal clearance, demonstrating the presence of an important and active nephrocalcinosis-clearing mechanism in both rat and man.