Animal models to study cognitive impairment of chronic kidney disease.

Mild cognitive impairment (MCI) is common in people with chronic kidney disease (CKD), and its prevalence increases with progressive loss of kidney function. MCI is characterized by a decline in cognitive performance greater than expected for an individual age and education level but with minimal impairment of instrumental activities of daily living. Deterioration can affect one or several cognitive domains (attention, memory, executive functions, language, and perceptual motor or social cognition). Given the increasing prevalence of kidney disease, more and more people with CKD will also develop MCI causing an enormous disease burden for these individuals, their relatives, and society. However, the underlying pathomechanisms are poorly understood, and current therapies mostly aim at supporting patients in their daily lives. This illustrates the urgent need to elucidate the pathogenesis and potential therapeutic targets and test novel therapies in appropriate preclinical models. Here, we will outline the necessary criteria for experimental modeling of cognitive disorders in CKD. We discuss the use of mice, rats, and zebrafish as model systems and present valuable techniques through which kidney function and cognitive impairment can be assessed in this setting. Our objective is to enable researchers to overcome hurdles and accelerate preclinical research aimed at improving the therapy of people with CKD and MCI.

Role of proton-activated G protein-coupled receptors in pathophysiology.

Local acidification is a common feature of many disease processes such as inflammation, infarction, or solid tumor growth. Acidic pH is not merely a sequela of disease but contributes to recruitment and regulation of immune cells, modifies metabolism of parenchymal, immune and tumor cells, modulates fibrosis, vascular permeability, oxygen availability, and consumption, invasiveness of tumor cells, and impacts on cell survival. Thus, multiple pH-sensing mechanisms must exist in cells involved in these processes. These pH sensors play important roles in normal physiology and pathophysiology, and hence might be attractive targets for pharmacological interventions. Among the pH-sensing mechanisms, OGR1 (GPR68), GPR4 (GPR4), and TDAG8 (GPR65) have emerged as important molecules. These G protein-coupled receptors are widely expressed, upregulated in inflammation and tumors, sense changes in extracellular pH in the range between pH 8 and 6, and are involved in modulating key processes in inflammation, tumor biology, and fibrosis. This review discusses key features of these receptors and highlights important disease states and pathways affected by their activity.

Physiological relevance of proton-activated GPCRs.

The detection of H+ concentration variations in the extracellular milieu is accomplished by a series of specialized and non-specialized pH-sensing mechanisms. The proton-activated G protein-coupled receptors (GPCRs) GPR4 (Gpr4), TDAG8 (Gpr65), and OGR1 (Gpr68) form a subfamily of proteins capable of triggering intracellular signaling in response to alterations in extracellular pH around physiological values, i.e., in the range between pH 7.5 and 6.5. Expression of these receptors is widespread for GPR4 and OGR1 with particularly high levels in endothelial cells and vascular smooth muscle cells, respectively, while expression of TDAG8 appears to be more restricted to the immune compartment. These receptors have been linked to several well-studied pH-dependent physiological activities including central control of respiration, renal adaption to changes in acid-base status, secretion of insulin and peripheral responsiveness to insulin, mechanosensation, and cellular chemotaxis. Their role in pathological processes such as the genesis and progression of several inflammatory diseases (asthma, inflammatory bowel disease), and tumor cell metabolism and invasiveness, is increasingly receiving more attention and makes these receptors novel and interesting targets for therapy. In this review, we cover the role of these receptors in physiological processes and will briefly discuss some implications for disease processes.

New Therapeutic Approach for Intestinal Fibrosis Through Inhibition of pH-Sensing Receptor GPR4.

BACKGROUND: Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of pH-sensing receptors compared with non-IBD controls. Acidification leads to angiogenesis and extracellular matrix remodeling. We aimed to determine the expression of pH-sensing G protein-coupled receptor 4 (GPR4) in fibrotic lesions in Crohn's disease (CD) patients. We further evaluated the effect of deficiency in Gpr4 or its pharmacologic inhibition. METHODS: Paired samples from fibrotic and nonfibrotic terminal ileum were obtained from CD patients undergoing ileocaecal resection. The effects of Gpr4 deficiency were assessed in the spontaneous Il-10-/- and the chronic dextran sodium sulfate (DSS) murine colitis model. The effects of Gpr4 deficiency and a GPR4 antagonist (39c) were assessed in the heterotopic intestinal transplantation model. RESULTS: In human terminal ileum, increased expression of fibrosis markers was accompanied by an increase in GPR4 expression. A positive correlation between the expression of procollagens and GPR4 was observed. In murine disease models, Gpr4 deficiency was associated with a decrease in angiogenesis and fibrogenesis evidenced by decreased vessel length and expression of Edn, Vegfα, and procollagens. The heterotopic animal model for intestinal fibrosis, transplanted with terminal ileum from Gpr4-/- mice, revealed a decrease in mRNA expression of fibrosis markers and a decrease in collagen content and layer thickness compared with grafts from wild type mice. The GPR4 antagonist decreased collagen deposition. The GPR4 expression was also observed in human and murine intestinal fibroblasts. The GPR4 inhibition reduced markers of fibroblast activation stimulated by low pH, notably Acta2 and cTgf. CONCLUSIONS: Expression of GPR4 positively correlates with the expression of profibrotic genes and collagen. Deficiency of Gpr4 is associated with a decrease in angiogenesis and fibrogenesis. The GPR4 antagonist decreases collagen deposition. Targeting GPR4 with specific inhibitors may constitute a new treatment option for IBD-associated fibrosis.

Acidosis, cognitive dysfunction and motor impairments in patients with kidney disease.

Metabolic acidosis, defined as a plasma or serum bicarbonate concentration <22 mmol/L, is a frequent consequence of chronic kidney disease (CKD) and occurs in ~10-30% of patients with advanced stages of CKD. Likewise, in patients with a kidney transplant, prevalence rates of metabolic acidosis range from 20% to 50%. CKD has recently been associated with cognitive dysfunction, including mild cognitive impairment with memory and attention deficits, reduced executive functions and morphological damage detectable with imaging. Also, impaired motor functions and loss of muscle strength are often found in patients with advanced CKD, which in part may be attributed to altered central nervous system (CNS) functions. While the exact mechanisms of how CKD may cause cognitive dysfunction and reduced motor functions are still debated, recent data point towards the possibility that acidosis is one modifiable contributor to cognitive dysfunction. This review summarizes recent evidence for an association between acidosis and cognitive dysfunction in patients with CKD and discusses potential mechanisms by which acidosis may impact CNS functions. The review also identifies important open questions to be answered to improve prevention and therapy of cognitive dysfunction in the setting of metabolic acidosis in patients with CKD.

Acidosis and alkali therapy in patients with kidney transplant is associated with transcriptional changes and altered abundance of genes involved in cell metabolism and acid-base balance.

BACKGROUND: Metabolic acidosis occurs frequently in patients with kidney transplant and is associated with a higher risk for and accelerated loss of graft function. To date, it is not known whether alkali therapy in these patients improves kidney function and whether acidosis and its therapy are associated with altered expression of proteins involved in renal acid-base metabolism. METHODS: We retrospectively collected kidney biopsies from 22 patients. Of these patients, nine had no acidosis, nine had metabolic acidosis [plasma bicarbonate (HCO3- <22 mmol/L) and four had acidosis and received alkali therapy. We performed transcriptome analysis and immunohistochemistry for proteins involved in renal acid-base handling. RESULTS: We found that the expression of 40 transcripts significantly changed between kidneys from non-acidotic and acidotic patients. These genes are mostly involved in proximal tubule (PT) amino acid and lipid metabolism and energy homoeostasis. Three transcripts were fully recovered by alkali therapy: the Kir4.2 potassium channel, an important regulator of PT HCO3- metabolism and transport, acyl-CoA dehydrogenase short/branched chain and serine hydroxymethyltransferase 1, genes involved in beta oxidation and methionine metabolism. Immunohistochemistry showed reduced staining for the PT NBCe1 HCO3- transporter in kidneys from acidotic patients who recovered with alkali therapy. In addition, the HCO3- exchanger pendrin was affected by acidosis and alkali therapy. CONCLUSIONS: Metabolic acidosis in kidney transplant recipients is associated with alterations in the renal transcriptome that are partly restored by alkali therapy. Acid-base transport proteins mostly from PT were also affected by acidosis and alkali therapy, suggesting that the downregulation of critical players contributes to metabolic acidosis in these patients.

Intestinal epithelial ablation of Pit-2/Slc20a2 in mice leads to sustained elevation of vitamin D3 upon dietary restriction of phosphate.

AIM: Several Na+ -dependent phosphate cotransporters, namely NaPi-IIb/SLC34A2, Pit-1/SLC20A1 and Pit-2/SLC20A2, are expressed at the apical membrane of enterocytes but their contribution to active absorption of phosphate is unclear. The aim of this study was to compare their pattern of mRNA expression along the small and large intestine and to analyse the effect of intestinal depletion of Pit-2 on phosphate homeostasis. METHODS: Intestinal epithelial Pit-2-deficient mice were generated by crossing floxed Pit-2 with villin-Cre mice. Mice were fed 2 weeks standard or low phosphate diets. Stool, urine, plasma and intestinal and renal tissue were collected. Concentration of electrolytes and hormones, expression of mRNAs and proteins and intestinal transport of tracers were analysed. RESULTS: Intestinal mRNA expression of NaPi-IIb and Pit-1 is segment-specific, whereas the abundance of Pit-2 mRNA is more homogeneous. In ileum, NaPi-IIb mRNA expression is restricted to enterocytes, whereas Pit-2 mRNA is found in epithelial and non-epithelial cells. Overall, their mRNA expression is not regulated by dietary phosphate. The absence of Pit-2 from intestinal epithelial cells does not affect systemic phosphate homeostasis under normal dietary conditions. However, in response to dietary phosphate restriction, Pit-2-deficient mice showed exacerbated hypercalciuria and sustained elevation of 1,25(OH)2 vitamin D3 . CONCLUSIONS: In mice, the intestinal Na+ /phosphate cotransporters are not coexpressed in all segments. NaPi-IIb but not Pit-2 mRNA is restricted to epithelial cells. Intestinal epithelial Pit-2 does not contribute significantly to absorption of phosphate under normal dietary conditions. However, it may play a more significant role upon dietary phosphate restriction.

Potassium channels in control of renal function.

Potassium channels are important to control membrane potential and drive epithelial transport processes. In this issue of Kidney International, Bignon et al. report the role of the Kir4.2 K+-channel, localized at the basolateral membrane of proximal tubules, in the reabsorption of bicarbonate and the modulation of renal ammoniagenesis. The findings have implications for our understanding of how the kidney reacts to hypokalemia, an acid load, and the metabolic acidosis of patients with advanced stages of chronic kidney disease.

Molecular Pathophysiology of Acid-Base Disorders.

Acid-base balance is critical for normal life. Acute and chronic disturbances impact cellular energy metabolism, endocrine signaling, ion channel activity, neuronal activity, and cardiovascular functions such as cardiac contractility and vascular blood flow. Maintenance and adaptation of acid-base homeostasis are mostly controlled by respiration and kidney. The kidney contributes to acid-base balance by reabsorbing filtered bicarbonate, regenerating bicarbonate through ammoniagenesis and generation of protons, and by excreting acid. This review focuses on acid-base disorders caused by renal processes, both inherited and acquired. Distinct rare inherited monogenic diseases affecting acid-base handling in the proximal tubule and collecting duct have been identified. In the proximal tubule, mutations of solute carrier 4A4 (SLC4A4) (electrogenic Na+/HCO3--cotransporter Na+/bicarbonate cotransporter e1 [NBCe1]) and other genes such as CLCN5 (Cl-/H+-antiporter), SLC2A2 (GLUT2 glucose transporter), or EHHADH (enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase) causing more generalized proximal tubule dysfunction can cause proximal renal tubular acidosis resulting from bicarbonate wasting and reduced ammoniagenesis. Mutations in adenosine triphosphate ATP6V1 (B1 H+-ATPase subunit), ATPV0A4 (a4 H+-ATPase subunit), SLC4A1 (anion exchanger 1), and FOXI1 (forkhead transcription factor) cause distal renal tubular acidosis type I. Carbonic anhydrase II mutations affect several nephron segments and give rise to a mixed proximal and distal phenotype. Finally, mutations in genes affecting aldosterone synthesis, signaling, or downstream targets can lead to hyperkalemic variants of renal tubular acidosis (type IV). More common forms of renal acidosis are found in patients with advanced stages of chronic kidney disease and are owing, at least in part, to a reduced capacity for ammoniagenesis.

Intestinal Activation of pH-Sensing Receptor OGR1 [GPR68] Contributes to Fibrogenesis.

BACKGROUND AND AIMS: pH-sensing ovarian cancer G-protein coupled receptor-1 [OGR1/GPR68] is regulated by key inflammatory cytokines. Patients suffering from inflammatory bowel diseases [IBDs] express increased mucosal levels of OGR1 compared with non-IBD controls. pH-sensing may be relevant for progression of fibrosis, as extracellular acidification leads to fibroblast activation and extracellular matrix remodelling. We aimed to determine OGR1 expression in fibrotic lesions in the intestine of Crohn's disease [CD] patients, and the effect of Ogr1 deficiency in fibrogenesis. METHODS: Human fibrotic and non-fibrotic terminal ileum was obtained from CD patients undergoing ileocaecal resection due to stenosis. Gene expression of fibrosis markers and pH-sensing receptors was analysed. For the initiation of fibrosis in vivo, spontaneous colitis by Il10-/-, dextran sodium sulfate [DSS]-induced chronic colitis and the heterotopic intestinal transplantation model were used. RESULTS: Increased expression of fibrosis markers was accompanied by an increase in OGR1 [2.71 ± 0.69 vs 1.18 ± 0.03, p = 0.016] in fibrosis-affected human terminal ileum, compared with the non-fibrotic resection margin. Positive correlation between OGR1 expression and pro-fibrotic cytokines [TGFB1 and CTGF] and pro-collagens was observed. The heterotopic animal model for intestinal fibrosis transplanted with terminal ileum from Ogr1-/- mice showed a decrease in mRNA expression of fibrosis markers as well as a decrease in collagen layer thickness and hydroxyproline compared with grafts from wild-type mice. CONCLUSIONS: OGR1 expression was correlated with increased expression levels of pro-fibrotic genes and collagen deposition. Ogr1 deficiency was associated with a decrease in fibrosis formation. Targeting OGR1 may be a potential new treatment option for IBD-associated fibrosis.

Erythropoietin stimulates fibroblast growth factor 23 (FGF23) in mice and men.

Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH)2 vitamin D3 metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH)2 vitamin D3, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH)2 vitamin D3 are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24 h) C-terminal FGF23 but iFGF23 only after 4 days without effects on PTH and plasma phosphate. 1,25 (OH)2 D3 levels and αklotho expression in the kidney decrease after 4 days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71+ erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH)2 vitamin D3 levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism.

NRF2 regulates the glutamine transporter Slc38a3 (SNAT3) in kidney in response to metabolic acidosis.

Expression of the glutamine transporter SNAT3 increases in kidney during metabolic acidosis, suggesting a role during ammoniagenesis. Microarray analysis of Nrf2 knock-out (KO) mouse kidney identified Snat3 as the most significantly down-regulated transcript compared to wild-type (WT). We hypothesized that in the absence of NRF2 the kidney would be unable to induce SNAT3 under conditions of metabolic acidosis and therefore reduce the availability of glutamine for ammoniagenesis. Metabolic acidosis was induced for 7 days in WT and Nrf2 KO mice. Nrf2 KO mice failed to induce Snat3 mRNA and protein expression during metabolic acidosis. However, there were no differences in blood pH, bicarbonate, pCO2, chloride and calcium or urinary pH, ammonium and phosphate levels. Normal induction of ammoniagenic enzymes was observed whereas several amino acid transporters showed differential regulation. Moreover, Nrf2 KO mice during acidosis showed increased expression of renal markers of oxidative stress and injury and NRF2 activity was increased during metabolic acidosis in WT kidney. We conclude that NRF2 is required to adapt the levels of SNAT3 in response to metabolic acidosis. In the absence of NRF2 and SNAT3, the kidney does not have any major acid handling defect; however, increased oxidative stress and renal injury may occur.

The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation.

BACKGROUND AND AIMS: During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. METHODS: The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. RESULTS: Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. CONCLUSIONS: Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

And the fat lady sings about phosphate and calcium.

Adipose tissue has been long recognized as secreting various endocrine factors. Emerging evidence demonstrates that adipokines play a role in modulating systemic mineral homeostasis through endocrine loops involving interleukin-6, leptin, and now also adiponectin, which all interact with FGF23 and vitamin D and thereby change the renal control of calcium and phosphate metabolism. Understanding these regulatory loops may shed light on a complex interorgan crosstalk controlling mineral homeostasis and its dysregulation in diseases associated with obesity.