Crystalline nephropathy in mice: the role of Tamm-Horsfall protein in Acute Kidney Injury and its progression to Chronic Kidney Disease

Abstract

Crystalline nephropathy (crystals in the kidneys) is associated with Acute Kidney Injury and recurrence, but there are no data about the progression to the chronic phase. In addition, the appropriate treatment for this pathology is not known. Recent studies of our group have shown a significant decrease in the expression of Tamm-Horsfall Protein (THP) in the ascending thick limb of the loop of Henle, associated with renal injury in the acute phase of experimental crystalline nephropathy. Thus, we hypothesize that in physiological conditions THP can modulate the innate immune system and play a renoprotective role. However, in Acute Kidney Injury induced by crystalline nephropathy, reduced THP synthesis may induce the increased activity of resident immune system cells, resulting in severe inflammation. In this condition, inflammatory factors may become relevant molecules in the progression of renal injury from the acute to the chronic phase, as well as in the recurrence phase. This study aims to indicate the function of THP in Acute Kidney Injury, progression of this to Chronic Kidney Disease and recurrence, associated with crystalline nephropathy. To this end, the following will be investigated: 1) renal function and morphology, as well as the signaling pathways associated with tissue injury in the acute, progressive, and after recurrence phases; 2) the action of THP in a possible cross-talk between the thick ascending limb of the loop of Henle and other kidney compartments (proximal tubule and glomeruli), especially observing the behavior of pro-inflammatory factors; 3) role of the hepsin enzyme in the effects promoted by THP. Thus, in vivo and in vitro experiments will be carried out. C57BL/6J mice with 08 weeks [control, treated with sodium oxalate (11 mg/100g, single injection i.p. to induce crystalline nephropathy) or treated with synthetic THP (5 ¼g/animal, single injection i.p.)]. Cell cocultures of the ascending thick limb of the loop of Henle, dendritic cells, macrophages, cells of the proximal tubule, and glomeruli will be treated with calcium oxalate suspension (200µg/mL) and/or THP (10 µM) or leupeptin (10 µM, hepsin inhibitor) for 24 and 48 hours. With this will be possible to know how THP modulates immune cells and interferes in the crosstalk between the different components of the nephron, contributing to the discovery of therapeutic targets that can be used to attenuate the renal injury resulting from crystalline nephropathy. The techniques used include clearance and renal function, immunofluorescence, western blotting, quantitative PCR, microscopy imaging analysis, and flow cytometry. Proposal challenges: standardize the experimental protocol, since there are not many studies about the progression of renal injury in crystalline nephropathy and the use of exogenous THP as a renoprotective molecule after the injury. In addition, methodological challenges should be addressed to investigate the role of THP in the crosstalk between the different segments of the nephron and for genomic analysis of the inflammatory factors involved in this model (BEPE proposal). (AU)