Biogem

Translational Nephrology

Director: prof. Francesco Trepiccione and dr. Anna Iervolino

Research areas

Our Translational Nephrology laboratory aims at deepening the knowledge of renal pathophysiology, in order to subsequently develop new therapeutic targets. The current research interests of our laboratory are directed to the study of arterial hypertension, diabetes mellitus, diabetes insipidus, polyuric syndromes, renal drug toxicity, renal cystic diseases and models of acute renal failure, as well as rare kidney pathologies. Our Translational Nephrology laboratory works on both genetically modified animal models, in which renal pathology or its alterations are reproduced, and mouse models, in which the pathological state is induced with nephrotoxic substances. These animal models are a valid tool for reproducing and studying renal pathologies, thus allowing for the development of molecules with a pharmaceutical action that might be used in humans, too.

Technologies used / developed

In order to achieve the aforementioned objectives, in vivo and in vitro studies are used.

Basic metabolic parameters: mice or rats are placed individually in metabolic cages that allow, after three days of adaptation, to monitor the consumption of water and food, the measurement of electrolytes (Na, Cl, Ca, K, Mg) and proteinuria, after sampling urine and hematocrit, and of blood metabolites (pH, pO2, pCO2, HCO3), after arterial or venous blood sampling.

Histological evaluation of renal morphology: mouse or rat kidneys are collected following renal perfusion with paraformaldehyde, via the abdominal aorta. They are then embedded in paraffin and cut into thin sections for the morphological evaluation of the organ or the expression of some proteins, through immunohistochemical assays.

Macroscopic and microscopic dissection of the kidney districts: once collected, the kidneys are decapsulated and dissected, in order to obtain a portion of the cortex, one of the external medulla and one of the internal medulla. The different segments of the nephron (glomerulus, proximal tubule, Henle’s loop, distal tubule and collecting duct) can be isolated by digesting the renal portion of interest with a solution containing collagenase and proteinase. The renal tubules, thus isolated, allow for an efficient extraction of RNA and proteins.

Renal micro-puncture: it is a highly specialized method that allows the evaluation of renal flow directly in the individual portions of the nephron. Thanks to the use of micropipettes, a perfusion liquid is injected upstream of the tubule of interest. This liquid is then collected downstream of the renal segment and analyzed. In this way, information is obtained on the renal flow of the tubule studied, on the extent of absorption and secretion, as well as on the mechanisms induced by any injected drugs.

Two-photon microscopy: this is the technique of choice for physiopathology studies in organs of living animals, at the cellular and sub-cellular level. It allows for real-time simultaneous acquisition of various physiological parameters. In nephrology, two-photon microscopy helps deepening the knowledge of many diseases, including acute renal failure, hypertension, or diabetes mellitus, thanks to the possibility of using fluorescent probes that allow continuous measurements in real time. After the externalization of the kidney, the animal is placed under the two-photon microscope and the kidney compartments of interest are marked through venous access, with specific fluorescent probes. The analysis of cellular events, recorded under normal or pathological conditions, can be carried out using IT approaches.

Main results achieved

Cystogenesis in a cKO mouse model for Dicer in the whole kidney

The inactivation of Dicer in the cKO mouse model (Dicerflox / flox; Pax8Cre / +) causes an altered biogenesis of miRNAs in the kidney and thyroid, resulting in the formation of numerous cysts and interstitial fibrosis in the cKO kidneys after two months. From a functional point of view, cKOs show a marked defect in urine concentration and proteinuria. The formation of the cysts is related to a gradual disappearance of the primary cilium and is associated with an alteration of the GSK3β / β-catenin pathway.

β1 integrin is essential for the urine concentration process. To study β1 integrin, a receptor that mediates the interaction between cell and extracellular matrix, a cKO mouse model (Itgb1flox / flox; Pax8 Cre / +) was generated in which the protein is deleted throughout the nephron. The kidneys of cKO mice show marked hydronephrosis and dilation of the distal tubules, the Henle’s loop and the collecting ducts. In these animals we also identified a reduction in the expression of the AQP2 channel, which resulted in a strong defect in urine concentration.

miRNA: therapeutic targets for aquaresis. To investigate the role of miRNAs in collecting ducts, a cKO mouse model was generated in which there is a specific Dicer deletion in the main cells (Dicerflox / flox; AQP2 + / Cre). In this portion of the nephron, the AQP2 water channel mediates the process of urine concentration, to the point that the absence or mutations of this protein cause diabetes insipidus. We found marked polyuria in cKO mice of approximately two months, development of hydronephrosis and premature death within three months. The definition of new molecular targets in this process is underway.

Cellular plasticity of the collecting duct as a self-repair mechanism. Primary (PC) and intercalated (IC) cells constitute the two cell types of the collecting duct, and originate from unknown stem precursors. Intercalated cells can change their structure from acid to secretory base, showing extreme plasticity. From the literature it appears that the main cells can instead mutate into intercalated cells. We have previously shown that lithium induces the appearance of cell types having intermediate characteristics between PC and IC. We generated mice selectively expressing YFP in PCs to follow their morphological and functional changes during lithium treatment. The possible expression of markers of the ICs in the labeled YFP cells would prove the possible conversion of the PC to IC.

New molecular hypertension targets in a salt sensitive hypertension model (NA rats). Alpha adducin polymorphisms are linked to the development of arterial hypertension in humans. We are determining the pathogenetic mechanism that determines the development of arterial hypertension in rats carrying an alpha adducin mutation. A molecular interaction between alpha adducin and the thiazide sensitive Na / Cl transporter (NCC) is known from preliminary experiments. This interaction modifies the state of phosphorylation and activation of NCC.

Application of molecular methods in the diagnostics of rare genetic diseases. We are studying several rare genetic diseases, including Fanconi Bickel syndrome, Gitelman syndrome and β-thalassemia. In particular, we use an innovative molecular approach for the validation of possible prognostic markers on urine samples from affected patients.

Research

Internal seminar

YOKO SUZUMOTO PhD, 'Characterization of renal Paraoxonase 2 (PON2)' Monday 20th September

Paraoxonase 2 (PON2) is a membrane-associated enzyme that possesses high hydrolytic activity against bacterial signaling molecule 3-oxo-C12 homoserine lactone, thus exhibits the defense against pathogenic bacteria such as Pseudomonas aeruginosa. In addition, PON2 shows redox function leading to the prevention of atherosclerosis. Furthermore, accumulating data suggest the involvement of PON2 in several human diseases such as Diabetes Mellitus, Alzheimer’s disease and pancreatic cancer. In particular, recent report demonstrated the potential involvement of PON2 in the pathogenesis of venous thromboembolism in COVID-19 patients. In the present study, regulatory mechanisms and potential roles of renal PON2 were investigated utilizing both in vivo and in vitro systems. In Milan normotensive rats strain, under high aldosterone condition induced by low sodium diet, PON2 was up-regulated in parallel with proteolytic activation of alpha-ENaC in the renal cortex. These phenomena were through the activation of mineralocorticoid receptor, since aldosterone antagonist spironolactone blocked both ENaC activation and PON2 up-regulation. Furthermore, spironolactone and canrenone were shown to inhibit PON2 lactonase activity in vitro. Finally, aldosterone induced-upregulation of PON2 was accompanied by decreased reactive oxygen species (ROS) levels in the renal cortex, in particular superoxide levels. These results indicate hormone aldosterone as a novel regulator of PON2 in the kidney, and suggest that PON2 could preserve the kidney from aldosterone-induced ROS-related damages, such as fibrosis, through the modulation of ROS levels.

VINCENZO COSTANZO PhD, In vivo evaluation of renal function using intravital multiphoton microscopyMonday 27th September

2-photon microscopy (2PM) represents the “gold standard” technique to carry out in vivo renal pathophysiology studies. Indeed, compared to previous methods, 2PM offers greater laser penetration into tissues thanks to the reduced light scattering, low photo-toxicity that allows to monitor the samples over the time, and lowered fluorescence loss. Biogem has been equipped with a 2PM laboratory, thanks to the generous donation of the “Terzo Pilastro Internazionale” foundation, led by Prof. Emmanuele F.M. Emanuele. In this study we focused on 3 main research projects: the study of glomerular function by assessing the single nephron glomerular filtration rate (SNGFR), the study of tubular function by evaluating the reabsorption of glucose and proteins in the proximal tubules, and the detection and quantification of renal fibrosis in vivo and ex vivo. In the Translational Nephrology laboratory, an innovative approach to measure in vivo the SNGFR has been developed, since it represents one of the most important renal parameters capable of providing precise and real-time information about renal function. This approach has been validated in experimental models of acute kidney injury and it guarantees great precision and reliability in the measurements, offering results comparable to previous methods. For studies of tubular function, in particular the reabsorption of glucose in the proximal renal tubules, a GLUT2 cko mouse model mimicking the Fanconi Bickel syndrome has been used. It is a genetic disease characterized by hepato-renal glucose accumulation, impaired glucose and galactose metabolism, and proximal tubule dysfunction. By 2PM we demonstrated in vivo that GLUT2 cko mice have an impaired mechanism of glucose reabsorption compared to the control group. Finally, an approach to detect and quantify renal fibrosis both in animal models and tissue sections has been developed. This is possible by exploiting the optical phenomenon of the "second harmonic generation" (SHG), which is an intrinsic property of 2PM allowing the detection of specific tissue molecules, such as collagen fibers and myosin, without any exogenous labeling. This method guarantees high specificity reducing the variability due to the classic histological staining techniques. SHG coupled with machine learning software is able to perform image classification and represents a valid tool for quantifying renal fibrosis, not only in animal models but also for routinely clinical practice. The microscopy approaches developed in this project will increase the knowledge of many pathological conditions, such as acute renal failure and renal fibrosis, and may be used to develop new drugs able to restore physiological conditions.