Role OF NADPH Oxidase 4 (NOX4) in the pathophysiology of kidney diseases

Thesis

Reference

Role OF NADPH Oxidase 4 (NOX4) in the pathophysiology of kidney diseases

NLANDU KHODO, Stellor

Abstract

Le rein est un organe fortement vascularisé impliqué dans la régulation de l'équilibre hydro-électrolytique et l'excrétion de déchets. La maladie rénale chronique est une maladie progressive et multifactorielle. Des études cliniques ont montré une corrélation entre la progression de la maladie rénale chronique et l'apparition de la fibrose interstitielle indépendamment de l'atteinte primaire. La famille des NADPH oxydases est constituée de 7 protéines transmembranaires dont la fonction consiste en la production des radicaux libres (ROS). NOX4 est l'isoforme majoritaire dans le rein. Plusieurs études associent l'activation de NOX4 à la pathogenèse de pathologies chroniques notamment rénales. Cette étude a mis en évidence le rôle protecteur de NOX4 dans la cellule tubulaire rénale en régulant les voies de survie telles que la voie HIF/VEGF ainsi que l'axe NRF2/KEAP1/glutathion. D'autre part, nous avons montré que le rein distal participe bel et bien dans la pathogenèse de la fibrose induite par la protéinurie.

NLANDU KHODO, Stellor. Role OF NADPH Oxidase 4 (NOX4) in the pathophysiology of kidney diseases. Thèse de doctorat : Univ. Genève, 2014, no. Sc. 4665

URN : urn:nbn:ch:unige-366328

DOI : 10.13097/archive-ouverte/unige:36632

Available at:

http://archive-ouverte.unige.ch/unige:36632

Disclaimer: layout of this document may differ from the published version.

1 / 1

Département de Biologie Cellulaire FACULTE DES SCIENCES

Professeur Jean-Claude Martinou

Département de Médecine Interne FACULTE DE MEDECINE

Professeur Eric Féraille

Docteure Sophie de Seigneux

ROLE OF NADPH oxidase 4 (NOX4) in the pathophysiology of Kidney Diseases

THESE

Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

Par

Stellor Nlandu Khodo

De Kinshasa

République Démocratique du Congo

Thèse N°4665

GENEVE

2014

Département de Biologie Cellulaire FACULTE DES SCIENCES

Professeur Jean-Claude Martinou

Département de Médecine Interne FACULTE DE MEDECINE

Professeur Eric Féraille

Docteure Sophie de Seigneux

ROLE OF NADPH oxidase 4 (NOX4) in the pathophysiology of Kidney Diseases

THESE

Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

Par

Stellor Nlandu Khodo

De Kinshasa

République Démocratique du Congo

Thèse N°4665

GENEVE

2014

ACKNOWLEDGEMENTS

CHAPTER 0 LIST OF ABBREVIATIONS ABSTRACT (French)

CHAPTER 1 INTRODUCTION

I. The kidney

1.1 Organization of the urinary system

1.2 General anatomy and function

1.3 Nephron

1.3.1 Glomerulus

1.3.2 Tubular system

1.3.3 Juxtaglomerular apparatus (JGA)

1.3.4 Vascularization of the nephron

1.3.5 Innervation of the nephron

1.4 Glomerular filtration rate (GFR) and renal blood flow (RBF)

1.4.1 Glomerular filtration rate (GFR)

1.4.2 Renal blood flow (RBF)

1.5 Kidney oxygen metabolism

II. Kidney Diseases

2.1 Chronic kidney disease (CKD)

2.1.1 Chronic kidney disease (CKD): Definition and epidemiology

2.1.2 Pathophysiology of chronic kidney disease

2.1.3 Kidney fibrosis

2.1.3.1 Definition and epidemiology p22

2.1.3.2 Pathophysiology of kidney fibrosis p22

2.1.3.2.1 Chronic inflammation p23

2.1.3.2.2 Apoptosis p28

2.1.3.2.3 Chronic hypoxia and hypoxia inducible factor (HIF) p30

2.1.3.2.6 Proteinuria p40 2.1.3.3 Kidney fibrosis and redox sensitive signaling pathways p42 2.1.3.3.1 Reactive oxygen species and cell signaling p42 2.1.3.3.2 Examples of redox sensitive pathways p44

a) TGF-β1 pathway p44

b) HIF-1α pathway p47

c) NRF2 pathway p51

2.1.4. Examples of diseases associated to chronic kidney disease (CKD)

2.1.4.1 Diabetic nephropathy p53

2.1.4.2 Hypertensive nephropathy p54

2.1.4.3 Obstructive nephropathy p55

2.2 Acute kidney injury (AKI)

2.2.1 Acute kidney injury (AKI): Definition and epidemiology

2.2.2 Pathophysiology of acute kidney injury

2.2.2.1 Hemodynamic alterations p57

2.2.2.2 Vascular and tubular injuries p58

a) Glomerular and medullary micro vessels component p58

b) Tubular component p59

2.2.2.3 Inflammation p59

2.2.2.4 Parenchymal post injury repair p60

III. NADPH oxidases (NOXs)

3.1 NOXs family

3.1.1 Structure, topology and function

3.1.2 Post transcriptional and translational modifications

3.1.3 Activation and activity modulation

3.1.4 Tissue expression and subcellular localization

3.2 NADPH oxidase 4 (NOX4)

3.2.1 Structure, topology and function

3.2.2 Post transcriptional and translational modifications

3.2.4 Tissue and subcellular localization

3.2.5 NOX4 kidney expression pattern

3.2.6 Physiology and pathophysiology

IV. Aims of the work

CHAPTER 2 EXPERIMENTAL MODELS OF KIDNEYDISEASES

I. Animal models

1.1 Mice and genetic background

1.2 Model of chronic kidney disease (CKD) and kidney fibrosis:

Unilateral ureteral obstruction (UUO)

1.3 Model of acute kidney injury (AKI) and long term fibrosis

induction: Ischemia-reperfusion (IR)

II. Cellular models

2.1 Mouse cortical collecting duct cells (mCCDcl1) and primary proximal tubule cells from WT and NOX4 KO mice

2.2 Mouse embryonic fibroblasts (MEF) derived from WT and NOX4

KO mice

CHAPTER 3 RESULTS

I. Role of NOX4 in the pathophysiology of chronic kidney disease

1.1 Aim of the study

1.2 Summary of the results

Article Nlandu Khodo S et al.

II. Role of NOX4 in the pathophysiology of acute kidney injury (AKI) and the regulation NRF2-glutathione antioxidant system

1.1 Aim of the study

1.2 Summary of the results

III. The role of distal kidney (collecting duct cells) in the pathogenesis of albuminuria

1.1 Aim of the study

1.2 Summary of the results

Article Dizin et al.

CHAPTER 4 DISCUSSION AND CONCLUSIONS

I. Discussion and perspectives

II. Conclusion

REFERENCES

Francais

Après 4 années de thèse au laboratoire de Néphrologie de l’Université de Genève, j’aimerais remercier Docteur Sophie de Seigneux et Professeur Eric Féraille pour leur soutien tout au long de ce travail.

Je remercie aussi tous mes collègues du laboratoire de Néphrologie qui m’ont accompagné durant cette enrichissante aventure.

Je remercie les membres du jury de ma thèse notamment Professeur Rudolf Wutrich et Professeur Jean-Claude Martinou.

Mes remerciements s’adressent finalement à toute ma famille, mes parents et mes sœurs en particulier.

English

At the end of 4 years of my PhD studies in the laboratory of Nephrology Geneva University, I would like to thank Doctor Sophie de Seigneux and Professor Eric Féraille who welcomed me and gave me the opportunity to carry out my PhD.

I thank all the members of the laboratory of Nephrology who supported me during this wonderful life and professional experience.

I thank also all the members of my thesis jury, especially Professor Rudolf Wutrich and Professor Jean-Claude Martinou.

Finally, I would like to thank all my family, especially my parents and my sisters.

Le rein est un organe fortement vascularisé impliqué dans la régulation de l’équilibre hydro- électrolytique et l’excrétion de déchets. Il assure en plus des fonctions endocriniennes en sécrétant des hormones impliquées dans le métabolisme phospho-calcique, l’érythropoïèse ainsi que dans la régulation de la pression artérielle. Son unité fonctionnelle et structurelle, le néphron, comprend le glomérule, le système tubulaire (tubule contourné proximal, anse de Henle et tubule contourné distal) ainsi que le système collecteur (canaux connecteur et collecteur). Le glomérule filtre l’urine primitive.

Les cellules tubulaires rénales bordent la lumière des tubules rénaux reposant sur une membrane basale. Ces cellules sont spécialisées dans les échanges d’ions et de fluides grâce à l’expression de canaux ioniques (Na⁺, K⁺, Cl^-) et hydriques (aquaporines), de systèmes de cotransport ou d’antiport et d’ATPases.

La maladie rénale chronique est une maladie progressive multifactorielle. Elle est placée en première ligne dans les stratégies de santé publique de l’organisation mondiale de la santé (OMS) du fait de sa prévalence croissante mais aussi du coût élevé de sa prise en charge ainsi que de l'importante mortalité associée. Ce constat peut s’expliquer par l’augmentation des facteurs de risques tels que les maladies cardiovasculaires, le diabète, le vieillissement ainsi que la survenue de maladies rénales aigues au sein de la population. Des études cliniques ont montré une corrélation entre la progression de la maladie rénale chronique et l’apparition de la fibrose interstitielle indépendamment de l’atteinte primaire.

Malheureusement, la physiopathologie de la progression de la fibrose rénale reste énigmatique et peu de traitements existent pour la ralentir ou la corriger.

La fibrose rénale peut être définie comme un processus de réparation mal adaptée à la suite d’une lésion rénale (glomérulaire, vasculaire et tubulo-interstitielle) caractérisé par l’accumulation des protéines matricielles (collagènes, fibronectine, etc.) qui altèrent progressivement l’architecture normale du rein ainsi que sa fonction. Plusieurs facteurs semblent contribuer à l’induction de la fibrose rénale parmi lesquels l’inflammation chronique, le stress oxydatif, l’apoptose, l’hypoxie et la protéinurie occupent une importante place. L’ordre chronologique de ces différents facteurs n’est pas clairement défini et dépendrait du type de lésions primaires. Néanmoins, nombre d’études placent l’inflammation à l’initiation de la maladie rénale chronique qui induirait progressivement différents mécanismes délétères notamment l’apoptose tubulaire. Cependant, l’hypoxie chronique et le stress oxydatif sont également considérés comme des facteurs alimentant irréversiblement la progression de la fibrose rénale.

La famille des NADPH oxydases (NOXs) est constituée de 7 protéines (enzymes) transmembranaires dont la fonction consiste en la production des radicaux libres (ROS). NOX4 ou Renox est l’isoforme majoritaire dans le rein. Elle est fortement exprimée dans les cellules du tube contourné proximal avec

mésangium, l’endothelium glomérulaire et les podocytes. NOX4 est généralement associé au stress oxydatif mais aussi à la modulation de la disponibilité de l’oxygène et la régulation de l’activation des voies sensibles au statut redox tel que les voies HIF, NRF2 et TGβ1. Plusieurs études associent l’activation de NOX4 à la pathogenèse de pathologies chroniques notamment cardiovasculaire, rénale, hépatique, pulmonaire, cérébrale voire des cancers. NOX4 semble associée à la néphropathie diabétique en induisant un stress oxydatif favorable à l’accumulation des protéines matricielles.

Néanmoins plusieurs études ont rapportées son rôle protecteur dans l’insuffisance cardiaque provoquée par une surcharge de pression en induisant l’angiogenèse. Le rôle de la forte expression de NOX4 dans le compartiment tubulaire est inconnu.

Au vu de cette controverse au sujet du rôle de NOX4, ce travail a eu pour but de déterminer d’une part, le rôle de NOX4 dans la pathogénèse de la maladie rénale aigue et chronique dans deux modèles de stress tubulaire, l’obstruction urinaire unilatérale et l’ischémie reperfusion, mimant respectivement la maladie rénale chronique et aigue. D’autre part, nous avons étudié le rôle du néphron distal notamment le tube collecteur dans la pathogenèse de la protéinurie.

Nous avons émis l’hypothèse de départ selon laquelle NOX4 participerait dans la fibrose rénale induite par des lésions tubulaires. Par ailleurs nous émettons l’hypothèse que le tube collecteur jouerait, à l’instar du tube proximal, un rôle actif dans la pathophysiologie de la protéinurie. Pour ce faire, des souris WT et KO NOX4 présentant une invalidation constitutive de NOX4 ont été utilisées en collaboration avec le Professeur K-H Krause de l’Université de Genève. Nous avons aussi utilisé des souris NOX2 KO (Charles River) que nous avons accouplé avec les souris NOX4 KO pour générer les souris NOX4/NOX2 double KO afin de déterminer l’éventuel effet compensatoire de NOX2, la seconde isoforme exprimée dans le rein, sur l’inactivation de NOX4. Des rats protéinuriques ont été utilisés dans l’étude sur la protéinurie. Nous avons utilisé la ligné cellulaire rénale mCCDcl1, des cellules proximales et des fibroblastes embryonnaires (MEF) issus de nos souris WT et KO NOX4 dans l’approche in vitro de ce travail.

Nous avons montré que les souris KO NOX4 ne présentent pas de phénotype morphologique rénal en condition basale. De façon surprenante, les souris KO NOX4 soumises à 7 et 14 jours d’obstruction urinaire présentent une fibrose rénale augmentée comparées aux WT. Pour comprendre ce phénotype, nous nous sommes attelés à analyser les principaux mécanismes impliqués dans la fibrogénèse dont l’apoptose et la raréfaction des capillaires péritubulaires. L’apoptose tubulaire est augmentée chez les souris KO NOX4 comparées aux WT après 7 jours d’obstruction alors que la densité capillaire est diminuée. L’induction de l’apoptose tubulaire chez les souris KO NOX4 est aussi observée dans le modèle d’ischémie reperfusion suggérant une propriété propre de NOX4 dans la survie des cellules

proximales primaires ainsi que les cellules MEF dérivant des souris KO NOX4. L’expression de base du facteur anti-apoptotique Bcl-2 est aussi diminuée dans les conditions d’inhibition de NOX4 in vitro. La raréfaction capillaire est soutenue par une diminution de l’expression de HIF-1α et de VEGF chez les souris KO NOX4 après 7 jours d’obstruction urinaire et aussi in vitro sur des cellules mCCDcl1 traitées avec siNOX4. Nous avons aussi montré que le traitement au siNOX4 n’altère pas la translocation nucléaire de HIF-1α mais plutôt sa stabilisation en condition hypoxique dans les mCCDcl1. Puisque NOX4 est liée au stress oxydatif, nous nous avons examiné le stress oxydatif dans le souris WT et KO NOX4. Le niveau de stress oxydatif mesuré par le marquage de l’ADN oxydé est augmenté après 7 jours d’obstruction urinaire chez les souris KO NOX4 comparées aux WT. Ce résultat n’est pas expliqué par une éventuelle compensation par d’autres NOXs. Les souris KO NOX4/NOX2 présentent quasiment le même phénotype que les KO NOX4. Ceci nous a conduits à examiner le système antioxydant notamment l’expression de NRF2 et de ces gènes cibles.

L’expression protéique de NRF2 et du messager GSTα2 est diminuée de base et après 7 jours d’obstruction urinaire tout comme après l’ischémie reperfusion chez les KO NOX4. La glutathionylation des protéines est aussi diminuée chez les souris KO NOX4. Ce résultat est confirmé in vitro sur les cellules MEF KO NOX4 et mCCDcl1 transfectées avec des siRNA ciblant NOX4 suggérant un déficit des mécanismes antioxydant notamment l’axe NRF2-glutathion dans les conditions d’inhibition de NOX4. Nous avons montré que l’inhibition de NOX4 altère le cycle redox de KEAP1 concordant avec la diminution de base de NRF2 observée. En outre, l’inhibition de NOX4 provoque une diminution du potentiel membranaire mitochondrial corrélée à une diminution de NQO1 et TRX2, deux antioxydants des mitochondries; mais aussi une diminution des protéines chaperonnes du réticulum endoplasmique. La surexpression de NRF2 dans les cellules MEFs corrige l’expression de base de Bcl-2, des facteurs antioxydant et tend à reverser l’apoptose.

Par ailleurs, nous avons démontré une participation autocrine du rein distal, notamment le tube collecteur dans la pathogenèse des conséquences rénales de la protéinurie, en favorisant l’inflammation et la fibrose interstitielle via le rôle du récepteur 24p3 dans l’internalisation de l’albumine.

En conclusion, NOX4 joue un rôle protecteur physiologique dans la cellule tubulaire rénale en régulant les voies de survie telles que la voie HIF/VEGF ainsi que le système antioxydant notamment l’axe NRF2/KEAP1/glutathion. NOX4 semble jouer un rôle dans le maintien de l’homéostasie redox des mitochondries et du réticulum endoplasmique. Le rein distal participe bel et bien dans la pathogenèse de la fibrose induite par la protéinurie.

The kidney is a highly vascular organ involved in the regulation of body fluid and electrolytes homeostasis. It also ensures endocrine function by secreting hormones involved in calcium metabolism, erythropoiesis and in the regulation of blood pressure. Its structural and functional unit, the nephron, includes the glomerulus, the tubular system (proximal tubules, loop of Henle and distal tubules) and the collecting system (connecting tubules and collecting ducts). Renal tubular cells lining the kidney tubules lumen are attached on a basement membrane. These cells are specialized in the transfer of ions and fluid by expressing ion channels (Na+, K+, Cl-), water channels (aquaporins), cotransport and antiport systems and ATPases.

Chronic kidney disease (CKD) is a multifactorial and progressive disease. It is placed in the front line of public health strategies of the World Health Organization (WHO) because of its increasing prevalence in the last decades, but also the high cost of care and high associated mortality. This prevalence can be explained by the increase incidence of risk factors such as cardiovascular diseases, diabetes and aging as well as the occurrence of acute kidney injury in the population. Clinical studies have shown a clear correlation between the progression of chronic kidney disease and the onset of interstitial fibrosis rather than glomerular lesions, regardless of the primary injury. Unfortunately, the pathophysiology of the progression of renal fibrosis remains enigmatic and cannot be treated currently.

Kidney fibrosis can be defined as a maladaptive tissue repair process after injury (glomerular, vascular and tubulo-interstitial). It is characterized by the accumulation of ECM proteins (collagen, fibronectin, etc.). Fibrosis gradually alters the normal architecture of the kidney and its function. Several factors contribute to the induction of kidney fibrosis including chronic inflammation, oxidative stress, apoptosis, hypoxia and proteinuria. The chronological incidence of these factors is not clearly defined and may depend on the type of the primary injury. However, several studies place the inflammation in the initiation of chronic kidney disease inducing progressively various deleterious mechanisms such as tubular apoptosis, whereas chronic hypoxia and oxidative stress are placed at the late stage of renal disease, leading to irreversible kidney fibrosis.

NAPH oxidase (NOXS) family consists of 7 transmembrane proteins (enzymes) whose intrinsic function is the production of reactive oxygen species (ROS). NOX4 or Renox is the predominant isoform in the kidney, highly expressed in proximal tubule cells with an apparent S1-S3 expression gradient observed by in situ hybridization. Besides its high proximal expression, NOX4 is also expressed at lower levels throughout the tubular system, as well as in mesangium, podocytes and glomerular endothelium. NOX4 is generally associated to oxidative stress but also plays a role in the modulation of oxygen sensing as well as regulation of redox sensitive pathways such as HIF, NRF2 and TGβ1 pathways. Several studies linked NOX4 activation to the pathogenesis of various chronic

stress contributing to ECM proteins accumulation. In contrast, NOX4 activation has been reported to mediate protection against chronic load induced stress in the heart by enhancing angiogenesis.

Given this controversy, this work aimed to determine the role of NOX4 in the pathogenesis of acute and chronic kidney disease in two models of tubular stress, unilateral urinary obstruction (UUO) and ischemia reperfusion injury (IRI) both mimicking respectively chronic kidney disease (CKD) and acute kidney disease (AKI). In parallel of NOX4 study, we aimed to determine the involvement of the distal kidney in the pathogenesis of the consequences of proteinuria in CKD.

We hypothesized that NOX4 may participate in the pathogenesis of kidney fibrosis. Furthermore we postulated that the distal kidney may participate in the pathogenesis of albuminuria-induced CKD. WT and NOX4 KO mice displaying constitutive NOX4 invalidation were used in collaboration with Professor KH Krause of the University of Geneva. We also used NOX2 KO mice (Charles River) and generated NOX4/NOX2 KO mice to determine the possible compensatory effect of NOX2, the second isoform expressed in the kidney, on NOX4 inactivation. Proteinuric rats were used for the study of albuminuria. We used the mCCDcl1 kidney cell line, primary proximal cells and embryonic fibroblasts (MEF) derived from NOX4 KO mice for the in vitro approach of this work.

In this work, we have demonstrated that kidneys of NOX4 KO mice did display basal morphologic phenotype in the kidney. NOX4 KO mice subjected to 7 and 14 days UUO showed increased kidney fibrosis as compared to WT. To understand this surprising phenotype we set out to analyze the main mechanisms involved in fibrogenesis including apoptosis and peritubular capillary rarefaction.

Tubular apoptosis was increased in NOX4 KO mice compared to WT after 7 days of UUO while the capillary density was decreased. Induction of apoptosis in NOX4 KO mice was also observed in the model of IRI suggesting an intrinsic NOX4 property in tubular cell survival under stress conditions.

These observations were confirmed in vitro on mCCDcl1 cells transfected with siRNA targeting NOX4 (siNOX4), primary kidney and MEF cells derived from NOX4 KO mice. The basal protein expression of Bcl-2 anti-apoptotic factor is also down-regulated under NOX4 inactivation or down-regulation in vitro. Capillary rarefaction phenotype was strengthened by a decrease in expression of HIF-1α and VEGF in NOX4 KO mice after 7 days of UUO and also in vitro in mCCDcl1 cells treated with siNOX4. We also showed NOX4 down-regulation did not alter the nuclear translocation of HIF-1α but its stabilization under hypoxia. Since NOX4 is linked to oxidative stress, we decided to examine oxidative stress in WT and KO mouse NOX4. The level of oxidative stress measured by the staining of oxidized DNA was increased in NOX4 KO mice compared to WT after 7 days of UUO. This result was not explained by compensation by other NOXs. NOX4/NOX2 KO mice exhibited almost the same phenotype than NOX4 KO. This result led us to examine the antioxidant system, especially the

was also basally decreased in NOX4 KO mice. This result was confirmed in vitro in MEF KO NOX4 and mCCDcl1 cells treated with siNOX4 suggesting a defective antioxidant defense, especially the NRF2-glutathione axis under NOX4 inhibition. NOX4 inactivation also altered the redox cycle of KEAP1, the NRF2 cytosolic inhibitor, in line with the basal decrease of NRF2. Furthermore, inhibition of NOX4 caused a decrease of the mitochondrial membrane potential correlating with decreased NQO1 and TRX2, two mitochondrial ROS scavengers; but also a decrease in the endoplasmic reticulum (ER) chaperones expression. Overexpression of NRF2 in MEFs corrects the basal expression of Bcl-2 and the basal defect of the antioxidant system tending to decrease apoptosis.

In parallel to NOX4 study, we have demonstrated an autocrine participation of the distal kidney, especially the collecting duct in the pathogenesis of proteinuria induced CKD, by promoting inflammation and interstitial fibrosis involving 24p3R mediating albumin internalization.

In conclusion, NOX4 plays a protective role in the physiology of a tubular cell by regulating cell survival pathways such as HIF/VEGF pathway and the antioxidant system including NRF2/KEAP1/glutathione axis. NOX4 appears to play a role in the maintenance of mitochondria/ER redox homeostasis. The distal kidney participate in an autocrine manner in the pathogenesis of albuminuria induced CKD.

CHAPTER 1 INTRODUCTION

I. The kidney

1.1 Organization of the urinary system

The urinary system is composed of a pair of kidneys and ureters, the urinary bladder and the urethra.

The kidneys remove toxic compounds from the blood and produce urine which is collected in the renal pelvis and conducted by peristalsis through the pair of ureters to a muscular and distensible tank, the bladder. The urine is then excreted through a tube connecting the bladder to the genital organs, the urethra. The bladder receives both autonomic (sympathetic and parasympathetic) and somatic (voluntary) innervation which is involved in the storage as well as the release of urine^1,2.

Figure 1: Representative Scheme of the urinary system (from Kidney and Urology foundation of

AMERICA, INC.)

The urinary system is composed of the pairs of kidney and ureter, the bladder where the urine is stored and the urethra which allows the external excretion of the urine. The release of urine by the bladder during the micturition involves essentially the somatic nerve system control of the sphincters in adults.

1.2 General anatomy and function

The kidneys are paired, bean-shaped organs dwelling in the rear of the abdominal cavity, on each side of the vertebral column at the level of the 12^th thoracic vertebra to the 3^rd lumbar vertebra. The kidney is covered by a fibrous, non-elastic capsule and shows a convex lateral surface and a concave medial surface³.

In human adult, each kidney measures in average 13 cm of length, 6 cm of largeness and 4 cm of thickness; and weighs 125 to 170 g and 115 to 155g in men and women respectively. Kidney size is dependent on body height⁴.

The kidney can be divided in three parts, the cortical region, the medullar region and the renal sinus which connects the kidney to the ureter.

In a kidney section, the cortex is the granular outer layer surrounding the medullar region. It is composed of microscopic clusters of capillaries and highly convoluted epithelial tubules.

The medulla is the darker inner region of the kidney which can be subdivided into outer and inner medulla. It is composed of parallel tubules organized in a conical pyramid like structure, the renal pyramids, whose bases delimitate the cortico-medullary border. The renal pyramids converge to the renal papillae where the medulla connects to the renal sinus. The outer medulla presents two stripes, outer and inner stripes (W F Boron, Medical Physiology).

At the middle of the medial concave side of the kidney is found a snip, the hilus through where renal artery, vein, sympathetic nerves (efferent and afferent fibers), lymphatic and the ureter enter or exit the kidney.

The hilus opens into a hollow chamber, the renal sinus which constitutes the urine filled space of the kidney. This renal sinus begins from the upper end of the ureter including the renal pelvis and their internal extensions, the major and minor calyces respectively. The minor calyces are opened to the duct of Bellini located at the tip of the renal papillae allowing the urine arrival to the renal sinus.

Figure 2: The kidney and vascular network (from iBiology)

From the external convex side, the kidney is constituted of capsule, the cortical part including the arcuate veins and arteries, the medullary part including the renal pyramids surrounded by interlobular veins and arteries, the renal sinus which is composed of the minor and the major calyxes, and the renal pelvis, finally the renal vein and artery, and the ureter

The kidney is a highly vascularized organ receiving 20% of cardiac output^5-8. The renal vascular system comprises a renal artery and vein, respectively routes of entry and exit of blood to the kidney.

The renal artery, providing blood to the kidney, enters at the hilus and gives rise to the interlobar and the arcuate arteries. The arcuate arteries branch to ascending and descending interlobular arteries. The ascending interlobular arteries give rise to afferent arterioles which initiate the glomerular capillary networks (glomeruli). The glomerular capillary networks converge into one efferent arteriole originating further the peritubular capillary networks which supply oxygen and nutrients to the renal cortex. The descending interlobular arteries form the juxtamedullary glomerular capillary networks (juxtamedullary glomeruli) whose efferent arterioles sink into the medulla where they originate a hairpin like capillary networks involved in the urine concentration, the vasa recta.

The renal blood is drained out of the kidney by the renal vein of which branches of the same names (interlobar, arcuate and interlobular) follow the circuit of the renal artery.

The renal lymphatics drain the cortical interstitium and plays major role in protein clearance. They are absent in the medulla where concentrating extracellular fluid (ECF) is necessary for the concentration and efficient production of urine.

Figure 3: Representative scheme of the anatomy of the vasculature of the kidney (from cardiovascular

physiology, inkling)

The vascular network of the kidney originates from the renal artery which gives rise in the renal medulla to the interlobar arteries and to arcuate arteries, the latter delimitating the renal medulla and cortex and giving rise to the ascending and descending interlobular arteries in the renal cortex. The ascending interlobular arteries originate the afferent arterioles of the glomeruli which converge to efferent arterioles involved in the peritubular capillary network. The descending interlobular arteries

originate the afferent arterioles of the juxtamedullary glomeruli whose efferent arterioles enter the renal medulla and originate the vasa recta. The renal vein follows the same circuit than the renal artery, and allows blood to exit from the kidney.

The kidneys receive innervation from the renal plexus whose fibers, following the renal arteries circuit, include efferent sympathetic and sensory afferent nerves. The renal sympathetic system descending from the central nervous system (medulla oblongata, pons, hypothalamus) reaches the spinal cord T11-L2 (preganglionic cholinergic/nitrergic) and the ganglion coeliacum respectively before entering the kidney (postganglionic adrenergic)⁹. The renal afferent sensory system ascending from the kidney passes upon the dorsal root ganglion (T11-L2) and the spinal cord (T11-L2) before arriving to the central nervous system. The sympathetic fibers are distributed to all segment of renal vasculature, and at lesser extend to the renal epithelial cells while the sensory fibers lie in the pelvic region, the major vessels and the cortico-medullary connective tissue. The renal sympathetic innervation plays a major role in the control of renal blood flow, glomerular filtration as well as sodium (Na⁺) and water balance^10-13.

Each kidney comprises millions of nephrons (800000 to 1, 2 million) which represent their functional and structural units, composed of vascular and mesenchymo-epithelial structures; the glomerulus and the tubular system^14-21.

The kidneys ensure both exocrine and endocrine functions²². They filter the blood to eliminate toxic compounds, ensure homeostatic functions such as the maintenance of body fluid and electrolytes homeostasis, acid-base balance and regulation of blood pressure by monitoring water and sodium balance. Regarding endocrine function, the kidneys produce or participate to the activation of hormones involved in Ca²⁺ metabolism through vitamin D hydroxylation, in erythropoiesis or red blood cell generation through erythropoietin production, and to the regulation of blood pressure by producing renin^23-25.

1.3 Nephron

The nephron, the functional and structural unit of the kidney, consists in a glomerulus and the attached tubular system (W F Boron, Medical physiology).

Figure 4: Representative scheme of the Nephron (modified from Joseph V. Bonventre and Li Yang,

2011)

The nephron, surrounded by the renal interstitium and the vascular network, is composed of a vascular part, the glomerulus occluded in the Bownman capsule, and a tubular system including the proximal tubule, the loop of Henle (the thin descending limb, the tin ascending limb and the thick ascending limb), the distal tubule, the connecting tubule and the collecting duct.

1.3.1 Glomerulus

The glomerulus, the filtrating part of the nephron, is a cluster of specialized blood vessels delimitated by two high resistance arterioles (the afferent and efferent arterioles) and occluded into the Bowman’s capsule. The glomerular capillary endothelial cells (glomerular endothelium) possesses a high permeability, lays directly on a basement membrane (glomerular basement membrane) to form a fenestrated layer and constitute a high surface of filtration^26,27.

Figure5: The glomerulus (s973.photobucket.com/user/ald5044/media/anatomy/glomerulus.jpg.html)

The glomerulus is a cluster of blood vessels delimitated by the afferent and efferent arterioles. The vessel cluster is composed of fenestrated endothelium surrounded by podocytes whose interdigitations delimitate the split pores (visceral layer). At the beginning of the vessel cluster, the afferent arteriole is composed of juxtaglomerular cells (JG cells) which, in contact with the tubular system at the level of the macula densa, forms the juxtaglomerular apparatus (JGA).The vessel cluster is occluded into a monolayer capsule (parietal layer) originating the tubular system (PCT).

The glomerulus allows filtration of blood by the capillaries. The ultrafiltrate passes into the Bowman’s space which is a transition space circumscribed by the glomerulus (visceral layer) and the Bowman’s capsule (parietal layer). There are two types of glomeruli: the superficial glomeruli and the juxtamedullary glomeruli.

The superficial glomeruli are the most abundant (85%) in the renal cortex; their efferent arterioles originate the peritubular capillary networks which are involved in tubular system nutrients and oxygen supply, as well as in fluid retrieval. The juxtamedullary glomeruli, representing only 15% of all glomeruli, are located at the renal cortico-medullary boundary. Their efferent arterioles extend into the renal medulla and give birth to the vasa recta, which surround the medullary segments of the nephron and play a crucial role in the urine concentration process^28-33.

The glomerular capillaries present a specific structural organization forming the glomerular filtration barrier (GFB) which includes the glycocalyx, an anionic glycosaminoglycan, lining the lumen of endothelial cells; a fenestrated endothelial layer (70 nm holes), the glomerular basement membrane (GBM) and foot processing modified epithelial cells, the podocytes. The glomerular filtration barrier carrying a net negative charge, it exerts a coulombic restriction for anions^34-43.

The podocyte’s foot interdigitations on the basement membrane of the glomerular capillaries form the filtration slits. These slits which are connected by a slit diaphragm covered by negative charged glycoproteins (Nephrin, neph1, podocin for example) presenting pores of 4 to 14 nm size. Slits invigorate the glomerular filtration barrier, restricting its crossing by cellular element of the blood (erythrocytes) and anionic macromolecules (proteins), but staying permeable to water and small molecules (ions, small proteins…)^44,45.

1.3.2 Tubular system

The glomerular filtrate formed in the renal corpuscle reaches the tubular system via the Bowman’s capsule. From the Bowman’s space, the glomerular filtrate will travel along the sinuosity of the tubular system to become the final urine. During nephrogenesis, the metanephros, gives rise to the

ureteric bud whose interactions with loose mesenchyme leads to the branching of the ureteric bud and further to the formation of the collecting tubules; while the condensation and the differentiation of the mesenchyme after these interactions generates the nephron segments comprised between Bowman’s capsule and connecting tubules^46-50.

The tubular system includes the proximal convoluted tubule (PCT), the proximal straight tubule (PST), the thin descending and ascending limb of Henle’s loop (tDLH and tALH), the thick ascending limb of Henle’s loop (TAL), the distal convoluted tubule (DCT), the connecting tubule (CNT), the initial collecting tubules (ICT), the cortical collecting tubules (CCT), the outer medullary collecting duct (OMCD) and the inner medullary collecting duct (IMCD). Each segment is composed of specialized epithelial cells to meet its specific transport activities^51-55.

The proximal tubule is the first segment of the nephron which drains the Bowman’s chamber where high transport activities occur (2/3 of filtered water, 70% of Na⁺ reabsorption, NaHCO3

-, glucose, amino acids reabsorption and NH3

+ secretion)^56,57. This low resistance epithelium is constituted of cells with plasma membrane amplifications necessary to meet its high-level transport function: the apical brush border structure with a primary cilium and the interdigitations of the basolateral and lateral membranes. The cells are characterized by the presence of large mitochondria, and well developed endoplasmic reticulum and Golgi apparatus for ATP production and protein synthesis respectively.

The proximal tubule epithelial cells participate in the control of Ca²⁺ and phosphorus metabolism by converting the circulating 25 hydroxyvitamin D to the active 1, 25 dihydroxyvitamin D^58-69.

The proximal tubule is divided into 3 segments (S1, S2 and S3) whose structural complexity decreases from the S1 to the S3 segment. The S1 corresponds to the first half of the proximal convoluted tubules (PCT) dwelling exclusively in the renal cortex; the S2 segment includes the second half of the proximal convoluted tubule and the first half of the proximal straight tubule (PST) while the S3 is the resting half of the proximal straight tubule that lies down the medulla.

The S3 segment of the proximal tubule is followed by the loop of Henle which includes the thin descending, the thin ascending and the thick ascending limbs of Henle’s loop (tDLH, tALH, TAL).

The thin descending and ascending limbs of Henle’s loop (tDLH, tALH) are composed of less specialized, flattened epithelial cells while the thick ascending limb of Henle’s loop (TAL), following the thin ascending limb of Henle’s loop, is constituted of specialized epithelial cells showing tall membrane interdigitations and abundant mitochondria necessary for high active NaCl transport rate.

This last segment of the loop of Henle ends into the macula densa belonging to the juxtaglomerular apparatus (JGA), where it comes in close contact with the glomerular afferent arteriola. The TAL pumps NaCl into the medullary interstitium but is impermeable to water, thereby generating a medullary hyperosmotic gradient necessary for the urine concentration process^70,71.

The distal convoluted tubule (DCT) begins from the macula densa and ends at the connecting tubule.

It is composed of similar same cell type than the thick ascending limb of Henle’s loop, except for the expression of specific transporters (TAL).

The connecting tubule (CNT) is constituted of two cell types, a cuboid epithelial cell and the intercalated cells (α and β), which are specialized epithelial cells that does not display apical primary cilium.

The initial collecting tubule (ICT) is the last nephron segment allowing the connection of a single nephron to another collecting tubule or collecting duct of one or several nephrons respectively.

The collecting tubule is composed of two cell types, the principal cells and the intercalated cells representing one thirds of cells lining this segment. The principal cell, displays modest interdigitations of the basolateral membrane, less abundant mitochondria and an apical primary cilium. These cells reabsorb Na⁺ and secrete K⁺ whereas intercalated cells are specialized in acid base and Cl^- transport.

The medullary collecting duct (OMCD and IMCD) which extends into the medulla until the renal papillae, is constituted of tall epithelial cells with increasing height from the outer medulla to the duct of Bellini (W F Boron, Medical physiology).

The distal nephron (Distal and collecting tubules) is the point of tight hormonal regulation (Arginine Vasopressin and Aldosterone) of NaCl and water excretion^72-74. This mechanism is essential for urine

concentration allowing water reabsorption from the distal convoluted tubules (DCT) and the collecting ducts (CD) under the influence of the Arginine Vasopressin.

This water recovery is due to an active transport of NaCl (Na⁺/K⁺/2Cl^-) from the thick ascending limb (mTAL) to the interstitium creating a medullary osmotic gradient. The resulting hyperosmotic blood which moves to the opposite direction to the urine is further diluted before reaching the general circulation with water coming from the water permeable thin descending limb (tDLH)^75-83.

Figure 6: The tubular system (image from www.studyblue.com/notes/n/kidneys/deck/6707757)

The tubular system is the site of transport activities (active and passive transports) in order to maintain the body fluid and electrolytes homeostasis, and to generate urine. It is composed of the proximal tubule (1) which drains the Bowman space; the descending limb of loop of Henle (2), the thin and thick segments of ascending limb (3), the distal tubule (4) and the collecting duct.

1.3.3 Juxtaglomerular apparatus (JGA)

The juxtaglomerular apparatus (JGA) is a part of a nephron where the glomerulus contacts the tubular system. It includes the juxtaglomerular cells of the afferent arteriole, the extra glomerular mesangial cells, the renin secreting granular cells and the cells of the macula densa where the glomerulus contacts its thick ascending limb of Henle’s loop (TAL).

The juxtaglomerular apparatus is innervated by postganglionic sympathetic axons and plays two major regulatory roles: The control of glomerular filtration rate by sensing luminal [Na⁺] and [Cl^-] at the level of the macula densa (tubuloglomerular feedback) and the control of renin secretion by granular cells of afferent arterioles baroceptors that sense transmural pressure. Any increase in luminal [Na⁺] and [Cl^-] induces a constriction of the afferent arteriole and a decrease in GFR. Any decrease in arteriole transmural pressure induces renin secretion and activation of the renin-angiotensin- aldosterone system (W F Boron, Medical physiology).

Figure 7: The juxtaglomerular apparatus (From Yao J Am J Physiol Renal Physiol, 2008)

The juxtaglomerular apparatus includes the juxtaglomerular cells of the afferent arteriole, the extra glomerular mesangial cells, the granular renin-secreting cells and the cells of the macula densa at the

junction between the thick ascending limb of the loop of Henle and the distal tubule, where the afferent arteriole contacts its tubular system.

1.3.4 Vascularization of the nephron

The nephron displays a sophisticated vascular system constituted of two vascular beds, the glomerulus and the peritubular capillary network, both separated by two high resistance arterioles, the afferent and the efferent arterioles as mentioned above.

The afferent arteriole drains arcuate arteriole and supplies blood to the glomerulus where it is filtered before reaching the efferent arteriole, which supplies blood to the peritubular capillary network or the vasa recta.

Under normal conditions (steady state), the high pressure of the glomerular capillaries bed allows blood flow into the Bowman’s space while the low pressure of the peritubular capillaries favor fluid retrieval according to the Starling equations (the difference between the hydrostatic pressure and the oncotic pressure due the presence of proteins in the plasma) (W F Boron, Medical physiology).

1.3.5 Innervation of the nephron

The sympathetic and sensory fibers are distributed to different segments of the nephron. The sympathetic adrenergic fibers are distributed to all segment of renal vasculature and at lesser extend to the tubular system while the sensory peptidergic fibers lie essentially in the pelvic region, the major vessels and the corticomedullary connective tissue^84,85. The renal sympathetic innervation releases catecholamines (norepinephrine and dopamine) that play a major role in the control of renal blood flow, glomerular filtration rate and tubule reabsorption by triggering vasoconstriction, renin secretion (activation of renin-angiotensin-aldosterone axis) and Na⁺ reabsorption^86-93.

1.4 Glomerular filtration rate (GFR) and renal blood flow (RBF)

1.4.1 Glomerular filtration rate (GFR)

Renal filtration depends of several factors including the intrinsic permeability (structural integrity) of the glomerular filtration barrier (GFB), the total surface area of glomerular capillaries, the rate at which blood flows into the glomerular capillaries (Renal blood flow) as well as the net Starling driving force favoring ultrafiltration (the difference between the capillary hydrostatic and oncotic pressures and those of the Bowman’s spaces). This process is under tight nervous and hormonal control. The structural properties of the glomerular filtration barrier as well as the high net hydrostatic pressure of the glomerular capillaries network allows water and small molecule (ions, glucose, amino-acids...) to flow into the Bowman’s space but retain macromolecules and blood cells^94,95.

The glomerular filtration rate (GFR) of the two kidneys is on average 125 mL/min or 125mL/min/1.73m² reported to the body surface area of an average 70 Kg weight man. This value far exceeds analogous movement of fluid across walls of most systemic capillaries due to the higher intrinsic permeability, high net filtration driving force and total surface of filtration; and can vary with sex, weight and age (very low in newborn, normalized to the body surface area from the age of 2 years and gradually falls down with age).

Considering an ideal solute freely filtered from the plasma to the Bowman’s space (filtration fraction (FF) is 1) that is neither reabsorbed nor secreted nor metabolized through the tubular system, its final amount in the urine will equal its amount in the glomerular filtrate. The glomerular filtration rate corresponds to the virtual volume of plasma that would be totally cleared of this solute during a given time. Inulin, a polymer of fructose, displays these properties and therefore allows measurement of GFR. Nevertheless, inulin measurement is unsuitable for clinical practice due the heaviness of the protocol of execution that requires several hours of intraveinous infusion. To estimate GFR in clinical practice, physicians rely on the clearance of endogenous creatinine, a metabolite of creatine phosphate metabolism that is produced at fairly constant rate by skeletal muscles. In normal human adult, under

normal physiological conditions (steady state), the endogenous plasma concentration of creatinine is on average 1 mg/dL but depends on age and sex (W F Boron, Medical physiology).

In clinical practice, several formulas based solely on serum creatinine have been derived and validated from large population studies to evaluate GFR: the Cockcroft Gault equation, and more recently the MDRD and CKD-epi equations^96,97. The estimation of glomerular filtration rate (GFR) is an important tool used in clinic to estimate the renal function; therefore kidney damage during chronic kidney diseases (CKD).

1.4.2 Renal blood flow (RBF)

Under normal conditions, the volume of blood deliver to the kidney per unit of time or renal blood flow (RBF) is 1L/min; representing 20% of cardiac output (5L/min). This blood supply is heterogeneously distributed in the kidney, since 90% is directed to the renal cortex while only 10% is distributed to the renal medulla^98-100. This low medullary blood flow is important for lowering the wash out of the hypertonic medullary interstitium that is necessary for urine concentration. However, this low medullary blood flow imposes a relative medullary hypoxia as compared to the renal cortex¹⁰¹.

The renal blood flow (RBF) as well as the glomerular filtration rate (GFR) are regulated by several mechanisms: the kidney auto regulatory mechanism as well as nervous and hormonal systems^102-109. Autoregulation of the renal blood flow includes the myogenic response of the smooth muscle of the afferent arterioles through its ability to sense and adapt the vessel circumference by contracting or relaxing and the tubuloglomerular feedback (TGF) mediated by the juxtaglomerular apparatus (JGA) which sense (macula densa) the increases in luminal [Na⁺] or [Cl^-] and generates a signal resulting in changes of contraction level of the afferent artery. The sympathetic nervous system controls the renal blood flow by responding to a decrease in the effective circulating volume. Release of norepinephrine into the renal interstitium leads to increased afferent and efferent arterioles resistance, consequently generating a decrease of renal blow flow (RBF) as well as a decrease of glomerular filtrate rate (GFR).

Among factors regulating renal blood flow (RBF), the renin-angiotensin-aldosterone (RAA) system, the Arginine Vasopressin and the Atrial Natriuretic peptide (ANP) are the most important. The overall effect of the first two is to reduce renal blood flow (RBF) while the latest increases it^110-113.

Many other vasoactive agents with unclear renal physiological role are known to affect the renal blood flow (RBF) such other catecholamines (epinephrine, dopamine), endothelins, prostaglandins, leukotrienes, nitric oxide (NO).

1.5 Kidney oxygen metabolism

The kidneys receive a quarter of cardiac output amounting to 350 mL/min for each 100 g of tissue;

nevertheless the kidneys are inefficient in term of oxygen utilization, extracting relatively few oxygen (10%) in comparison to other organs with high metabolic activity such as the brain (34%) and the heart (65%)¹¹⁴.

One explanation for this discrepancy is the heterogeneous distribution of the kidney vascularization in the renal cortex and medulla. Indeed the bulk of renal blood flow (90 %) is directed to the renal cortex resulting in a relatively low oxygen tension in the medulla, around 10 to 20 mmHg (relative hypoxia), against approximately 30 to 50 mmHg in the renal cortex^115,116.

Figure 8: Anatomical and physiological features of the renal cortex and medulla (Mayer Brezis and

Seymour Rosen, 1995)

The kidney displays a cortico-medullary decreasing gradient of vascularization such as the cortical blood flow (4.2 ml/min/g) and the oxygen delivery (50 mm Hg pO2) is at least two times higher than the medullary ones (1.9 ml/min/g and 10-20 mm Hg respectively)

This relative medullary hypoxia is strengthened by the medullary countercurrent multiplier mechanism which requires energy consumption. The inefficiency in oxygen consumption is also due to the existence of an arterial-to-venous diffusional oxygen shunt (AV oxygen shunt) which allows direct oxygen diffusion from the renal artery to the renal vein bypassing the renal circulation. This unequal distribution of blood flow as well as oxygen supply in the kidney suggests a distinctive oxygen metabolism in different kidney regions; thereby a different susceptibility to hypoxia according to the cellular oxygen demands in each region^117-120.

Figure 9: Schematic representation of the arteriole-to- veinous oxygen shunt (modified from Imari mimura and Masaomi Nangaku, 2010)

The existence of an arteriole-to-veinous (AV) oxygen shunt enables oxygen to move directly from the artery to the vein strengthening the medullary relative hypoxia

The proximal tubular cells which are the most abundant in the renal cortex, display a high oxygen metabolism dependency to meet their high energy requirement for the transport activities (Na⁺ reabsorption). These cells, notably those from the S3 segment, exhibit a less efficient antioxidant defense system and are very sensitive to ischemic injuries in contrast to cells of the medullary thick ascending limb of Henle’s loop (mTAL) and collecting duct which can override hypoxia by using glycolysis¹²¹.

Under hypoxia, the site of renal injury depends of various factors, including the local oxygen tension, cell metabolic rate (energy requirements), and the intrinsic or adaptive cell resistance to hypoxia. This striking heterogeneous oxygen metabolism characterizing the kidney has important implications for renal pathophysiology and diseases.

II. Kidney diseases

2.1 Chronic kidney disease (CKD)

2.1.1 Chronic kidney disease (CKD): Definition and epidemiology

Chronic diseases are among the leading causes of death worldwide. It is an emerging disease that occupies an important place in world health care because of its increasing prevalence in the population and the cost of therapies and associated morbi-mortality^122,123.

Several professionals meetings have been organized and ruled to define chronic kidney disease as kidney damage (defines by structure alterations, proteinuria, hematuria) and/ or a glomerular filtration rate (GFR) < 90 ml/min per 1.73 m² for ≥ 3 months, irrespective of initial causes^124-128. GFR is usually estimated via one of the creatinine based formulas (CG, MDRD, and CKD-epi)^129,130. The prevalence of CKD in Switzerland using these definitions is approximately 10%^131-134.

Independently of the initial causes of CKD, kidney fibrosis remains the hallmark of the end stage of its progression and is associated to the loss of kidney function. In Switzerland, CKD constitutes a serious health problem as a corollary of the increase of predisposing conditions such as cardiovascular diseases, diabetes and aging (Swiss Society of Nephrology, 2008)¹³⁵.

CKD leads to several clinical complications^136-138. The first one is obviously the loss of renal function leading to a need for renal replacement therapy at late stages of the disease, such as dialysis or transplantation. Moreover, CKD is also associated to cardiovascular mortality and infectious complications, leading to high mortality even before reaching end stage renal disease^139-142.

2.1.2 Pathophysiology of chronic kidney disease (CKD)

Chronic kidney disease is a progressive and irreversible situation characterized by kidney structural and functional damage resulting from diverse causes of which diabetes and hypertension take a leading place^143-146. However, infectious glomerulonephritis, ureteral obstruction, autoimmune diseases, renal vascular disease as well as genetic diseases are common causes involved in CKD pathogenesis.

Kidney structural damage occurs in the entire nephron’s segments, including the glomerulus (glomerulopathy), the vessels (vascular disease) and the tubular system (tubular diseases) as well as the interstitial compartment (renal interstitial diseases) converging irreversibly, by various mechanisms, to kidney tubulo-interstitial fibrosis and glomerulosclerosis. From previous works, it appears that tubulo-intertitial fibrosis is a better predictor of GFR and GFR decline than glomerulosclerosis^147-149.

Several mechanisms meddle in the pathophysiology CKD independently of the initial glomerular or tubular injury. One of the striking events during CKD progression is the destruction of renal parenchyma (decrease of number of nephrons as well as the coefficient of glomerular ultrafiltration, Kf) which is gradually replaced by extracellular matrix (ECM) through a fibrotic process. To overcome this situation, the remaining nephrons increased their filtration rate to respond to the normal excretory capacity of the kidney. This response includes cellular hypertrophy, increased blood flow due to afferent and efferent vasodilatation as a mechanism increasing the glomerular filtration rate (GFR). Unfortunately, this adaptive and compensatory response increases intraglomerular pressure (glomerular hypertension) inducing glomerular distension and stretch as well as the injury of the glomerular filtration barrier (GFB); thereby accelerates the destruction of the remaining nephrons.

The resulting glomerular hypertension may lead to glomerulosclerosis accompanied by GFR reduction and proteinuria, leading finally to interstitial fibrosis which culminates to renal failure. The mechanisms are discussed in the part focused on the pathogenesis of fibrosis.

Macrophages (monocytes) as well as leucocytes infiltration also contribute to renal tissue scaring and ECM deposition (fibronectin, collagens I and IV, laminin, heparans, entactin…) process by multiple cell types (Fibroblast or myofibroblasts...) through a controverted epithelio-mesenchymal transition (EMT)^150-152.

2.1.3 Kidney fibrosis

2.1.3.1 Definition

Kidney fibrosis results from accumulation of extracellular matrix (ECM) proteins as a consequence of several mechanisms leading to the loss of the normal structure of the kidney (loss of renal parenchyma) as well as of its function. It is considered as a failed or maladaptive wound-healing and repair process of the kidney tissue after chronic injury.

Regardless of the underlying primary renal disease, tubulointerstitial fibrosis has been reported to better correlate to GFR decline than glomerulosclerosis and is associated with tubular atrophy, glomerulosclerosis, interstitial inflammation and loss of peritubular capillaries^153-156.

2.1.3.2 Pathophysiology of kidney fibrosis

The pathophysiology of kidney fibrosis as the hallmark of CKD involves several interplaying mechanisms such as chronic interstitial inflammation, oxidative stress, apoptosis, chronic hypoxia and cell reprograming processes^157-161. Given the complexity of the pathogenesis of kidney fibrosis, the chronology of different events involved in its progression remains unclear. Nevertheless, inflammation and oxidative stress seem to initiate the maladaptive response leading to kidney fibrosis. Chronic inflammation and associated oxidative stress have been reported to promote fibrogenesis by triggering epithelial cells apoptosis and extracellular matrix (ECM) proteins deposition such as collagens (I, IV) and fibronectin. Oxidative stress which is associated to several pathogenic conditions favors organ fibrosis by activating specific profibrotic and proinflammatory pathways such as TGF-β1 pathway, a major pathway mediating kidney fibrosis^162-169.

Chronic interstitial hypoxia is considered as the final step in the pathogenesis of kidney fibrosis which fuels inflammation, oxidative stress and cell apoptosis121,170,171

.

Figure 10: Schematic mechanism of kidney fibrogenesis

Kidney fibrosis results from the accumulation of extracellular matrix (ECM) proteins in the kidney interstitium as a maladaptive response to various renal injuries, involving several mechanisms such as chronic inflammation, oxidative stress, apoptosis and chronic hypoxia. Accumulation of ECM proteins leads to the disruption of the normal kidney architecture and the loss of the renal parenchyma or function.

2.1.3.2.1 Chronic inflammation

Inflammation is a mechanism by which the vasculature or tissues respond to injurious stimuli that could be a pathogen (bacteria, viruses…), damaged cells (cancer cells) and irritants (physical, chemical and biological), in acute or chronic manner.

Acute inflammation is the early response of the organism involving innate immunity which consists to the mobilization and recruitment of immune cells (neutrophils, monocytes/macrophages, NK cells and DCs) to the site of injury, and to the vascular response (vasodilatation, increased vascular permeability and blood flow) in order stop and repair the tissue. The prolongation of this protective process or chronic inflammation usually leads to a maladaptive immune response, involving both the innate and specialized immunity (antigen recognition). DCs maturation links the innate immunity to the adaptive one, by activating lymphocytes (T and B cells) through antigen presentation, leading to T and B cells interactions and proliferation. Chronic inflammation actively contributes to several pathologic states, including CKD¹⁷².

Figure 11: Innate and adaptive response during inflammation

Inflammation is natural response of the organism to external or pathogenic factors leading to nonspecific response (innate immunity) which involves dendritic cells (DCs) mediating macrophages and natural killer cells (NK Cells) activation. Maturation of DCs leads to an adaptive immune response through DCs antigen presentation to T and B cells

Independent of the nature of the initial kidney injury (metabolic, diabetic, hemodynamic, ischemic or toxic), the inflammatory response is the earliest mechanism activated in the kidney in order to participate to repair process and parenchymal cell regeneration. However, even though inflammation presents beneficial effects in term of tissue repair and cell regeneration, it may be detrimental when the repair process is disrupted, notably during prolonged injury, leading to uncontrolled extracellular matrix (ECM) proteins deposition and the subsequent destruction of the kidney architecture as well as function.

Increasing evidences place chronic inflammation at the center of the pathogenesis of CDK progression. Inflammation chronicity is maintained by several mediators, such as hypoalbuminemia, atherosclerosis, protein oxidation, advanced glycation end products (AGEs), the thiobarbituric acid system, leptin, iron, cytokines and others. This inflammatory state is worsened by autocrine and paracrine involvement of epithelial cells which bring an active participation by mediating inflammation through cytokines and chemokines release inducing recruitment of immune cells (macrophages, lymphocytes…); and the expression of adhesion molecules (CAMs), Tumor necrosis factor receptor (TNFR) as well as Toll like receptors (TLRs 2/4) and T cell co stimulatory (CD28) molecules which activate immune cells and amplify the inflammatory response^162,172.

This inflammatory vicious circle fuels epithelial injury and participates to the destruction of the renal parenchyma as well as resulting kidney fibrosis. Cytokines and chemokines play a determinant role in the fibrotic process by mediating myofibroblast activation and recruitment leading to ECM deposition^172-179.

Kidney fibrosis which marks the progression of CKD is very often preceded and closely associated with chronic interstitial inflammation in response to a prolonged kidney injury. However, the pathogenic role of chronic inflammation in kidney fibrosis is complex and involves glomerular and tubulo-interstitial infiltration by inflammatory cells (neutrophils, macrophages, leukocytes, dendritic cells) and the active immune response of kidney cells (epithelial cells, podocytes and mesengial cells).

Neutrophils are the first cells recruited, as they uptake cell debris and phagocyte apoptotic bodies.