Renal epithelial injury and fibrosis

    Renal epithelial injury and fibrosis Brigitte Kaissling, Michel LeHir, Wilhelm Kriz PII: DOI: Reference:

S0925-4439(13)00055-0 doi: 10.1016/j.bbadis.2013.02.010 BBADIS 63643

To appear in:

BBA - Molecular Basis of Disease

Received date: Revised date: Accepted date:

26 November 2012 7 February 2013 9 February 2013

Please cite this article as: Brigitte Kaissling, Michel LeHir, Wilhelm Kriz, Renal epithelial injury and fibrosis, BBA - Molecular Basis of Disease (2013), doi: 10.1016/j.bbadis.2013.02.010

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Renal epithelial injury and fibrosis

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Special issue of "BBA-Molecular Basis of Disease (ELSP)"

Brigitte Kaissling1, Michel LeHir1 and Wilhelm Kriz2

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1 Institute of Anatomy, University of Zurich, Zurich, Switzerland

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2 Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

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To whom correspondence should be directed: Wilhelm Kriz, MD 
 D 68167 Mannheim, Ludolf-Krehl-Str.13-17, Germany Phone: ++49 621 383 9941 Fax: ++49 621 383 9949 [email protected]

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ACCEPTED MANUSCRIPT ROAD MAP Introduction and question

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Fibrosis development

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Fibrosis development starting from glomerular diseases

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Tubular degeneration subsequent to glomerular injuries

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Peritubular response to tubule degeneration: the myofibroblast and fibrosis development

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Fibrosis development starting from tubular injuries

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Signalling underlying the tubulo-interstitial phenomena

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Relevance of fibrosis for progression of CKD?

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Relevance of fibrosis for the progression of CKD subsequent to glomerular diseases Relevance of fibrosis in the progression of a tubular injury to CKD Concluding Remarks

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Abstract

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Chronic kidney disease at a certain advanced stage inevitably

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progresses to end stage renal failure characterized by the

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progressing loss of nephrons accompanied by the increasing appearance of fibrotic tissue, called renal fibrosis. The urgent question is whether renal fibrosis is a response to injury or if

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fibrosis acquires a self-sustaining progressive potential that

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actively contributes to the deterioration of the kidney.

The present review distinguishes between renal fibrosis

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subsequent to a glomerular injury and fibrosis subsequent to a primary tubular injury. Glomerular diseases enter a progressing

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course after encroaching onto the tubule leading to what is generally called "tubulointerstitial fibrosis". The progression of

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the injury at the level of the tubulointerstitium appears to be fully dependent on the progression of the disease in the corresponding glomerulus

Primary tubular injuries have a very good chance of recovery. If they develop a local fibrotic process, this seems to be supportive for recovery. Cases in which recovery fails appear to secondarily initiate a glomerular disease accounting for a glomerulus-dependent vicious cycle to progression. Even if most researchers think of renal fibrosis as a process promoting the progression of the disease this review points out 3

ACCEPTED MANUSCRIPT that the available structural evidence speaks in favour of a protective role of fibrosis supporting recovery after acute tubular

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injury or, under progressing circumstances, providing a firm

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three-dimensional framework that permits still intact or partially

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damaged nephrons to survive.

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Keywords: tubulointerstitial fibrosis-tubular injury-glomerular injury-progression to CKD

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Highlights

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Fibrosis associated with a tubular injury is supportive for recovery

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Fibrosis associated with a glomerular disease fully depends on the progression of the glomerular injury.

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Fibrosis provides secured spaces for intact or partially injured nephrons to survive Fibrosis is no promoter of progression, in contrast, fibrosis is protective

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Introduction and question

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The present review deals with mechanisms of renal fibrosis

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development secondary to an epithelial injury, i.e. secondary to

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an injury to the glomerulus or to the tubule. Renal fibrosis derived from an interstitial or vascular disease is excluded. The

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present review predominantly relies on structural observations

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in animal models.

Nephrons degenerate individually and largely independent from

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neighbouring nephrons [1]. In case that a nephron undergoes

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complete degeneration a considerable micro volume of an

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estimated 40x106 m3 (rat) of epithelial tissue is lost. That volume will be contracted and filled by fibrous tissue. Since most of this volume is due to the loss of the tubule (an

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estimated 95% of a nephron is made up by the tubule), renal fibrosis is generally understood as "tubulointerstitial fibrosis", as a process that occurs in conjunction with tubule degeneration [2-4]. Glomerular fibrosis, i.e. glomerulosclerosis, is considered as an own disease entity, though, by inducing tubular degeneration, it represents the cause of tubulointerstitial fibrosis in the majority of cases progressing to end stage renal disease [1].

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ACCEPTED MANUSCRIPT The term "tubulointerstitial fibrosis" denominates the pathology with extensive scar formation encountered in autopsies/biopsies

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of end stage kidneys as well as all intermediate stages of

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fibrosis development, including early stages of focal nephron

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degeneration/atrophy locally associated with interstitial matrix production. Descriptively this term correctly characterizes the pathology, mechanistically, however, it is a biased term. Since it

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points to fibrosis instead of tubular injury as the central

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condition, it "imputes a degree of autonomy to fibrosis that neglects the tubular component" [5].

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The crucial question, whether the processes in the tubule or those in the interstitium are the primary driving force for

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progression of chronic kidney disease (CKD) remains a matter of intense discussion. In general it is simply assumed, without

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explicit justification, that fibrosis is harmful. According to that assumption, foci of "tubulointerstitial fibrosis", i.e. ongoing local inflammatory processes associated with injured nephrons, will not only accelerate the degeneration of those nephrons but they may also encroach onto neighbouring healthy tubules, thereby becoming an active contributor to progression. The present review will propose another point of view, whereby fibrosis develops secondary to tubular injury. In this concept fibrosis either participates as a healing process or it produces a framework of dense fibrotic tissue that supports the survival of healthy and partially damaged nephrons. 6

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Fibrosis development

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Diseases of the nephron that progress to fibrosis have to be

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subdivided into those beginning in the glomerulus and those beginning in the tubule. Glomerular diseases make up the overwhelming majority of diseases, which progress to CKD.

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Primary tubular diseases generally display as acute kidney

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injury (AKI or acute renal failure), in which the majority of affected tubules undergoes complete regeneration [6]. However, in a small fraction the regenerating processes may

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enter a prolonged, a chronic development also showing up as

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foci of "tubulointerstitial fibrosis

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Fibrosis development in diseases beginning in the glomerulus

A large number of inflammatory and degenerative glomerular diseases are prone to progress to CKD displaying different histopathological features and time courses of progression. It is of utmost importance to understand that, although many nephrons may be affected at one time, the evolution of the disease is specific for each individual nephron and solely dependant on the evolution in the respective glomerulus; the progression of the disease process in the tubule is secondary to that in the glomerulus.

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ACCEPTED MANUSCRIPT Progression of a glomerular disease starts with the loss of the separation between the tuft and Bowman's capsule, i.e. the

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formation of an adhesion of the tuft to Bowman's capsule. In

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degenerative conditions this process leads to focal segmental

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glomerulo-sclerosis (FSGS), in inflammatory conditions to crescentic glomerulonephritis. In both, the outcome with respect to progression is very similar [1]. Both processes start as injury

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of a glomerular segment and both progress within Bowman's

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capsule, i.e. between the parietal epithelium and the parietal basement membrane. Both processes may encroach on other lobules (eventually leading to global process) and/or on the

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glomerulo-tubular junction, leading to a narrowing and finally to a complete obstruction of the urinary orifice (Fig.1). The

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associated decrease of filtrate delivery, finally depriving the tubule from any workload, causes atrophy, finally degeneration

lost.

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of the corresponding tubule. In the end the entire nephron is

This process may favourably be studied in mouse or rat models of rapidly progressing glomerular diseases (e.g. DOCA-salt hypertension in rats [7]; THY-1 mediated glomerulonephritis [8] and anti-GBM nephritis in mice [9]). In these models, the degenerative process advances undelayed to full nephron degeneration and focal fibrosis. Comparing the advancement of the damage in individual nephrons which are caught in various 8

ACCEPTED MANUSCRIPT stages of disease provides a reliable basis for the

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understanding of the sequence of damage progression.

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In addition to the encroachment hypothesis two other

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mechanisms have been proposed to account for the transfer of the glomerular injury onto the tubule and the interstitium. First, the leakage of substances (proteins) through the damaged

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glomerular filter that exert toxic effects on the cells of the

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proximal tubule [10] leading to the development of fibrosis. However, recent studies [11,12] have shown that protein leakage induces proliferation of tubule cells, not degeneration.

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Second, it has been proposed, that sclerosis and loss of postglomerular microvasculature related to the affected

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glomerulus cause decreased peritubular perfusion of the corresponding tubule [13,14]. That hypothesis overlooks the

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fact, that the peritubular capillary network is commonly supplied by all glomeruli in the area [15]. Moreover, a recent study in von Hippel Lindau-knockout mice did not found supportive evidence for this hypothesis [16].

Tubular degeneration subsequent to glomerular injuries

A straightforward course of tubular degeneration is consistently seen after complete obstruction of the urinary orifice by a sclerotic or crescentic lesion [1] The deprivation of the tubule from any filtrate delivery leads to inactivity atrophy rapidly 9

ACCEPTED MANUSCRIPT progressing to complete decomposition of the tubule. The degenerative process starts in the most proximal tubule

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segments just behind the glomerulo-tubular junction and

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proceeds distally (Fig.2A). Tubules are cylindrical structures

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consisting of a single epithelial cell layer resting on the tubular basement membrane (TBM). During degeneration of a tubule the epithelial cells degenerate and disappear well before the

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dissolution of the TBM. Thus until their death, tubular cells

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remain separated from the interstitium by the TBM. The removal of the TBM occurs much later. As presented in detail in recent work in the UUO model [17] and in an injury model in which

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overproduction of TGFb1 caused cellular demise [18] autophagy represents the major mechanism of tubular epithelial

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cell death (Fig.2B), fratricide may participate. In response to direct toxic effects on tubular cells other mechanisms of cell

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death (necrosis, apoptosis) may prevail [19]. The decomposition and removal of the cells may finally lead to almost cell-free cylinders of the TBM, which become increasingly wrinkled and eventually collapsed.

The TBM maintains its continuity for considerable time even after collapsing and after disappearance of any cellular elements inside these cylinders. Tubular cells crossing the TBM border of degenerating tubules were not encountered in our studies nor documented by others. Even in case of a complete degeneration of a tubule, removal of its remnants and formation 10

ACCEPTED MANUSCRIPT of a scar, the glomerular remnants may survive for some time

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as inoperable structures [8,20,21]

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Extended segmental sclerosis without encroachment on the

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glomerulo-tubular junction, or an incomplete obstruction of the urinary orifice will result in a significant decrease of filtration and the workload of the tubule will be markedly reduced. Under

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those conditions tubular cells do not completely degenerate but

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undergo atrophy with loss of features characteristic of a differentiated phenotype [22]. Such intermediate stages of tubule degeneration appear to cause very similar, if not identical

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responses of the surrounding interstitium - like they are seen after straightforward degeneration of the tubule (see below).

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They may obviously persist over extended periods of time in humans. The rapid progression in most animal models does not

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provide the opportunity to observe that type of lesions. Therefore details of their evolution are unknown.

Peritubular response to tubule degeneration: myofibroblasts and fibrosis development

Tubule degeneration evokes a vivid peritubular reaction. The injured tubule cells initiate a local inflammation, which will finally lead to the removal of all tubular remnants and the formation of a local area of fibrosis, finally a scar. The outstanding feature of the local inflammatory process surrounding a degenerating 11

ACCEPTED MANUSCRIPT tubule consists of the massive accumulation of myofibroblasts. The myofibroblast is a central player in fibrosis development.

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According to our results, the only relevant source for these

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myofibroblasts are the resident interstitial cells [23,24] (Fig.3),

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which are generally called interstitial fibroblasts, some authors call them perivascular fibroblasts or pericytes [25]. The much discussed proposal that myofibroblasts may also develop from

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injured tubular cells by a process called "Epithelial to

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Mesenchymal Transition (EMT)" has not found supporting evidence in recent studies [18,23,25]. Also a contribution of bone-marrow derived "fibrocytes" is generally considered as

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marginal, at best [26]. Myofibroblasts organize the interstitial fibrosis by synthesizing type I collagen and finally by contraction

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of the affected area to a scar.

Thus, the differentiation of resident interstitial fibroblasts into myofibroblasts is central in fibrosis development. Interstitial fibroblasts are branched cells with partially very long cellular projections that are stuffed with bundles of actin filaments. The processes are attached (by a kind of adherens junctions) to the basement membrane of tubules and peritubular capillaries as well as to processes and cell bodies of other fibroblasts [27], forming a continuous network throughout the interstitium (Fig.3).

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ACCEPTED MANUSCRIPT Upon activation by profibrotic cytokines (see below) or mechanical stress [28] resident interstitial fibroblasts

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progressively gain the myofibroblast phenotype as consistently

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seen in various disease models including the UUO model [24].

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Within one day after UUO the interstitial fibroblasts started to proliferate in the vicinity of injured tubules [24], alpha smooth muscle actin (αSMA), the hallmark of myofibroblasts, became

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detectable by immunofluorescence in interstitial fibroblasts and

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after two days bundles of alpha-SMA traversed the cells in all directions and accumulated in their processes. The contacts to neighbouring fibroblasts / myofibroblast increased in number,

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the area of contacts to injured tubules broadened, partially encircling a tubule (Fig. 2B). Thus, the network of fibroblasts

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developed into a network of myofibroblasts (Fig.4) suggesting that this conversion is not simply a response of individual cells

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but of a cellular network. The cells produce abundant amounts of matrix and collagen fibrils that fill the meshes of this network. In total this newly emerged fibrous tissue forms an area with strong mechanical properties centripetally interconnecting all structures of this area

In case of complete tubular degeneration the tubular remnants finally disappeared and the girdles of fibrotic tissue formed around the degenerating tubules were combined to areas of scar tissue as routinely seen in biopsies/autopsies of human endstage kidneys. In case that the degeneration of the tubule is 13

ACCEPTED MANUSCRIPT prolonged with maintenance of atrophic tubules for some time, the peritubular inflammatory response persists as well.

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Unfortunately, such advanced stages of fibrosis development

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have never been studied in detail in an animal model.

Fibrosis development starting from tubular injuries

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Acute kidney injury (AKI) as clinical disease most frequently

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occurs in conjunction with multiple organ failure. Many factors (hemodynamic, inflammatory, endothelial, toxic, metabolic) may converge to cause the tubular injury. Here, we will only review

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the disease mechanisms subsequent to primary tubular injuries as they are encountered in response to direct hypoxic (ischemic

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reperfusion injury) or toxic/metabolic challenges and as they

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have been studied in animal models.

Under such experimental conditions the entire population of nephrons is affected, however, the severity of the damage may be very different among nephrons. The immediate loss of a nephron occurs only in the rare cases that the tubular basement membrane is disrupted (tubulorrhexis) [29]. Generally, even after severe insults the TBM maintains its continuity providing the scaffold for epithelial regeneration.

The epithelial damage is generally heterogeneous among segments within a nephron and among cells within a segment 14

ACCEPTED MANUSCRIPT of the nephron. Some cells are lethally injured and undergo necrosis or apoptosis (the cell debris seems to be washed away

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by urine flow) [30,31] others survive the attack with various

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degrees of injury. The latter start to proliferate vigorously,

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replacing lost cells and finally differentiate into functioning tubular cells re-establishing a functional tubule in most cases.

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Ischemic events after ligation of the renal artery and some toxic

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events (e.g. uranyl acetate [32], cisplatin [33], mercury chloride (HgCl2) [34] predominantly attack the S3 segments of the proximal tubules. Distal tubular segments are targeted by

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agents that interfere with the segment-specific function (Fig.5). For instance, uromodulin-associated kidney disease [35] affects

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specifically the thick ascending limb of Henle’s loop (TAL), the site of uromodulin synthesis, thiazide treatment in rats [30,31]

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affects exclusively the distal convoluted tubule (DCT; Fig. 5), the location of the thiazide-sensitive neutral sodium cotranporter (NCC) and distal tubules and collecting ducts are injured by lithium [36] which interferes with the function of these segments.

With respect to recovery of a tubular injury the question arises whether there is a certain fraction of cells capable of regeneration and another fraction that is not. Considering the high degree of structural homogeneity among cells in the proximal tubule, the TAL and DCT it is unlikely that two separate cell populations might co-exist, with different capacity 15

ACCEPTED MANUSCRIPT to survive insults and to proliferate. The heterogeneity in cell fate observed after injury likely results from differences in the

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cycling stage of individual cells. Differentiated tubular cells are

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cycling at a slow rate, even in the adult [37,38]. As shown

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previously the proximal tubule contains a large fraction of cells arrested in G1 that constitutes a "ready-to-go" pool of cells to immediately start proliferation in case of a tubular injury [38]. It

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was also shown [39] that proximal tubular injury by HgCl2

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induced expression of the nuclear factor NFATc1 in some cells, which thereby became more resistant to cell death (apoptosis). Fate tracing showed that those cells were largely responsible

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for the regeneration of the tubular epithelium. Thus, there seem to be relevant differences between tubular cells concerning their

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stage in the cell cycle and in their vulnerability to toxic or ischemic challenges resulting in a fraction of cells being prone

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for regenerative proliferation. A very recent study in rats uncovered the emergence of a population of CD24, CD133 and vimentin positive cells in proximal tubules undergoing regeneration [40] confirming previous studies showing that the expression of vimentin characterizes a fraction of regenerating proximal tubule cells [34] On the other hand, two recent studies [5,6,41] show that there is or develops a fraction of cells that does not properly participate in the process of regeneration. These cells are arrested in G2/M delaying proliferation and produce JNK (c-jun NH2-terminal kinase) dependent profibrotic factors including 16

ACCEPTED MANUSCRIPT connective tissue growth factor (CTGF) and transforming growth factor beta (TGFß) [41]. In the other study such cells

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were shown to be characterized by deficiency or loss of

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phosphatase and tensin homolog (PTEN) likely evoked by

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TGFß in autocrine or paracrine fashion [5]. They are likely the cause of the failure of some tubules to regenerate,

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consequently the triggers of the peritubular inflammation.

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In summary, injured tubules seem to contain a fraction of cells, dependant on their stage in the cell cycle that accounts for the great regeneration capacity of tubular epithelia whereas other

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cells do not have these outstanding capabilities for proliferation

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and redifferentiation.

In case that tubules fail to recover, a signalling activity is

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maintained that evokes a local peritubular inflammation. Interstitial fibroblast start to proliferate and to convert into myofibroblasts followed by matrix deposition leading to foci of "tubulointerstitial fibrosis". When comparing figures 2B and 5B it is evident that the inflammatory response to a primary tubular injury is structurally identical to that associated with a glomerular-dependant tubular injury.

In a series of elegant studies in the uranyl acetate-induced AKI in rats of the Akira Hishida group [32,42-44] it was shown that the interstitial response is spatially very precisely correlated to 17

ACCEPTED MANUSCRIPT the affected tubular segment and cells, and disappears precisely at loci where the tubular cells recovered. This strongly

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suggests that the injured tubular cells in small groups or even

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individually are responsible for the inflammation and fibrosis in

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their immediate surroundings. Moreover, the authors did not find any evidence that this peritubular response is harmful to the tubule. In contrast, the authors came to the conclusion that the

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interstitial inflammation supports the recovery of the tubule.

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They argue as follows: In early stages of acute tubular injury, tubules undergo expansion caused by elevated intratubular pressures. Myofibroblasts gather around such tubules in a

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circular arrangement tightly fixing to the TBM. These contractile cells might counteract further expansion of the tubule and

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thereby favour tubular recovery. Our results in the UUO model [24] and observations in drug-induced tubular injury in rat distal

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tubules by thiazides [30,31], by lithium [36] and thick ascending limbs in a transgenic mouse model for uromodulin-associated kidney diseases [35], are in full agreement with these observations

Signalling underlying the tubulointerstitial phenomena To discuss this issue it is necessary to distinguish between signals to the interstitium and those directed to tubular cells. Many studies were undertaken to figure out the factors secreted by injured and/or regenerating tubule cells acting on the 18

ACCEPTED MANUSCRIPT surroundings and being likely responsible for the peritubular reactions. Recent reviews by Venkatachalam and colleagues in

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2010 [2] and Bonventre and Yang in 2011 [6] have discussed

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this issue in detail. A confusingly large array of signalling

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pathways are involved and there is no certain knowledge

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whether they are protective, injurious or restorative.

There is agreement that the interstitial process is started by

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paracrine stimulation from injured tubular cells. The main factor is the production of TGFß [2,5,6,18,41,45-47]

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TGFß induced loss of PTEN appears to contribute to the failure

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of regenerating epithelial cells to redifferentiate, thereby causing the retention of proliferative signalling, and in this

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fashion, give rise to the production of profibrotic peptides [5]. TGF stimulates fibrosis not only via its direct effects on

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fibroblasts but additionally by inducing the production of other molecules like Notch [48], and CTGF [49] Yang 2012) or PDGF [2,43,46]. In addition, TGFß mediates upregulation of osteopontin (OPN) expression in injured epithelial cells [50] and triggers a focal inflammatory process with immigration of neutrophils (Fig. 4a), mononuclear cells [51,52] and macrophages [53]. The resident dendritic cells adopt a proinflammatory phenotype and activate T-cells, but do not directly contribute to fibrosis [54]. These factors taken together initiate the proliferation of interstitial fibroblasts and enhance the 19

ACCEPTED MANUSCRIPT differentiation of fibroblasts into myofibroblasts [55] with the

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effects described above

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Additionally, changes in the capillary network are widely

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believed to be involved. As a consequence of tubular injury, peritubular capillaries may be deprived from survival factors of tubular origin, first of all VEGF [2] leading to capillary

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rarefaction. The potentially resulting focal hypoxia is thought to

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contribute to inflammation and fibrosis. However, no direct evidence of a vascular contribution is available. Inflammation and the emergence of myofibroblasts in the peritubular

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interstitium have been observed already after one to two days following initiation of injury in UUO [24] and injection of uranyl

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acetate [32] or a of thiazide diuretic [30,31]. Remodelling of the capillary network would hardly have taken place in such short

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periods of time. In addition, after injury with uranyl acetate or thiazide the interstitium was affected precisely around the specifically targeted nephron segment, the S3 segment of the proximal tubule and the distal convoluted tubule, respectively. Any signal produced by peritubular capillaries would have affected a broader area.

Whereas considerable effort has been devoted to identify signals from injured tubules affecting the interstitium, little is known about signals from the interstitium, in particular from

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ACCEPTED MANUSCRIPT fibroblasts or myofibroblasts, which might influence the fate of

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injured tubular cells.

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Much less is known about signals that regulate the regeneration

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/ degeneration of injured tubular cells. Above we discussed the hypothesis that failed differentiation and growth arrest of a certain fraction of regenerating tubular cells after an acute

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tubular injury is responsible for the maintenance of the

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tubulointerstitial process [2].. Here the question arises, whether this failure depends exclusively on signalling inside the injured

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tubule or whether signals from outside may also be relevant.

No candidates for signals from myofibroblasts to the tubule

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have ever been proposed. As noted by Lan and colleagues [5] individual tubule cells showing signs of failed differentiation and

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growth arrest can be found amidst regenerating cells . Such a pattern cannot be the consequence of signalling from outside the tubule but appears to be based on inherent properties of those cells. Paracrine and/or autocrine effects of TGF might be involved [5].

Relevance of fibrosis for progression of CKD?

Above we considered fibrosis as a healing process, which results in the formation of a scar in the place of a lost nephron or supports the healing of a nephron. This concept contrasts 21

ACCEPTED MANUSCRIPT with the prevalent view that renal fibrosis has a progressive potential which significantly contributes to the deterioration of

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the kidney in CKD [56,57]. Here we will discuss whether fibrosis

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may acquire self-sustaining properties contributing to the

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destruction of so far healthy or partially damaged nephrons or whether any sustained fibrotic activity is the consequence of persisting disease processes within the nephron. Let us start

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with fibrosis subsequent to a primary glomerular disease.

Relevance of fibrosis for the progression of CKD subsequent to glomerular diseases

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The hypothesis of an active contribution of fibrosis to progression is essentially based on the observation that the

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decline in renal function in chronic renal disease correlates more closely with interstitial fibrosis than with glomerular

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damage [58-61].

This correlation is taken as argument in favour of a genuine interstitial mechanism of progression. Thereby one overlooks a crucial fact: As animal models [1] and studies in the aging human kidneys show [62,63] the remnants of a degenerating nephron, including glomeruli, may be completely removed and replaced by fibrous tissue. Thus, with the degeneration of nephrons, the interstitial damage score will increase, while the glomerular damage score will remain constant or even decrease due to complete disappearance of sclerotic glomeruli. 22

ACCEPTED MANUSCRIPT More recent studies clearly show that the decline in renal function in chronic renal disease correlates best with the

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number of remaining nephrons [64-66]. Thus, the evidence for

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the fibrosis hypothesis from these studies is far from being

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conclusive.

Our own observations in a variety of animal models do not

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support the hypothesis that fibrosis may become a self-

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sustained process that injures healthy nephrons. We consistently found that tubule segments of healthy nephrons, even if completely surrounded by degenerating tubules or

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embedded in fibrotic tissue, preserve their normal structure. Figure 6 shows an area of extensive nephron degeneration

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from an experimental fibrosis model (overexpression of TGFß; [18] that contains a single structurally intact nephron completely

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trapped into an area of tubulo-interstitial fibrosis. Surely, such examples do not prove that on the long run such a hostile environment will not exert damaging effects to this nephron. However, in autopsies or biopsies of endstage kidneys groups of structurally intact tubular profiles are frequently encountered surrounded by scar tissue. If harmful signals from fibrotic areas or local hypoxia due to capillary rarefication (as frequently suggested) were the major cause of tubular injury, all nephrons in that area would display a similar degree of damage.

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ACCEPTED MANUSCRIPT The pathophysiological situation in a stage of advanced fibrosis consisting of proliferating and fibrotic areas, of dead, of

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damaged and of some intact (but mostly hypertrophied)

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nephrons is complex and chaotic. The chance to clearly

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decipher profibrotic and antifibrotic, destructive and protective mechanisms is small, at best, as is the chance for any successful therapeutic interventions. The fight against

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progression of glomerular disease has to start much earlier at

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stages when the process is still confined to the glomerulus. Regarding glomerular injuries a vicious cycle has been documented, in which podocyte loss increases the probability of

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further podocyte loss [1]. That mechanism underlies the glomerular overload hypothesis for progression, first formulated

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by Brenner [67,68]. An equally evidence-supported hypothesis is not available for fibrosis as a self-sustained damaging

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mechanism.

Relevance of fibrosis in the progression of a tubular injury to CKD The issue of how acute kidney injury may enter a chronic course eventually leading to end-stage renal failure is currently of great interest [69]. As discussed above, the accumulation of myofibroblasts around injured tubules during AKI is not likely to provoke further tubular injury but rather plays a supportive role. Indeed, as may be seen from the work of Fujigaki and colleagues [32] and others (see above) such tubules may 24

ACCEPTED MANUSCRIPT undergo regeneration accompanied by the disappearance of

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the peritubular changes, i.e. the myofibroblasts.

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On the other hand these pathologies may progress to the loss

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of the entire nephron and to CKD as shown in a recent study by Grgic and colleagues [19]. In an inducible transgenic mouse model a single toxin insult to proximal tubules was promptly

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followed by peritubular inflammation, tubule cell proliferation

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and recovery. However, three insults at one-week intervals led to a progressing tubulo-interstitial disease that encroached upon the glomerulus. Glomerulosclerosis significantly correlated

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with interstitial fibrosis. The authors suggested several mechanisms responsible for the transfer of the disease on the

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glomerulus, among them that obstructive damage to the initial segment of the proximal tubule initiated sclerosis development

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in the glomerulus, leading to sclerotic, likely atubular glomeruli as commonly observed in obstructive nephropathy [21]. In case that this happens in a sufficiently large fraction of nephrons overload will be imposed on the remaining nephrons starting the vicious cycle to progression to CKD proposed by Brenner [67,68]. In that way, an originally tubular disease may enter a progressing course due to the involvement of glomeruli.

This discussion leads to the insight that the regenerative potential is fundamentally different between foci of "tubulointerstitial fibrosis" derived from a primary glomerular 25

ACCEPTED MANUSCRIPT disease and those of primary tubular origin. Descriptively, both cases of persistent "tubulointerstitial fibrosis" are similar.

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However, they differ with respect to the underlying mechanisms

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and to the possible outcome. Primary tubular injuries leave a

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chance of healing and the transient fibrosis seems to play a supportive role. Tubular injuries derived from a glomerular disease do not have this chance: they fully depend on the

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disease in the corresponding glomerulus. This is not expected

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to revert since full structural and functional recovery of a sclerotic or crescentic injury is unlikely to occur. On the contrary, such glomerular lesions tend to progress. Since

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progression proceeds at different rates in different glomeruli some nephrons will still be operative while others have

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degenerated. The maintenance of a residual function obviously requires the structural integrity of the still perfused tubules.

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Fibrosis may play an important role in that respect. Nephrons are fragile microanatomical entities, which are not supported by connective tissue boundaries as found in most other organs (e. g. glands, lung). The tubular coils of adjacent nephrons come very close to each other and to glomeruli and the shape of a nephron is largely maintained by its relationships to neighbouring nephrons. The tenuous network of interstitial fibroblasts acts as a link between neighbouring structures. How to maintain a mechanical support for a nephron when adjacent nephrons undergo decomposition? It seems plausible that fibrosis provides a stable three-dimensional framework 26

ACCEPTED MANUSCRIPT permitting the survival and residual function of still intact or

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partially damaged nephrons.

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Concluding remarks

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Renal fibrosis is consistently associated with chronic renal disease. It develops in conjunction with tubular degeneration,

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therefore the generally used but vague term "tubulointerstitial fibrosis”. This designation obscures the individual roles of the

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tubular and the interstitial component in this process and led to

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the widely accepted hypothesis that fibrosis causes the degeneration of the tubule. However, any reliable evidence

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speaks in favour of the opposite mechanism: the progressing

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glomerular and tubular injury drives the fibrosis.

Progression of fibrosis accompanied with a glomerular disease crucially depends on the progression of the injury in the glomerulus. If this process has passed the point of no return, i.e the loss of separation between the tuft and Bowman`s capsule, the only option to control the course of the disease consist of slowing down the progression of the glomerular injury and, consequently, that of the tubule. In case of progression to nephron loss, the resulting replacement of the nephron by fibrosis may well play a protective role for the remaining 27

ACCEPTED MANUSCRIPT nephrons providing them mechanically secured spaces to

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survive.

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The situation of fibrosis subsequent to a primary tubular disease

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is different. The dominating process associated with a tubular injury is regeneration. This is accompanied by a transient peritubular inflammation and fibrosis that appear to support the

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recovery of the tubule. Peritubular fibrosis persists when tubules

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fail to undergo full regeneration and differentiation. This failure, the causes of which are largely unknown, may eventually lead to the involvement of the glomerulus and to the glomerulus

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dependent vicious circle resulting in CKD. If a sufficient number of nephrons is concerned, the glomerular damage-dependant

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vicious cycle to progression to CKD will be started. Thus, the entry of a tubular disease into a progressing course likely

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depends on its mutation into a glomerular disease.

Acknowledgements The authors would like to thank Brunhilde Hähnel and Hiltraud Hosser for tireless technical and organizational help. The authors gratefully acknowledge the continuous support of our work by the "Gotthard Schettler Gesellschaft fuer Herz- und Kreislauf-forschung" and the "Professor Dr. Karl und Gerhard Schiller Stiftung".

Figure legends 28

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Fig.1 Encroachment of a glomerular injury onto the tubulointerstitium Two injured glomeruli and the corresponding tubules are highlighted in yellow. The crescentic glomerular injury

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(asterisks) encroaches inside the basement membrane (arrows) on the proximal tubule. The tubular epithelium is severely

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damaged, most advanced in proximal tubules immediately after the glomerulo-tubular junction (star), less advanced in distal

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tubule segments (triangles). Around injured glomeruli and tubules a vivid proliferation of interstitial cells is seen. Tubular

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profiles belonging to glomeruli with a patent urinary orifice are

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intact (circles; verified by tracing in serial sections).

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Thy-1 mediated glomerulonephritis in rat, day 9; 1µm Epon section; bar: 20 µm. Reproduced with permission from Kriz et

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al. (2003) [8]

Fig.2 Tubule degeneration A: Overview of a group of degenerating proximal tubules. The severity of epithelial changes increases downstream; upstream segments (1, 2, 3, 4) exhibit epithelial desintegration with cells filled with autophagic vacuoles (stars), in downstream segments (5, 6) the epithelial integrity is still preserved. The surrounding interstitium is expanded, rich in cells and fluid, and contains ample capillaries. Proximal tubules (asterisks) of unaffected nephrons exhibit an intact epithelium. 29

ACCEPTED MANUSCRIPT B: Collapsed profile of proximal tubule in an advanced stage of destruction. Cells contain assemblies of autophagic vacuoles

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(asterisks) and start to disintegrate (thick arrows).The tubular

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basement membrane (shown in yellow) is wrinkled

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(arrowheads). The interstitium contains many myofibroblasts, processes of which (arrows) form an encircling envelope. A: Thy-1 mediated glomerulonephritis in rat, 2 weeks;

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transmission electron micrograph (TEM), bar: 3 µm.

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Reproduced with permission from Kriz et al. (2003) [8] B: Anti-GBM glomerulonephritis, day 10; bar: 5 µm.

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Unpublished TEM from [9].

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Fig.3 Renal cortical interstitium

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A: Schematic illustration. Interstitial fibroblasts are shown in green, a dendritic cell in yellow, tubules in violet, a capillary is

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labelled with C.

Fibroblasts have multiple projections that bridge the interstitial space terminating in focal adhesions at tubules, capillaries and processes of other fibroblasts (arrowheads). They contain thin assemblies of f-actin filaments that run through the cell bodies (located immediately under the plasmalemm) and extend into the processes. Thus, renal fibroblasts form a network that interconnects all structures of renal cortex. Dendritic cells show more rounded cell bodies and lack the dense f-actin

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ACCEPTED MANUSCRIPT assemblies; their generally thin cell projections are intermingled with fibroblast processes.

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B: Three-dimensional view of the renal cortical interstitium of rat

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by scanning electron microscopy, after digestion of tubular and

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vascular basement membranes. Fibroblast cell bodies (S) give rise to attenuated and perforated leaflet-like processes that touch each other at many sites. Dendritic cells (asterisk),

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enclosed into the network of fibroblasts, form broad cytoplasmic

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projections (arrows) as well as filiform processes (arrow heads).

PT, proximal tubule; C, capillary; bar: 10 µm.

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Reproduced with permission from Takahashi-Iwanaga (1991)

Fig.4

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[70].

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Rat renal cortex after 4 days of ureter ligation. Immunofluorescence: ecto-5- nucleotidase (5-NT)— red , smooth muscle actin (SMA)—green; chromatin staining by DAPI—blue channel. 5’NT labels all cortical interstitial fibroblasts, the brush border of proximal tubules (P), and intercalated cells in a connecting tubule (CN). In areas around proximal tubules with advanced degeneration (asterisks) SMA is strongly upregulated in fibroblasts while 5’NT is faintly detectable; in fibroblasts in areas with less advanced injury of proximal tubules (P) SMA is 31

ACCEPTED MANUSCRIPT weakly upregulated while 5’NT is distinctly detcetable: A, glomerular arteriole; 3 µm cryostat section; bar: 50 µm.

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Reproduced with permission from Picard et al. (2008) [24]

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Peritubular response to tubular injury

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Fig. 5

Tubular damge and interstitial response induced by thiazide diuretics; thiazides inhibit specifically the neutral sodium co-

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transporter (NCC) in the apical plasma membrane of cells of the

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distal convoluted tubule (DCT). In rats the treatment induces damage exclusively in DCT profiles [30] A: DCT profile (D) 24 hours after a single dose of thiazide-like

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diuretic metolazon®; tubular injury becomes evident by upregulation of osteopontin (red; thin arrrows) in DCT cells. At

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this stage of injury neutrophil leucocytes (labelled by OX 1 antibody directed against common leucocyte antigen; green;

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thick arrow) adhere to the injured cells. 1 µm cryostat section; bar: 10 µm.

B: After 36 hours cells in the DCT (D) are apoptotic and the peritubular interstitial fibroblasts have transformed to myofibroblasts (arrows) and encapsulate the injured tubule. 1 µm Epon section; bar: 10 µm. C: After 72 hours massive up-regulation of SMA in the interstitium immediately around the injured tubule (D); the strength of fluorescence for SMA decreases towards more distant areas (arrows). 3 µm cryostat section; bar: 20 µm.

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ACCEPTED MANUSCRIPT A and B: unpublished micrographs from [30] ; C: reproduced

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with permission from Kaissling and Le Hir (2008) [27]

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Fig. 6

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Advanced Fibrosis

A: Area of extensive nephron degeneration and fibrosis housing a single intact nephron (shown in yellow). The higher

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magnification in (B) shows that the glomerulus, the glomerulo-

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tubular junction and the corresponding tubule segments do not exhibit any damage.

Over-expression of TGFβ in renal tubules in mouse [18] , after 6

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weeks. 1 µm Epon section; bars: A: 40 µm, B: 20 µm.

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Unpublished light micrographs from [18]

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ACCEPTED MANUSCRIPT Highlights

Fibrosis associated with a tubular injury is supportive for recovery

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Fibrosis associated with a glomerular disease fully depends on the progression of the glomerular injury.

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Fibrosis provides secured spaces for intact or partially injured nephrons to survive

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Fibrosis is no promoter of progression, in contrast, fibrosis is protective

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