Accepted Manuscript A Possible Mechanism for the Progression of Chronic Renal Disease AND Congestive Heart Failure Richard N. Re, M.D PII:

S1933-1711(14)00774-8

DOI:

10.1016/j.jash.2014.09.016

Reference:

JASH 584

To appear in:

Journal of the American Society of Hypertension

Received Date: 1 September 2014 Revised Date:

11 September 2014

Accepted Date: 13 September 2014

Please cite this article as: Re RN, A Possible Mechanism for the Progression of Chronic Renal Disease AND Congestive Heart Failure, Journal of the American Society of Hypertension (2014), doi: 10.1016/ j.jash.2014.09.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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AND CONGESTIVE HEART FAILURE

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Richard N. Re, M.D.

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A POSSIBLE MECHANISM FOR THE PROGRESSION OF CHRONIC RENAL DISEASE

Ochsner Clinic Foundation 1514 Jefferson Highway

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New Orleans, LA 70121 504-842-3700

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[email protected]

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Key Words: Neurodegenerative diseases; chronic renal disease, congestive heart failure; the reninangiotensin system; intracrine. Short Title: Cardiorenal disease progression Word count: 4967

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ABSTRACT Chronic neurological diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, as well as various forms of chronic renal disease and systolic congestive heart failure are

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among the most common progressive degenerative disorders encountered in medicine. Each disease follows a nearly relentless course, albeit at varying rates, driven by progressive cell dysfunction and drop out. The neurological diseases are characterized by the progressive spread of disease-causing proteins

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(prion-like proteins) from cell to cell. Recent evidence indicates that cell autonomous renin angiotensin systems (RASs) operate in heart and kidney, and it is known that functional intracrine proteins can also

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spread between cells. This then suggests that certain progressive degenerative cardiovascular disorders such as forms of chronic renal insufficiency and systolic congestive heart failure result from

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dysfunctional RAS intracrine action spreading in kidney or myocardium.

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BACKGROUND Many apparently non-infectious chronic illnesses demonstrate a clinical course characterized by progressive organ dysfunction and cell loss. For example, neurodegenerative diseases such as

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Alzheimer’s disease, Parkinson’s disease and others typically progress over years to decades as cells become dysfunctional and die 1,2. Similarly, systolic congestive heart failure is characterized by a

progressive loss of cardiac contractility associated with cardiomyocyte drop out 3. And it is well known

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that chronic renal insufficiency, be it the result of diabetes, hypertension, or other factors, follows a progressive downhill course in spite of the best available therapy 4, 5. This kind of progression is

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characteristic of infectious and neoplastic diseases, although in those cases the time course of progression is, in general, more rapid than in the above cardiovascular and neurologic disorders. However, it has become apparent that in the case of the neurodegenerative disorder Creutzfeldt-Jakob disease (CJD), as well as bovine encephalopathy (mad cow disease), kuru, and a variety of other so-

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called prion-diseases, an infectious protein unassociated with nucleic acid is the likely causative agent, capable of spreading between individuals on contaminated neurosurgical instruments and capable of spreading through the nervous system by an essentially infectious process 1,2 . Bovine encephalopathy

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and kuru have an even more obvious infectious capability demonstrated by spread to humans via consumption of infected tissue. The proteinaceous infectious particles involved in these disorders

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(prions) are mis-folded normal proteins that produce disease in target cells by inducing a mis-folding of their normal homologues in those cells (along with secondary aggregation of mis-folded prions), resulting in trafficking, by one mode or another, of the newly created mis-folded protein to nearby cells, intracellular aggregation of the prion protein, and cell death. Of note, several neurodegenerative disorders that are apparently not easily transmissible between individuals, do nonetheless exhibit the intercellular spread of mis-folded prion-like proteins that produce aggregation and progressive disease. Included in this category are Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and

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others 1,2. This then suggests the possibility that a similar process could be operative in non-neurologic chronic degenerative diseases and, in particular, in systolic congestive heart failure (CHF) and chronic

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renal failure (CRF).

This proposal at first seems unlikely to be correct. Although CHF and CRF are progressive, there is no evidence of a transmissible agent operative in either of them. Rather progression seems to result from

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the persistence of an underlying cause—for example ongoing ischemia in the case of CHF, and diabetes or hypertension in the case of CRF—coupled with one or more secondary physical or pathogenic factors

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--–for example ventricular dilatation in the case of CHF and glomerular hypertension and hyperfiltration in the case of CRF—that perpetuate tissue stress and continued cell loss. Physical processes and diseaseassociated pathophysiologic factors seems to produce progression, not a quasi-infectious agent. Also, the fact that renal function normally declines with age suggests the possibility that progressive renal

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disease represents an acceleration of an expected decline in function. Finally, while the progression of the prion-like neurodegenerative diseases appears to depend on the inter-neuronal spread of prion-like proteins, the initiating cause of these diseases can be genetic (usually involving the over-production of

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normal prion-like protein which increases the chance of random mis-folding, or the synthesis of an abnormal aggregation-prone form of the protein), infectious, or environmental, as in the case of

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neurotoxin exposure and Parkinson’s disease. In the case of CHF and CRF initiating causes are more clearly defined. Moreover, interventions directed against those causes and the physical and physiological factors associated with progression have been shown to ameliorate disease. For example, glucose and blood pressure control are beneficial in CRF and renin angiotensin system (RAS) blockade is partially effective in both CRF and CHF secondary to systolic dysfunction, although in spite of considerable effort, attempts at halting disease progression by modifying these pathophysiologic factors have not been successful. 3-5. At the same time, the partial effectiveness of RAS blockade in these

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cardiorenal disorders could point to a nexus between the protein components of the RAS and prion-like proteins.

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SIGNALING PROTEINS AND CHRONIC DISEASE PROGRESSION

Just as prions and prion-like proteins can spread between cells, be internalized, and act in cell interiors, so also can many physiological signaling proteins and peptides. These moieties, called intracrines, can

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traffick between cells either following secretion, atypical secretion, via exosomes, or possibly even via nanotubes 6-12. They can signal at classical cell surface receptors or after internalization, and their

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intracellular signaling can be non-canonical—that is independent of classical receptors—or canonical— mediated by classical receptors located in the intracellular space 12. Some intracrines (for example parathyroid hormone related protein, PTHrP) also function in their cells of synthesis and in that circumstance frequently one or more intracrine transcripts lacks a secretory signal and therefore its

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product is retained within the cell, while other transcripts encode proteins which are secreted. Intracrines, while sharing these common functionalities are structurally diverse: hormones, growth factors, cytokines, DNA binding proteins, and enzymes, among other moieties, can act in an intracrine

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mode. Also of note, these signaling proteins, when acting in target cells, often up-regulate their own synthesis or that of their signaling cascades and thereby produce a feed-forward loop which produces

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what might be called an active form of differentiation in target cells: the action of the up-regulated internalized intracrine renders the cell in a new state of responsiveness/differentiation which persists even when extracellular intracrine is no longer present 6-12. For example, homeodomain transcription factors can be secreted by cells, taken up by target cells, up-regulate their own synthesis, and then spread from cell to cell. This kind of active differentiation has been reported for the homeodomain proteins PDX1 and Pax6, although virtually all homeodomain proteins contain an internalization signal and are intracrine 13, 14.

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There are similarities between the action of the prion-like proteins involved in neurodegenerative disorders and intracrines 15. Indeed, the normal forms of some prion-like proteins function as intracrines

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under normal physiological circumstances. For example, native alpha-synuclein, the protein which in a mis-folded form is a prion-like protein involved in Parkinson’s disease, is an intracrine: it trafficks

between cells; after internalization, it trafficks in the intracellular space to microtubules, nucleus, and

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mitochondria; it has physiologic functions in target cells after internalization. The intracrine-like

properties of prion-like proteins have already been touched on: they can traffick between cells after

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secretion, release from dead cells, or via exosomes or nanotubes; they are internalized by target cells where they produce disease because of one or another aberrant function usually involving aggregation 8, 15

. There is some evidence of up-regulation of the underlying normal protein form as a means of

enhancing disease propagation. These observations further suggest a nexus between the pathogenic

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mechanism involved in prion-like neurodegenerative disorders and intracrine functionality 15. They further raise the possible that intracrines, many of which are active in cardiovascular disease, could, either as result of their normal function or of abnormal action, produce progressive disease in CHF, CRF,

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and possibly other cardiovascular disorders. The renin-angiotensin system (RAS) is an exemplar of a

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possible link between intracrine biology and progressive cardiovascular disease.

THE RAS AND PROGRESSIVE DISEASE It is well established that inhibitors of angiotensin II action, be they converting-enzyme inhibitors (CEIs) or AT1 receptor blockers (ARBs), blunt cardiac left ventricular hypertrophy, diminish inappropriate ventricular dilatation following myocardial infarction, and modestly prolong life in CHF patients 3. Similarly, they blunt, but do not halt, the progressive decline in renal function associated with diabetic nephropathy and certain other renal diseases 4-6. Presumably, inhibitors of renin enzymatic activity,

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direct renin inhibitors, will have the same effects. However, when attempts were made to improve the effect of RAS inhibition on renal functional decline by treating with two inhibitors such as a convertingenzyme inhibitor and an ARB, no additional benefit was achieved and side effects, likely secondary to

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diminished cardiovascular pressor reserves, became manifest 16. Assuming that residual RAS activity is at least partially responsible for disease progression after RAS blockade, several explanation for the failure of dual blockade have been proposed. First, there is evidence for stretch-induced stimulation of AT1

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receptor (AT-1R) signaling independent of angiotensin II. This could permit ongoing cell pathology unimpeded by angiotensin receptor blockade 17. However, candesartan, an ARB that is an AT-1R reverse

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agonist, inhibits this kind of signaling but there is no evidence that this receptor blocker is more effective in terms of dampening disease progression than any others 18. Another suggestion is that because renin can act directly at specific renin receptors on cells; the direct action of renin or prorenin on these receptors, unmitigated by available forms of RAS inhibition, could promote disease progression

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in spite of inhibition of angiotensin II formation or of its receptor binding 19. This idea can only be tested with the development of an effective inhibitor of the renin receptor. An alternative explanation is that RAS components acting in cell interiors are responsible for RAS action unattenuated by RAS blockade 6,7,10

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The latter two explanations are not mutually exclusive. Components of the RAS are intracrines, including angiotensin II (AII), angiotensisn 1-7 (Ang 1-7), angiotensinogen, angiotensin converting enzyme, and renin 6-12, 19-29. For example, angiotensin II is secreted by a variety of cells, signals at cell membrane receptors, can be internalized and traffick to intracellular sites such as nucleus, acts directly at nucleus to regulate transcription of various RAS components, and generates its usual second messengers at nucleus and mitochondrion. AT1 and AT2 receptors are associated with nucleus and mitochondria; binding at these organelles up-regulates nitric oxide. There is also evidence for intracellular synthesis of

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angiotensin II in some cells Likewise, renin is both an enzyme that cleaves angiotensin I from angiotensinogen but also is a hormone in its own right acting at specific cell receptors 11, 19-29. A renin transcript is also found in some cells which lacks the secretory signal and is expected to produce an

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active intracellular renin as opposed to a prorenin moiety. Intracellular renin potentially can generate angiotensin II in the intracellular space and also it may be that some functional renin receptors exist in the intracellular space 19. Renin is also internalized by cells; although the extent to which internalized

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renin is physiologically active or simply degraded is unclear, it does appear that some internalized renin is active 10. The fact that angiotensin II, renin, and other RAS components are intracrines raises the

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possibility that intracrine RAS action in the intracellular space could account for the inability of RAS blockade to blunt cardiorenal disease progression. If this is true and if angiotensin II is the operative RAS intracrine, the development of effective intracellular blockers of all modes of angiotensin II action should halt the progression of these cardiorenal disorders even if they involve cell autonomous RAS up-

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regulation driven by physiological factors such as ventricular wall-stress or glomerular hyperfiltration. However, the intracrine nature of RAS components also raises the possibility that intracrine upregulation of one or more RAS components leads to enhanced intracrine trafficking to nearby cells and

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the subsequent spread of cellular dysfunction. In this regard, it can be noted that intracrine angiotensin II, acting at nucleus, up-regulates renin and angiotensinogen 25. This provides the substrate for intracrine

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amplification of angiotensin II synthesis and therefore could support persistent pathology in affected cells as well as disease spread to target cells after known deleterious physiological factors have been corrected or mitigated .

INTRACELLULAR AND NON-CANONICAL INTRACRINE RAS ACTION If intracrine function is to be fully addressed, attention must be paid to the intracellular actions of these peptides. For example, the intracellular actions of angiotensin II could well be important in RAS driven

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pathology. Some RAS blockers such as the ARB losartan seem to enter cells and inhibit canonical angiotensin II action more than others, but a comprehensive evaluation of the intracellular actions of currently available drugs has not been undertaken 27. Similarly, the potential intracellular actions of

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other RAS components such as (pro)renin, angiotensin (1-7), angiotensinogen, and converting enzyme should be further explored.

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At the same time, attention must be paid to non-canonical and other atypical actions of angiotensin II and other RAS components whether that action takes place at cell membrane or in the cell 5,6,19, 22-26, 29-34.

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By non-canonical is meant action independent of established receptors and second messenger signaling systems; atypical action implies an unappreciated action associated with traditional receptor binding. As an example of non-canonical angiotensin II action at the cell membrane one can point to angiotensin binding to cell surface angiotensin converting enzyme with the latter protein serving to transduce a

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signal to the intracellular space 33. Intracellular non-canonical angiotensin II action is exemplified by direct binding of the peptide to electron transport chain proteins in mitochondria with secondary effects on the generation of reactive oxygen species; similarly the AT-1R independent actions of angiotensin II

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in cadiomyocytes exposed to high glucose likely represent another example 29, 31, 32 . Because intracellular concentrations of intracrines are lower their extracellular concentrations, and because

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intracellular non-canonical and atypical actions, such as effects on mitochondrial electron chain proteins and chromatin remodeling, likely are stochastic, it is to be expected that the time course of these actions is much slower than the seconds to minutes responses associated with canonical membrane binding 23, 24, 30, 31. As already noted, there are variations on canonical and non-canonical intracrine action. Some RAS signaling modes are mixed or atypical. For example, AT1 receptors undergo spontaneous slow cleavage of their cytoplasmic tails, generating an intracellular carboxy-terminal fragment 34. This process is somewhat reminiscent of the generation of beta-amyloid in Alzheimer’s

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disease and of the cleavage of other membrane receptors 15. AT-1R cleavage is enhanced by angiotensin II binding. Moreover, the carboxy-terminal fragment trafficks to nucleus and thereafter induces apoptosis 30, 34. This atypical canonical angiotensin II action (it employs a classical receptor but a

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previously unrecognized second messenger) is potentially pathological and could contribute to cell death in both heart failure and various forms of renal insufficiency. It is important to note that AT1 receptors, like AT2 receptors, have been identified on intracellular organelles such as nucleus and

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mitochondria, and angiotensin II binding to these receptors can generate canonical second messengers 20, 21, 23, 26, 27

. Therefore, it seems likely that AT1 receptor cleavage can also occur at these intracellular loci

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resulting in the generation of a pro-apoptotic, and potentially, pathogenic, carboxy-terminal receptor fragment. Thus, the development of binders/inhibitors of intracellular canonical, non-canonical, and atypical mediators potentially becomes important. Finally, although angiotensin II non-canonical action is highlighted here, it is possible that investigation of potential non-canonical actions of other RAS

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components could be productive.

HYPOTHESIS

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Given that : (i) RAS components, and in particular AII, can act as both extracellular and intracellular regulators of cell function, acting in the interiors of theirs cells of synthesis or in target cells after

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internalization; (ii) Angiotensin II, and perhaps other RAS components, can up-regulate its own synthesis through intracellular action; (iii) Interruption of angiotensin II action at the cell surface produces modest protection form progressive disease, indicating a pathological role for the peptide, It is hypothesized that, analogous to the pathogenicity of prion-like proteins, angiotensin II, and perhaps other RAS components, act in an intracrine fashion whereby their internalization is followed by persistent intracrine up-regulation followed by enhanced trafficking to nearby cells with the result that pathological over-stimulation occurs in the intracellular spaces of an expanding field of parenchymal

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cells. Cell death ensues and chronic tissue degeneration occurs. The intracellular actions of angiotensin II, and perhaps other RAS components, can be either canonical, non-canonical, or atypical, and novel Inhibitors of the RAS capable of working in cell interiors and capable of blocking all three modes of

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action will be required to achieve full RAS suppression.

That is to say, if a stress such as elevated glucose or high pressure persists for a sufficient time, not only

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is the intracellular RAS activated in random parenchymal cells, but feed-forward loops develop in those cells and these loops most likely are angiotensin II-driven. In that case, elimination of the stress or

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application of an AT-1R blocker would not eliminate ongoing intracellular angiotensin II up-regulation and the development of pathology over time in affected cells, although it would lessen the pathological effects of extracellular angiotensin II. Persistent secretion of the peptide over time would cause activation of the RAS in nearby cells. In the case of angiotensin II this spreading activation, but not

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persistent intracellular action, could be blunted, but likely not eliminated by most currently available ARBs, thereby slowing progression of disease. To the extent angiotensin II trafficked via exosomes, ARBs acting at the cell membrane would be ineffective in preventing intercellular spread. To the extent that

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any intracrine actions of other RAS components such as angiotensinogen or renin (either of which could indirectly up-regulate intracellular angiotensin II) are involved, cell surface AT-1 blockade would be of no

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benefit in limiting disease spread.

There are two distinct facets to this hypothesis. First, the intracellular actions of angiotensin II or other RAS components participate in producing cell pathology and death. Second, up-regulation (amplification) of angiotensin II or other RAS components occurs and is a critical element in producing ongoing disease in affected cells after initiating factors are removed and in producing ongoing disease spread. For example, elevated glucose up-regulates RAS synthesis in mesangial cells and podocytes and

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by the first aspect of the hypothesis this causes pathology as a result of higher than normal intracellular angiotensin II 35-37. RAS up-regulation with subsequent intercellular trafficking explains the fact that disease progresses/spreads, albeit more slowly, even if glucose and blood pressure—the initiating

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stimuli-- are controlled. RAS inhibitors capable of acting within the cell would block these effects, unlike simply lowering glucose.

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INTRACRINE FEED-FORWARD LOOPS AND RAS UPREGULATION

The contention that intracrines can generate feed-forward regulatory loops is borne out by a great deal

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of experimental evidence. For example, homeodomain transcription factors induce their own synthesis in nearby cells and in so doing produce a differentiated cell state as evidenced by the actions of PDX1 to induce beta cell differentiation and Pax6 to promote eye development; angiotensin II acting at nucleus up-regulates angiotensinogen and renin; dynorphin B up-regulates its own synthesis in the course of

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cardiac development; vascular endothelial growth factor (VEGF) up-regulates its own synthesis in myeloma cells and hematologic stem cells; parenthetically, VEGF is also up-regulated in podocytes exposed to high glucose and could participate as an additional pathogenic intracrine loop in kidney

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disease 6, 9, 13, 14, 25, 32, 38, 39. Many other examples could be provided 6-12.

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At the same time, components of the RAS are up-regulated in podocytes and mesangial cells in high glucose conditions and other simulated renal insults. High glucose up-regulates podocyte angiotensinogen and angiotensin II synthesis in cell culture. Angiotensinogen and AT1 receptor expression are up-regulated in a subset of podocytes and mesangial cells in the glomeruli of streptozotocin-treated diabetic rats 35, 37. Stretch both up-regulates podocyte angiotensin II and produces angiotensin II mediated apoptosis 35-37. These findings are relevant because podocyte dropout is a feature of diabetic nephropathy and a variety of other renal disorders associated with

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proteinuria—the types of renal disease shown to benefit from RAS inhibition 40, 41. It should be noted that podocyte injury upregulates hepatic angiotensinogen synthesis, but for the purpose of establishing intracellular feed-forward loops it makes little difference if internalized hepatic protein or locally

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synthesized angiotensinogen serves as precursor. Moreover, depending on conditions, high glucose, for example, can upregulate either podocyte angiotensinogen or renin to generate a feed-forward loop, and so the possible availability of hepatically synthesized precursor angiotensinogen for the support of feed-

(assessed by mRNA or protein) in a cell-autonomous fashion

42-44

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forward loops doesn’t negate the fact that both renin and angiotensinogen can be upregulated

. Similarly, mesangial expansion

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characterized by deposition of extracellular matrix (ECM) and mesangial cell hyperplasia/hypertrophy are characteristic of diabetic renal disease. Angiotensin II induces mesangial cell production of ECM by stimulating Transforming Growth Factor-beta1 (TGF-B1) and it also stimulates cell proliferation. Of note, there is evidence indicating that in some renal cells angiotensin II can act directly at nucleus to upregulate TGF-B1; parenthetically, TGF-B1 is itself an intracrine and like VEGF could participate in the

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creation of intracrine loops in the kidney 6,7, 24,40-46. High glucose up-regulates mesangial cell angiotensin

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II synthesis 36, 37.

Similarly, in cultured neonatal cardiac myocytes high glucose up-regulates RAS components—apparently

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with the formation of an angiotensin II feed-forward loop: high glucose up-regulates angiotensinogen and renin and secondarily increases angiotensin II, but an intracellularly active renin-inhibitor, aliskiren, blocks angiotensin II and angiotensinogen up-regulation, thereby revealing an angiotensin II/angiotensinogen feed-forward loop 32. Follow-on studies by this group in cardiac myocytes derived from diabetic rats indicate that up-regulated angiotensin II and angiotensinogen are normalized by aliskiren. 32. These innovative studies did not addresses how long RAS up-regulation could persist if, after long term exposure, high glucose is removed. Nor conversely, do the results directly address

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whether renin protein is down-regulated by suppressing the intracellular RAS with aliskiren. But, these studies demonstrate that a feed-forward loop is required for angiotensin II formation in high glucose conditions. These results are similar to those obtained in other models. Another group also suggested a

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feed-forward angiotensin II loop in adult cardiomyocytes derived from rats with streptozotocin –induced diabetes. In this study treatment with losartan (which can act within cells) down-regulated

cardiomyocyte angiotensin II formation and apoptosis. This again suggests that although diabetes

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initiated RAS up-regulation, a feed-forward loop was required to maintain it. Similarly, these

investigators found an angiotensin II feed-forward loop in cardiomyoctes from adult rat ventricles

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subjected to stretch 47, 48. Cardiomyocyte angiotensin II is markedly elevated in myocardial biopsies of patients with diabetic cardiomyopathy even when they are treated with oral hypoglycemic agents and angiotensin converting enzyme inhibitors 49. In summary, there is strong evidence for RAS up-regulation in models of progressive cardiorenal diseases, and this is, at least in part, secondary to feed-forward

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loops. Feed-forward loops in cardiomyocytes support the reasonableness of the present proposal. As expected, RAS up-regulation is more exuberant and uniform across cells in the cell culture models than in vivo. The operative in vivo RAS feed-forward loop in CRF and CHF likely is angiotensin II-driven

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angiotensinogen synthesis, but up-regulation of other RAS components and of other intracrine loops

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(VEGF, TGF-B1) could also play a role.

The principal intracrine model of disease progression presented here is dependent upon cell autonomous synthesis of angiotensin II. Message for (pro)renin, angiotensinogen, ACE, and AT1 receptor has been detected in a variety of cells and the proteins themselves have been detected (for example, in cardiomyocytes and podocytes), consistent with the capacity for cellular synthesis of angiotensin II 6, 7, 10, 32, 44, 50-54. Cells of the anterior pituitary contain prorenin, renin, and angiotensin II colocalized in secretory granules 51. This suggests that angiotensin II can be produced in secretory vacuoles

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from which it could enter the intracellular space either before or after secretion. But the existence of renin and ACE in the nucleus and mitochondria raises the possibility of intracellular angiotensin II synthesis outside secretory vacuoles 19, 22, 50. This is contentious given that angiotensinogen is a secretory

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protein. Nor does there appear to be an angiotensinogen transcript encoding a non-secreted form of the protein 19. However, cultured cardiomyocytes express message for renin and angiotensinogen, and accumulate or synthesize angiotensinogen. They synthesize angiotensin II and this appears to occur

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both in the cytoplasm and in secretory vacuoles 32. Juxtaglomerular cells, which do not express

angiotensinogen, nonetheless, synthesize intracellular angiotensin II apparently from internalized

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angiotensinogen 55. Moreover, angiotensinogen binding to cells with subsequent internalization has been reported 56. Finally, in astrocytes angiotensinogen has been reported in cytoplasm and nucleus 57. Thus, although angiotensin II produced in secretory vacuoles could be the driver of an intracrine system,

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the cytoplasmic synthesis of angiotensin II likely occurs.

An argument against the present hypothesis is that in cell cultures of podocytes and neonatal cardiac myocytes the up-regulation of cellular angiotensin II by high glucose is prevented by the direct renin

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inhibitor aliskiren which can enter cells 32, 43. Therefore one could argue that if the present hypothesis is correct, aliskiren should markedly slow the progression of diabetic renal disease and diabetic

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cardiomyopathy. Although definitive studies have not been carried out---usually aliskiren has been studied as an add-on to another RAS inhibitor leading to a variety of potentially harmful side effects and the degree to which the drug actually blunted intracellular angiotensin II in patients could not be determined—no such beneficial effects of aliskiren over usual RAS inhibition have been seen 58. However, it is important to note that in cell culture studies, aliskiren was administered before, or at the same time as the introduction of high glucose, and in control cells aliskiren reduced baseline angiotensin II synthesis either minimally or not at all. Rather, the drug only prevented the glucose mediated

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increase in angiotensin II 32, 43. This then raises the issues of whether the inhibition of intracellular renin was sufficient in the clinical studies to adequately lower intracellular angiotensin II, and whether the drug can break a positive feed-forward loop in human cells once the loop has been established 59. There

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are related issues that bear on the effects of aliskiren in human disease. The drug’s efficacy in

suppressing intracellular angiotensin II likely is different in cell culture than in vivo; in an in vivo model of diabetes, RAS up-regulation occurred in only a subset of podocytes and mesangial cells at a given time,

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so the efficacy of a drug like aliskiren is dependent on its ability to reach and act on those glomerular cells (as opposed, for example, to being removed from the circulation by renin-rich juxtaglomerular cells

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in the afferent arteriole or binding to renin released by those cells) 35, 37, 59, 60. Also, angiotensin II can be formed in the intracellular space independent of renin through the cleavage of angiotensin (1-12). It is possible that a feed-forward loop driven by up-regulation of angiotensinogen with secondary generation, possibly by kallikrein, of angiotensin (1-12) and then of angiotensin II---a pathway not

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dependent on renin---could be operative in human disease 32, 61, 62. To the extent this occurred, aliskiren would not be effective. To the extent that angiotensinogen is up-regulated in human disease and trafficks between cells, intracellular angiotensin II could remain elevated in treated patients given that

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aliskiren produces only partial renin inhibition 32, 42-44, 59. Moreover, although possibly model-related, there are data showing that aliskiren therapy can prevent, and even reverse, renal disease in transgenic

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mice over-expressing renin, consistent with the possibility that there is species variability in the response to the drug and possibly to the ARB losartan which also can act in the intracellular space and is very effective in another rodent renal disease model 27,,63. Similarly, aliskiren appeared to better reduce the intracellular angiotensin II of cardiomyocytes of diabetic rats than did other RAS blockers, and this was associated with reduced oxidative stress, apoptosis, and fibrosis; whether these effects would improve benefit, or arrest disease, over time is unknown 64. However, in these and other in vivo studies, it is necessary to distinguish aliskiren’s effect on perturbation-driven processes, such as those produced

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by on-going hyperglycemia, as distinct from slow-to-develop, longer-term progression in the absence of the underlying insult. And, as already noted, it is in general necessary when studying responses to aliskiren to distinguish drug effects in cell culture from those in vivo, and effects in rodents and from

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those in man. Further study will be needed to better define the in vivo actions of aliskiren and other RAS inhibitors on the human intracellular RASs before conclusions can be reached based on the clinical

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response to these agents.

CONCLUSION

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Here it is suggested that intracrine biology can explain progressive renal disease and cardiac insufficiency. The processes involved could mirror those operative in several common neurodegenerative disorders caused by prion-like-proteins. But there are differences. First, in the case of disease caused by prion-like proteins, pathology is caused by the action of a mis-folded form of a

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prion-like protein and even if the normal form of the prion-like protein is an intracrine (as they often are), the action of the mis-folded protein differs from that of its normal homologue. Indeed, because the mis-folded form usually results in aggregation, a portion of its pathological effects are arguably

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secondary to loss of its normal functionality. Thus, the process involved in prion-like neurodegenerative disease properly should be deemed aberrant intracrine action or intracrine-like, rather than intracrine

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per se. Second, alternative modes of disease progression secondary to established physiological risk factors such as blood pressure are more apparent in the case of the cardiovascular disorders. However, the distinction between established physiological factors and intracrine mediators of disease progression may be more apparent than real. One thread linking these mechanisms is the evidence that stretch (a physical promoter of disease) activates the RAS in multiple cell type; the two mechanisms may actually be overlapping 35, 37, 47, 48. Third, the absence of protein aggregation and mis-folding in congestive heart failure and chronic progressive renal disease further distinguishes the neurological

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disorders from the cardiovascular pathologies. And here it is proposed that the persistent abnormal upregulation of a normal RAS intracellular function produces cellular dysfunction in cardiorenal disease rather than the effects of an abnormal protein. Nonetheless, there are similarities between the two

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disorders. Both the prion-like neurodegenerative diseases and the proposed intracrine mechanism of cadiorenal disease involve disease spread by proteins capable of up-regulating their own synthesis by one means or another and then producing intracellular pathology. Exploring these similarities,

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especially the twin notions of cell autonomous protein amplification and intercellular spread, could inform medicine’s understanding of both and lead to more effective therapies directed at canonical,

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non-canonical, and atypical intracrine action. Irrespective of the correctness of the suggestions that intracrine amplification and trafficking play a role in progressive cardiorenal disease, investigating such unexpected angiotensin II actions as the production of pro-apoptotic AT-1R fragments, and the direct binding of angiotensin II to mitochondrial electron transport chain proteins, would likely be informative. 27, 64-67

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.

Funding: This work was supported by the Ochsner Clinic Foundation

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Disclosures: None

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FIGURE LEGEND

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Top panel: High glucose can up-regulate angiotensinogen (Aogen) and/or renin in a subset of cells, resulting in increased angiotensin II (AII) synthesis. Middle panel: After persistent stimulation a feedforward loop is formed resulting in sustained RAS activation even when high glucose is no longer present. Lower panel: In time intracrines such as angiotensinogen or angiotensin II traffick to nearby

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cells and produce feed-forward loops in them resulting in the spread of intracellular pathological action

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(IPA). This causes progressive disease.

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