Insulin Signalling to the Kidney in Health and Desease

Clinical Science (2013) 124, 351–370 (Printed in Great Britain) doi: 10.1042/CS20120378

Insulin signalling to the kidney in healthand diseaseLorna J. HALE and Richard J. M. COWARD

Academic and Children’s Renal Unit, University of Bristol, Learning and Research, Southmead Hospital, Bristol BS10 5NB, U.K.

AbstractNinety-one years ago insulin was discovered, which was one of the most important medical discoveries in the pastcentury, transforming the lives of millions of diabetic patients. Initially insulin was considered only important forrapid control of blood glucose by its action on a restricted number of tissues; however, it has now become clear thatthis hormone controls an array of cellular processes in many different tissues. The present review will focus on therole of insulin in the kidney in health and disease.

Key words: diabetes, diabetic nephropathy, insulin, intracellular signalling, kidney, metabolic syndrome

OVERVIEW OF CELLULAR INSULINSIGNALLING

The role of insulin in the human body has been an active sub-ject of interest since the discovery of insulin in 1921 by Banting,Best, Collip and Macleod. The critical importance of this find-ing was recognized by the Nobel Committee in 1923 when theyawarded Banting and Macleod the Nobel Prize in Physiology orMedicine [1] just 2 years after their discovery. In the 50 years thatfollowed the effects of insulin were intensely studied and revealedits glucose-controlling effects focusing on the liver, muscle andadipose tissue [2]. Insulin is a highly potent physiological ana-bolic hormone that promotes the synthesis and storage of lipids,carbohydrates and proteins, while also inhibiting their degrada-tion and release back into the circulation. In mammals insulin isthe main hormone controlling blood glucose; it achieves this bystimulating glucose influx and metabolism in muscles and adipo-cytes, and by inhibiting gluconeogenesis by the liver. These tis-sues have always been considered the classically insulin-sensitiveorgans of the body. However, insulin has the ability to modify theexpression and/or activity of an assortment of enzymes and trans-port systems in a wide variety of cell types [3], as the presentreview will describe.

Abbreviations: BK channel, large-conductance Ca2 + -activated K+ channel; BP, blood pressure; CAP, Cbl-associated protein; DM, diabetes mellitus; DN, diabetic nephropathy; DOK,downstream of kinase; ECM, extracellular matrix; eNOS, endothelial NO synthase; ERK, extracellular-signal-regulated kinase; ESRD, end-stage renal disease; FSGS, focal segmentalglomerulosclerosis; GBM, glomerular basement membrane; GFB, glomerular filtration barrier; GFR, glomerular filtration rate; GLUT, glucose transporter; GEnC, glomerular endothelialcell; Grb2, growth-factor-receptor-bound protein 2; IGF, insulin-like growth factor; IGF-IR, IGF-I receptor; IL, interleukin; IR, insulin receptor; IRS, insulin receptor substrate; ksp,kidney-specific; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MPGN, membranoproliferative glomerulonephritis; mTOR, mammalian target of rapamycin;mTORC, mTOR complex; OMIM, Online Mendelian Inheritance in Man®; PH, pleckstrin homology; PI3K phosphoinositide 3-kinase; podIRKO mouse, podocyte-specific IR-deficienttransgenic mouse; PPARγ , peroxisome-proliferator-activated receptor γ ; PTB, phosphotyrosine-binding; Raptor, regulatory associated protein of mTOR; RBF, renal blood flow; SGK1,serum- and glucocorticoid-induced protein kinase 1; SH2, Src homology 2; SOS, Son of Sevenless; TRPC, transient receptor potential cation channel; TSC, tuberous sclerosis complex.

Correspondence: Dr Richard Coward (email [email protected]).

INSULIN AND IGF (INSULIN-LIKE GROWTHFACTOR) RECEPTORS

When discussing the receptors that insulin can signal throughit is important to consider another closely related collection ofhormones, namely the IGF family. The reason for this is that theIGF hormones, IGF-I and IGF-II, have structural similarity to in-sulin and their major functional receptor, IGF-IR (IGF-I receptor)[4,5], is also structurally similar to the IR (insulin receptor). Thesignificance of this is that insulin can signal through the IGF-IR and likewise IGF-I/-II can signal via the IR, although withdiffering affinities. Indeed, it is even more complicated than thisas hybrid receptors are formed, by combinations of the IR andIGF-IR, through which all of the hormones can signal but withdiffering affinities (Table 1). Insulin has the greatest affinity forthe IR, so the rest of the present review will predominantly focuson this receptor.

The IR in humans is located on chromosome 19 and is en-coded by a gene containing 22 exons and 21 introns spanning120 kb [6,7]. It is a heterotetrameric receptor consisting of two α

and two β subunits [8], which are linked by disulfide bonds in aβ-α-α-β configuration (Figure 1). The α subunits are extracellu-lar and have the insulin-binding domain, whereas the β subunits

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Table 1 Receptor subtypes of the insulin/IGF system in mammalsVariable assembly of receptors showing the primary ligand-binding affinities for each.

Ligand(s)

Receptor Name Level of affinity . . . High-level Mid-level Low-level

Homotetramers

IR-B/IR-B IR-B Insulin IGF-II IGF-I

IR-A/IR-A IR-A Insulin and IGF-II IGF-I

IGF-IR/IGF-IR IGF-IR IGF-I and IGF-II Insulin

Heterotetramers (hybrids)

IR-B/IR-A HIR-AB Insulin and IGF-II

IR-B/IGF-IR HR-B IGF-I IGF-II

IR-A/IGF-IR HR-A IGF-I, IGF-II and insulin

have three compartmental domains: extracellular, transmembraneand cytosolic domains. Tyrosine residues in the cytosolic domainof β subunits are involved in signal transduction and are auto-phosphorylated when insulin binds to the receptor or by exogen-ous tyrosine kinase activity [8].

There is a further level of complexity within the IR as it existsin two different isoforms, A and B, which are formed due to theinclusion or exclusion of exon 11 of the IR gene [9–11]. IR-Alacks exon 11, whereas IR-B includes it. IR-A is widely expressedthroughout the body but is importantly up-regulated during pre-natal development and when cells become cancerous [7]. IR-Bis expressed largely in the classically insulin-sensitive tissues ofliver, skeletal muscle and adipose tissue. Interestingly IR-B isalso expressed in the kidney [12,13]. The IR isoforms dimerizeand can form either ‘pure’ or ‘hybrid’ receptors with each otheror the IGF-IR. The receptor make-up dictates the affinity of thecell for insulin and/or the IGF ligands, as the different receptorshave differing affinities for each of these molecules (Table 1). Itshould also be noted that IRs are not solely located in glucose-regulating insulin target tissues, but in many other tissue types,suggesting other functional roles of insulin signalling in multiplebiological systems distinct from glucose homoeostasis.

The IR and IGF-IR mediate the actions of IGF-I, IGF-II andinsulin. The IGF-IR shares a high degree of homology with theIR [14,15] (Figure 1). It is therefore unsurprising that insulin iscapable of activating the IGF-IR and vice versa. IGF-I has thegreatest affinity for the IGF-IR, followed by IGF-II, with insulinhaving a 500-fold lower affinity in comparison with its primaryligands [14].

CELLULAR INSULIN SIGNALLING PATHWAYS

The majority of work in this field has been performed on adipo-cytes, liver and skeletal muscle, as these are crucial for post-prandial glucose regulation in response to insulin.

The insulin signal transduction pathway is highly conservedand responsible for the regulation of a number of aspects of cel-lular physiology, most notable of which is the metabolic effectsof glucose uptake and its utilization within the cell. Followinga meal, increased levels of insulin encourage enhanced glucoseuptake, metabolism and storage within muscle and adipose cells

[16]. Insulin levels rapidly increase approximately 10-fold aftera meal from a basal level of approximately 50 pmol to 600 pmol[17]. GLUTs (glucose transporters) are energy-independent andallow glucose to enter or leave the cell, passively down a con-centration gradient, when they are incorporated into the cellmembrane. The classic insulin-responsive glucose transporter isGLUT4 [18], which translocates from a cytoplasmic vesicularpool to the plasma membrane in response to insulin. This is thesignature molecule of rapidly insulin-sensitive cells that absorbglucose. However, there is also robust evidence that GLUT1 [19]can also translocate in a similar manner from an intracellularpool to the plasma membrane and rapidly increase its plasmamembrane concentration in response to insulin. GLUT1 is alsoa constitutional transporter in many cells [20]. Here it sits at theplasma membrane of cells continuously and allows a constantdelivery of glucose for cellular function.

The IR differs from many other receptor tyrosine kinasesin that, instead of recruiting downstream effector molecules toits phosphorylated cytoplasmic domains, when activated it phos-phorylates a number of scaffolding proteins which then in turn areresponsible for recruiting various downstream effector proteins[21]. A number of intracellular substrates have been discovered,including the IRS (insulin receptor substrate) family (IRS1–IRS4), IRS5/DOK4 (downstream of kinase 4), IRS/DOK5, Gab1,Cbl, APS [adaptor protein with PH (pleckstrin homology) andSH2 (Src homology 2) domains] and Shc isoforms, and SIRP(signal regulatory protein) family members [22,23]. The bestcharacterized have been the IRS family of proteins [24]. IRS pro-teins do not possess intrinsic catalytic activities, and are insteadcomposed of multiple interaction domains and phosphorylationmotifs. Four IRS proteins have been identified (IRS1–IRS4), withIRS1 and IRS2 being the most widely expressed. Each IRS pro-tein has the distinct characteristics of an N-terminus PH domainadjacent to a PTB (phosphotyrosine-binding) domain, ending in avariable length C-terminus. The C-terminal tail of each IRS pro-tein contains tyrosine phosphorylation sites that serve as on/offswitches and recruit the downstream signalling proteins. IRS1and IRS2 have the longest tail therefore providing them with agreater number of possible phosphorylation sites (20) in com-parison with IRS3 and IRS4 [24]. The IRS proteins form animportant node of control for the regulation of insulin and IGFsignal transduction in cells.

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Insulin and the kidney

Figure 1 Structure of the IR and IGF-IRBoth the IR and the IGF-IR are transmembrane tyrosine kinase cell-surface receptors that mediate the actions of IGF-I,IGF-II and insulin [5]. The IGF-IR shares a high degree of homology with the IR [14] and, like the IR, forms a β -α-α-βtetramer composed of two α and two β subunits joined by disulfide bonds [15]. This Figure was adapted and reprintedfrom Brain Research Reviews, 44(2–3), Hawkes, C., and Kar, S., The insulin-like growth factor-II/mannose-6-phosphatereceptor: structure, distribution and function in the central nervous system, 117–140, Copyright (2004), with permissionfrom Elsevier.

When the intrinsic tyrosine kinase activity of the receptoris triggered by insulin binding, three major signalling path-ways have been described that are propagated in response: (i)CAP (Cbl-associated protein), (ii) the PI3K (phosphoinositide3-kinase) pathway, and (iii) the MAPK (mitogen-activated pro-tein kinase) pathway (Figure 2). The PI3K pathway is one ofthe best characterized downstream effectors of the IRS proteinsand activates many of the metabolic functions of insulin. Asso-ciation of the p85 regulatory subunit with IRS proteins leads tothe activation of further downstream molecules, including Aktsubstrates (which form another functional node in the pathway)and mTOR (mammalian target of rapamycin) [25,26], eventuallyresulting in PI3K being targeted to the plasma membrane [22].There is evidence that mTOR is important for kidney functionand this will be discussed in more detail later in the presentreview.

The stimulation of glucose uptake by insulin is mediated byPI3K-dependent and PI3K-independent pathways, which play avital role in the translocation of GLUT4. This is highlighted byuse of the PI3K inhibitor wortmannin, which is able to com-pletely block the uptake of glucose into cells upon insulin stimu-lation [27]. However, despite the critical role of PI3K in insulin-stimulated glucose uptake, activation of at least a second pathwaydistinct from PI3K is also necessary [28]. This is evident froma number of studies that have examined different elements ofthe pathway during insulin signalling. By overexpressing a con-stitutively active membrane-bound form of Akt in 3T3L1 adipo-

cytes, glucose transport and GLUT4 translocation increases inthe absence of insulin [29]; conversely, the insulin-stimulatedtranslocation of GLUT4 is inhibited by the expression of adominant-negative Akt mutant [30]. These results indicate thatAkt is required for insulin signalling. However, if the PI3K path-way is activated by factors other than insulin, such as PDGF(platelet-derived growth factor) or IL (interleukin)-4, althoughthese factors can robustly activate PI3K and Akt, they do notpossess insulin’s ability to stimulate GLUT4 translocation andglucose uptake [28]. This suggests that other pathways also needto be activated by insulin to elicit an effect. A number of studieshave suggested that a separate signalling pathway exists for theIR in microdomains within the cell, such as lipid rafts [22]. It hasbeen proposed that insulin can also activate the GTPase TC10,via lipid-raft localization of the CAP–Cbl–Crk complex and theguanine-nucleotide-exchange factor C3G [31], and initiate gluc-ose uptake in cells. This process occurs independently of PI3Kand has been shown to be crucial to insulin-stimulated GLUT4translocation [32,33]. However, in contrast with this, findingsby Mitra et al. [34] have reported that Cbl/CAP isoforms arein fact not required components of insulin signalling to GLUT4transporters; therefore the precise role of this pathway remainscontentious.

The final pathway through which insulin can act is the MAPKpathway. This pathway controls a range of cellular activities,including differentiation, proliferation, transformation, survivaland death [35–37]. The mammalian MAPK family consists of

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Figure 2 Substrates of the IR family and the major cellular signalling pathways evokedThere are a number of critical nodes which regulate the biological response to each stimulus. These include the CAP/Cblpathway (1), the PI3K pathway (2) via Akt and the MAPKs (3). AS160, Akt substrate of 160 kDa; FOXO1, forkhead box O1;GSK-3, glycogen synthase-3; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate;PDK1, phosphoinositide-dependent kinase 1; PKB, protein kinase B; PTP1B, protein tyrosine phosphatase 1B; pY, tyrosinephosphorylation.

p38, ERK (extracellular-signal-regulated kinase) and JNK (c-JunN-terminal kinase), each of which exist in a number of isoforms:p38-α, -β, -γ and –δ, ERK1–8 and JNK1–3 [35,38]. In termsof insulin signalling, the MAPK pathway is activated follow-ing the binding of Grb2 (growth-factor-receptor-bound protein2) and SOS (Son of Sevenless) to phosphotyrosine residues onShc and Gab1 [39]. Phosphorylation of certain tyrosine residueson Gab1 are required for binding to and activation of the proteintyrosine phosphatase SHP2 (SH2 domain-containing protein tyr-osine phosphatase 2) [40,41], whereas phosphorylation of certaintyrosine residues on Shc allow the binding of Grb2/SOS to thesesites [42]. This binding initiates activation of the GTPase Ras,followed shortly by Raf, leading to a kinase cascade resulting inthe phosphorylation and activation of the MAPK pathway [39]. Itis important to note that the p85/p110 PI3K complex also bindsto Ras, thereby connecting two pathways which are often con-sidered to be separate [43]. With regard to insulin signalling, theMAPK pathway is primarily associated with the regulation ofmitogenesis [44]. Again, stimulation of the MAPK pathway inisolation is not able to induce glucose uptake in fat or muscle.

In summary, insulin transduces its signal through at least threedifferent cellular pathways. There are also a number of criticalnodes involved in insulin signalling, including the IR–IGF-IRcomplexes, IRS molecules and Akt/MAPK substrates that areable to modify the biological effects of a ligand on a particularcell type. What is currently unclear is how cells are able to controlwhich pathways are activated in response to specific ligands. Thiscould be of great therapeutic benefit as it may enable novel waysof overcoming cellular resistance or hyperstimulation of eitherthe insulin or IGF pathways.

INSULIN SIGNALLING IS NOT ALL ABOUTGLUCOSE HOMOEOSTASIS

IR, IGF-IR and hybrid receptors are expressed throughout thebody in most tissues and are not restricted to the classic insulin-sensitive glucose uptake tissues, such as liver, muscle andfat [45]. It is therefore not surprising that insulin has other



important biological effects on many different tissues independ-ent of glucose uptake, as evolution rarely allows redundantsystems to persist. Two good examples of important non-glucose-controlling actions of insulin are found in the brain and the car-diovascular system. In the brain, insulin signalling is crucial forcontrolling appetite, regulating food intake [46–48] and prevent-ing obesity and infertility [45]. These are all features associatedwith insulin resistance. Similarly, within the cardiovascular sys-tem, insulin can directly signal to both the heart dynamicallyregulating metabolism [49] and also the peripheral vasculaturecontrolling tone. Cardiovascular tone is controlled both centrally[50] and through local modulation of endothelial NO release byinsulin [51]. This is potentially relevant in the cardiovascularmorbidity and mortality associated with insulin-resistant states.

INSULIN AND THE KIDNEY

The kidney is the major organ that regulates fluid balance, BP(blood pressure), acid–base status, haemoglobin production, elec-trolyte control and waste removal in the body. It is a highly vascu-lar structure that receives 20 % of the circulating blood volume permin through the renal artery: the RBF (renal blood flow). Fromhere, glomeruli within the kidney are perfused and their filtrateper min is defined as the GFR (glomerular filtration rate). Withineach kidney are approximately 1 million nephrons. These are thefunctional units of the kidney. Each nephron has a primary filter-ing unit, the glomerulus, and a tubule, which is able to modifythe primary filtrate produced from the glomeruli. There is nowaccumulating evidence that both the glomeruli and tubules areinsulin sensitive.

INSULIN AND THE KIDNEY: IN HEALTH

The active insulin molecule, after loss of the c-peptide, existsas a monomer; it contains 51 amino acids and is approximately6 kDa in size, allowing it to freely traverse the GFB (glomerularfiltration barrier) and pass into the tubular lumen [6]. This isimportant as it allows insulin rapid access to all of the cells inthe glomerulus and nephron after it has been secreted into thecirculation and passes through the renal artery.

The major high-affinity receptor for insulin, the IR, is locatedthroughout the kidney in all of the cells of the glomerulus [52–55]and the entire length of the renal tubule, from the proximal tubuleto the collecting ducts [56–59]. Recently, it has also been shownthat kidney, as a whole, abundantly expresses IR isoform B [60],which is the isoform found in the classically insulin-responsive,glucose-regulating, tissues of fat, skeletal muscle and liver. It isnow also clear that insulin is involved in a number of homoeostaticphysiological responses throughout the kidney and are describedbelow.

Renal gluconeogenesisTwo main processes, glycogenolysis and gluconeogenesis, resultin the release of glucose, both of which involve the hydrolysis of

glucose 6-phosphate by glucose-6-phosphatase. Originally, it wasthought that the liver was the only organ in the body that couldrelease glucose in times of starvation and stress. However, it isnow clear that the kidneys are also capable of gluconeogenesis.This explains why patients with fulminant liver failure can main-tain their circulating glucose concentrations [61]. It transpiresthat both the liver and the kidney possess sufficient gluconeo-genic enzyme and glucose 6-phosphate activity to facilitate thisproduction [62]. Furthermore, the kidney not only releases gluc-ose in times of acidosis or after prolonged fasting as initiallythought, but is also able to release significant amounts of gluc-ose in normal post-absorptive individuals [63]. Insulin reducesrenal gluconeogenesis [64,65], so is an important factor in thecontrol of glucose release from here. The importance of renalgluconeogenesis is discussed in depth in a review by Gerich etal. [62] and highlights that the kidney may be equally as import-ant in the production and regulation of gluconeogenesis as theliver. The cellular location of gluconeogenesis in the kidney isnot entirely clear; however, the proximal tubules have been shownto express glucose-6-phosphatase as have the parietal cells andpodocytes [66]. Clinically, this may be relevant as congenitalloss of glucose-6-phosphatase causes glycogen storage diseasetype I. These children have developmental delay, episodes ofhypoglycaemia and, from a renal perspective, proximal renal tu-bular dysfunction and glomerular damage as illustrated by FSGS(focal segmental glomerulosclerosis) on renal biopsy [67]. There-fore, interestingly, the renal phenotype of this condition revolvesaround the two cell types that are known to express glucose-6-phosphatase in the kidney.

Haemodynamic control of renal organ blood flowand local glomerular blood flow: the role of insulinThere are conflicting findings on the direct role of insulin in RBFthrough the major vessels and local glomerular blood flow withinthe kidney, which appears to be species-dependent. Early workon conscious dogs suggested that insulin decreased RBF and thatthis was independent of the effects of insulin on the sympatheticnervous system [68]. However, in humans, a number of studies[69–72], but not all [73,74], have shown that RBF actually in-creases in response to insulin. It is possible that the differencesreported in these studies are due to dual actions of insulin. In-sulin is able to act systemically by stimulating a catecholamineresponse and activating the sympathetic nervous system [75].However, it can also cause a local renal vasodilatory effect in thekidney.

A more consistent finding is that insulin increases the GFRin insulin-sensitive subjects. This occurs through local renal vas-odilation and is mediated by a prostoglandin-dependent pathwaythat can be blocked with indomethacin [76] and regulated byeNOS (endothelial NO synthase) [77]. Interestingly, a numberof groups have shown that insulin increases the GFR in normalsubjects, but this response is lost in insulin-resistant subjects[72,78,79]. However, and in contrast, a recent study has identi-fied a human polymorphism in the IRS1 gene which is associatedwith an increased GFR [80], and the authors speculate that lossof renal insulin signalling may, in fact, be responsible for anincreased GFR.

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Figure 3 The glomerulusThe diagram in the upper panel is a simplified view of the glomerulus.This is shown in elegant detail using electron microscopy (lower panel),where the specific cell types and distinct areas of the glomerulus arehighlighted in a mouse glomerulus from our laboratory. The upper panelwas reproduced from Postgraduate Medical Journal, Vinen, C.S., andOliveira, D.B, 79, 206–213, 2003 with permission from BMJ PublishingGroup Ltd.

Specific cellular actions of insulin throughout thenephronGlomerulusGlomeruli are composed of three different resident cell types:podocytes, GEnCs (glomerular endothelial cells) and mesangialcells. It is now clear that all of these cells respond to insulin,but in different ways. Podocytes and GEnCs are separated bythe GBM (glomerular basement membrane) and constitute theGFB. Mesangial cells are specialized smooth-muscle-like cellsthat are able to contract and regulate blood flow to the glomerulus(Figure 3). The glomeruli filter as much as 5 million litres of

Figure 4 Transmission electron micrograph of the glomerularcapillary cell wallThree interacting layers make up the glomerular capillary cell wall:GEnCs, whose fenestrations are denoted by �, the GBM, and podo-cytes, whose tertiary foot processes are denoted by FP. Examples of aslit diaphragm between the foot processes are indicated by the thickarrows.

primary urine across the GFB during an average human lifetimeand, as the primary urine is practically protein-free, this meansthat more than 200 000 kg of albumin has to be prevented fromcrossing [81].

PodocytesPodocytes are unique cells found on the urinary side of the GFB(Figure 4). In the past 15 years, it has become clear that theyare critically important in maintaining the integrity of the GFBand preventing leakage of albumin into the urine. There are nowmore than ten inherited human genetic mutations, all of whichcause nephrotic syndrome and all of which code for proteinsfound predominantly in the podocyte (Table 2). Podocytes areembryonically derived from mesenchyme and are classified bymost as epithelial cells, as they are polarized and sit on a basementmembrane. However, they also have features of other cell types,including smooth muscle, as they are contractile and expresssmooth muscle markers [82], and neurons due to their processes,secretory capacity and, for the most part, inability to replicatewhen fully formed [83]. Podocytes adhere to the GBM through anetwork of anchoring proteins [84–86] and have specialized mod-ified adherens junctions, called slit diaphragms, formed betweentheir foot processes. These contain a set of proteins that arecrucial in maintaining the integrity of the GFB [81,87]. It isnow clear that the podocyte depends on its actin cytoskeleton tomaintain its structure and the integrity of the GFB and many ofthe slit diaphragm proteins are linked to this [88,89]. Although themajority of disease-causing mutations in the podocyte are relatedto actin-regulating functions, it is of note that gain-of-functionmutations in the Ca2 + channel modulating TRPC6 [TRPC



Table 2 Podocyte mutations associated with disease

Gene names Protein(s) encoded Consequence(s) of mutation

NPHS1 Nephrin Massive proteinuria in utero, nephrosis at birth: congenital nephrotic syndrome ofthe Finnish type (NPHS1) [202]

NEPH1 Structurally related protein to nephrin Proteinuria and perinatal lethality in mice at 8 weeks [203]

ACTN4 α-Actinin-4 Results in autosomal-domininant form of FSGS; adult onset nephrotic syndrome[204]

WT1 Wilms tumour protein Linked to Denys–Drash syndrome, pseudohermaphroditism; proteinuria occurs early[205]

LMX1B LIM homeobox transcription factor 1-β Childhood-onset proteinuria, regulates expression of multiple podocyte genesrequired for differentiation and function; causes nail-patella syndrome [206]

CD2AP CD2-associated protein Congenital nephrotic syndrome [207]

NHPS2 Podocin Steroid-resistant nephrotic syndrome; presents in early childhood [208]

TRPC6 Transient receptor potential cationchannel, subfamily C, member 6

Autosomal-dominant FSGS; normally presents in late childhood/early adulthood [90]

INF2 Formin Autosomal-dominant FSGS; adolescence or adulthood presentation: often mildproteinuria initially [209]

PLCE1 Phospholipase C, ε1 Early onset nephrotic syndrome with ESRD [93]

COQ6 Co-enzyme Q10 biosynthesismono-oxygenase 6

Early onset nephrotic syndrome, sensorineural deafness and early lethality in some[92]

FAT Protocadherin Fat 1 Proteinuria and perinatal lethality in mice [210]

(transient receptor potential cation channel), subfamily C, mem-ber 6] has also been discovered to cause nephrotic syndrome[90,91], as well as mitochondrial proteins [92] and enzymaticproteins [PLCE1 (phospholipase C, ε1)] [93].

In recent years, our group [52,94] and others [95] have shownthat the podocyte is a rapidly insulin-responsive cell. Our initialwork employed a conditionally immortalized human podocytecell line [96] to study insulin responses in this cell. This washelpful as the cell line contains a temperature-sensitive transgenethat enables the cells to replicate and proliferate ad infinitumat 33 ◦C but when they are thermo-switched to 37 ◦C they exitthe cell cycle and are able to differentiate and express many ofthe markers of maturity. This is important as the thermo-switchedcells resemble mature podocytes found in the normal glomerulus.We found that differentiated podocytes rapidly respond to insulinby doubling their glucose uptake within 15 min [97]. This issimilar to the kinetics observed in muscle, which is not surprisinggiven the muscle-like features and markers that podocytes exhibit[82]. Importantly proliferative immature 33 ◦C human podocytes,human proximal tubular cells and human GEnCs did not respondto insulin in respect to glucose uptake. We went on to showthat this process was dependent on the actin cytoskeleton andactivated translocation of the glucose transporters GLUT1 andGLUT4 from cytoplasmic vesicles to the plasma membrane ofthe cell [97].

We extended these observations to show that the podocyteprotein nephrin was also important in insulin signalling in thepodocyte [98]. This was achieved by studying conditionally im-mortalized podocytes derived from children with the most severeform of congenital nephrotic syndrome called Finnish type con-genital nephrotic syndrome. This occurs secondary to mutationsin the protein nephrin. We developed a number of conditionallyimmortalized natural human knockout cell lines from nephrin-deficient (no nephrin protein made) or nephrin-mutant (protein

made but unable to target to the plasma membrane) kidneys. Wefound that these cells were completely unresponsive to insulin inrespect to glucose uptake, but could be rescued by genetically re-constituting nephrin back into them. Mechanistically this was dueto a failure of GLUT-rich vesicles to dock and become incorpor-ated into the plasma membrane of the nephrin-deficient/mutantpodocytes. We went on to show that the C-terminus of neph-rin was able to form a protein–protein association with VAMP2(vesicle-associated membrane 2), which is important for vesicledocking with the plasma membrane through SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein re-ceptor) processes [98]. Although nephrin is only expressed in thepodocyte in the kidney it is also found in pancreatic β-cells inthe body, where it has also been shown to be potentially involvedin insulin release here in response to glucose [99].

Interestingly given the possible role of nephrin in both insulinrelease and its cellular action, children with nephrin mutationswho receive kidney transplants do not appear to develop overt DM(diabetes mellitus). This suggests that, although nephrin may beinvolved in the control of insulin release, it is not critical.

Recently, we have generated a podocyte-specific IR-deficienttransgenic (podIRKO) mouse, which has proven to be highlyinformative [94]. podIRKO mice develop albuminuria and lossof foot process architecture by 8 weeks of age. In light of this,we have also found that insulin is able to rapidly remodel thefilamentous actin cytoskeleton of podocytes. This is via the IR andthrough modulation of small GTPases, which act as molecularswitches for actin remodelling in cells. RhoA is activated andCDC42 is inhibited [94]. We have also shown that in the podocyteinsulin stimulates the PI3K and MAPK signalling cascades, butnot the CAP/Cbl pathway.

Another recent exciting advance in our understanding of thehomoeostatic role of insulin on podocyte biology has come fromDryer’s group. They have discovered that insulin rapidly increases

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Figure 5 Post-prandial actions of insulin in the podocyteInsulin is released into the circulation by the pancreas and then reaches the podocyte by passing through the GFB. It issmall (6 kDa), so freely passes through here. It then causes the podocyte changes depicted in a short time frame of lessthan 15 min.

the membrane expression of Ca2 + and K+ channels in the podo-cyte [100]. They have demonstrated that, within minutes, in-sulin causes TRPC6 to increase on the surface of podocytes, butTRPC5 channels decrease. Functionally this causes an increasedinflux of cations, particularly Ca2 + , into the cell. This elegantstudy went on to show that the effects of insulin on TRPC6 weremediated through the production of ROS (reactive oxygen spe-cies) via activation of NADPH oxidases [100]. The same grouphave also shown that, in addition to modulating TRPC channelsin podocytes, insulin also rapidly causes Ca2 + -regulated K+

channels to locate to the plasma membrane of this cell [101].Collectively these findings suggest that, when insulin stimulatesthe podocyte, as occurs after a meal, it causes the podocyte torapidly take up a readily usable energy source, glucose, remodelits actin cytoskeleton and contract, which is facilitated by ionicflux into the cell. We think this makes biological sense in orderfor this cell to ‘brace’ itself for the increased work this cell isrequired to perform at this time (Figure 5).

GEnCsGEnCs line the capillaries of the glomerulus (Figure 4). Theyare highly fenestrated and therefore freely permeable to an arrayof molecules. These cells express IRs [55,102], IGF-IRs [103]and hybrid receptors [104], so have the receptor apparatus forinsulin signalling. Early work has suggested that their primaryrole was to remove insulin from the circulation and degrade it[105]. However, we have shown that insulin activates the PI3Kpathway rapidly in human GEnCs [94], but it does not have thesame biological actions in these cells as in podocytes and doesnot cause rapid glucose uptake [52] or actin remodelling [94].Using human GEnCs that have been conditionally immortalized

[106] in the same way as podocytes, we have found that insulinrapidly induces the production of eNOS (J. Hurcombe and R.J.M.Coward, unpublished work). This complements work by Mimaet al. [107], who have demonstrated that glomeruli isolated fromrats are insulin-sensitive and insulin also rapidly induces eNOS.

Recently, it has been shown that reduced endothelial insulinsignalling elsewhere in the body reduces local eNOS productionand impairs the physical delivery of insulin to extra-capillary sites[108]. As insulin is secreted into the circulation from the pan-creatic β-cells, it needs to traverse the endothelial layer of bloodvessels to reach extra-vascular targets. However, in the glom-erulus, the GEnC is highly fenestrated, which may protect theunderlying podocytes from insulin deficiency when the GEnC isinsulin-resistant. Elucidating the functional importance of insulinsignalling in the GEnC in the intact glomerulus is currently chal-lenging. This is partly due to a lack of transgenic tools to be able togenetically manipulate the GEnC. Specifically, there is currentlyno way of targeting cre recombinase to this cell within the kidney,which is important when developing cell-specific-knockout micemodels using cre-loxp technology [109]. However, there is nowpromise that this may be rectified in the future, as specific GEnCgenes have recently been identified within the kidney which maybe beneficial in developing these mice [110] and genetically ma-nipulating the GEnC in the intact glomerulus in the future.

Mesangial cellsMesangial cells are specialized contractile cells that support theglomerular capillaries. Their contractile properties show similar-ities to vascular smooth muscle cells [111,112], with the release ofCa2 + from stores within the ER (endoplasmic reticulum) uponthe initiation of contraction. The released Ca2 + activates Cl−



channels, which then depolarize the cell membrane and activateVOCCs (voltage-operated Ca2 + channels), resulting in an in-crease in intracellular Ca2 + levels and the subsequent activationof BK (large-conductance Ca2 + -activated K+ ) channels. Activ-ation of BK channels then causes the cell membrane potential tohyperpolarize [113,114].

Both the IR and IGF-IR are expressed in mesangial cells,although at differing levels, with the IGF-IR being predomin-ant [54]. Despite this, insulin at high levels is able to stimulatemesangial cell proliferation through activation of either receptor[115]. BK channels are expressed in abundance in mesangialcells, where they contribute to the relaxation of the cell and as aresult an increased GFR [116]. Similar to podocytes, insulin hasbeen shown to increase the density of BK channels in the plasmamembrane of human mesangial cells via activation of the MAPKpathway [117].

Unlike the podocyte, mesangial cells do not rapidly increaseglucose in response to insulin and, as a result, the intracellularglucose level observed more directly reflects its plasma con-centration. Excessive extracelluar glucose in the diabetic mileucan enter mesangial cells with ease via GLUT1 in an insulin-independent manner, which can result in glucotoxicity. Highglucose levels have been shown to enhance GLUT1 expression inmesangial cells, which may result in progressive damage [118]; inaddition, these glucose-induced effects can be mimicked in ‘nor-mal’ glucose conditions by specifically overexpressing GLUT1[119], resulting in excessive production of ECM (extracellularmatrix) proteins [120].

Apoptosis is an important mechanism within a number oforgans, tissues and cells, and the glomerulus is no exception.Apoptosis of glomerular cells is a closely regulated process; itcan be beneficial in allowing the removal of excess cells in orderto resolve glomerular injury, but can also be detrimental if ex-cessive apoptosis is allowed to occur leading to hypocellularity[121]. Insulin has been shown to be a pro-survival factor via ac-tivation of PI3K, which in turn allows the recruitment of Akt tothe plasma membrane, where it phosphorylates a number of mo-lecules to suppress apoptosis [122,123]. Both insulin and IGF-Iprotect mesangial cells from a variety of apoptotic triggers viathe PI3K/Akt pathway using this mechanism [124]; in conjunc-tion with these findings, insulin has also been shown to reduceERK1/2 activation and increase levels of the cyclin-dependentkinase inhibitor p21 during apoptosis, providing an additionallevel of protection.

Renal tubulesThese are segregated into a number of defined regions, includ-ing the proximal tubule, loop of Henle, distal convoluted tubuleand the collecting ducts. This part of the kidney is able to modu-late the primary filtrate from the glomerulus by reabsorbing ionsback into the blood or secreting ions into the urine. Important ionsthat are regulated include glucose, Na+ and HCO3

− resorptionin the proximal tubule, Na+ and water resorption in the loop ofHenle, and K+ and H+ secretion in the distal convoluted tubule,together with water retention in the collecting ducts. Collectively,the tubules are able to control acid–base status, Na+ and waterresorption and hence BP regulation in the body.

The tubule consists of a number of different specialized po-larized epithelial cells that are able to transport molecules to andfrom the tubular lumen into and from the circulation. Numer-ous groups have shown that the IR, IGF-IR and hybrid receptorsare expressed throughout the tubule [56,59,125,126], includingthe proximal tubule, loop of Henle, distal convoluted tubule andcollecting ducts. More insulin binds to the tubules in compar-ison with the glomeruli, although with less affinity as reportedby some [127], but not all groups [57]. In our initial work, westudied immortalized proximal tubular cells (HK2) and foundthat they did not respond to insulin in respect to glucose uptake[52]. However, it is clear that the tubules are insulin-responsive,but that insulin elicits different cellular effects in this part of thekidney. Elegant work by Mima et al. [107] has shown that ex vivorenal tubules from rats are insulin-responsive in respect to activ-ation of the PI3K and MAPK pathways. Interestingly, this insulinresponse is not lost in established diabetes, as was found withinsulin responses in the glomeruli in these studies. Furthermore,in vitro studies of isolated tubular cell types suggest that insulincan rapidly modify a variety of transporter systems throughoutthe tubule [128–131]. These include the NHE3 (Na+ /H+ ex-changer type III) [132,133], which is the major Na+ transporterin the proximal tubule and responsible for 65 % of Na+ resorp-tion here. Modulation of this channel is also able to alter theacid–base status in the body. Insulin also augments Na+ resorp-tion through other transporters throughout the tubules, includingthe loop of Henle, via the butamide-sensitive Na+ –K+ –2Cl−

channels [131], and ENaC (epithelial Na+ channel), Na+ /K+ -ATPase and recently the Na+ –Cl− co-transporter [134] in thecollecting ducts. A number of studies examining the distal tu-bule have demonstrated that insulin binds to the IR and activ-ates the PI3K pathway, which then phosphorylates and stimu-lates SGK1 (serum- and glucocorticoid-induced protein kinase1) phosphorylation [135] that inhibits the breakdown of transport-ers through endocytic retrieval pathways and may also directlyphosphorylate transporters resulting in enhanced actions [136].This is interesting, as SGK1 seems to be a connection throughwhich mineralocorticoids and insulin can modify Na+ retentionin the distal part of the nephron. In addition to Na+ , insulin canalso modulate the resorption of other ions in the proximal tubule,including PO4

3 − (phosphate ion) through the Na+ – PO43 − co-

transporter type-II in the proximal tubule [137] and Mg2 + in thedistal convoluted tubules [138]. A comprehensive review ofthe role of insulin in the renal tubules was performed in 2007and we would recommend reading this excellent article [139].

INSULIN SIGNALLING AND THE KIDNEY:DISEASE

Systemic insulin-resistant statesInsulin-resistant states are a major global healthcare problem inthe 21st century, with an estimated 171 million diabetics presentin the world. However, owing to current sedentary lifestyles,population aging and urbanization, the anticipated number ofcases is predicted to more than double in the next 15 years [140].The incidence of the insulin-resistant metabolic syndrome is even

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Figure 6 Histological changes in the kidney from a patient with DMThese images demonstrate the characteristic changes observed in the glomeruli of patients with DN compared withthose of a healthy patient. Upper panels show a normal glomerulus. Light microscopy Periodic Acid–Schiff staining(left-hand panel), PAAg (Periodic Acid silver) staining (middle panel) and transmission electron microscopy (TEM) at ×2900magnification (right-hand panel). Lower panels show the classic features of DN. Glomerulosclerosis, mesangial matrix,mesangial hypercellularity and Kimmelstein–Wilson lesions (arrowed) are shown in the light microscopy pictures. Thickeningof basement membrane are shown in the transmission electron microscopy pictures on the right. These pictures weregenerated and supplied by Dr Tibor Toth, Department of Pathology, Southmead Hospital, Bristol, U.K.

more pronounced, with an estimated prevalence of 20 % in thepeople over 20 years of age in the U.S.A. rising to over 40 % whenover 60 years of age being reported [141].

Approximately one-third of all new cases of ESRD (end-stagerenal disease) worldwide is accounted for by DM [142] and, in theU.S.A., this is even higher with over 50 % of new patients havingDM (U.S. Renal Data System, USRDS 2011 Annual Data Report;http://www.usrds.org). Type 2 DM is due to insulin resistance ofperipheral tissues, in contrast with Type 1 DM which that occurssecondary to insulin deficiency caused by destruction of the β-cells of the pancreas [143]. DN (diabetic nephropathy) is the mostcommon microvascular chronic complication of DM [144]. DNis a progressive disease which takes several years to develop; itoccurs in 30–40 % of patients with Type 1 DM and 8–10 % ofpatients with Type 2 DM [87]. Its natural history is dominated byprogressive albuminuria.

There is now accumulating evidence that a loss of insulinresponses in the kidney may contribute to a number of the com-plications that occur in insulin-resistance states, including albu-minuric glomerular disease and hypertension.

The glomerulus, insulin, diabetes and the metabolicsyndromeEarly renal manifestations of DN are focused on the glom-erulus in the kidney consisting of glomerular hyperfiltrationand microalbuminuria, alongside other changes, including GBMthickening, mesangial expansion and accumulation of ECM pro-teins such as laminin, collagen and fibronectin [144]. AdvancedDN is characterized by increased albuminuria (macroalbumin-

uria), glomerulosclerosis, interstitial fibrosis and ESRD [87,144](Figure 6). In recent years, the podocyte has become an intensefocus of research into this field as loss of this cell has been foundto be the best histological predictor of progression in DN [145].Furthermore, as progressive albuminuria dominates the naturalhistory of DN, this also makes the podocyte an attractive targetcell in DN, because of its crucial role in preventing albuminuria,as discussed above (Table 2) [146].

The metabolic syndrome is also associated with microalbu-minuria; indeed, it is part of the diagnostic criteria in some clas-sifications, including that of the World Health Organization.

As described above, we have shown that the human podocyteis an insulin-sensitive cell [52] and we have developed podIRKOmice. These mice were highly informative as they developeda number of features of DN, including albuminuria, glomer-ulosclerosis, matrix accumulation (including type-IV collagen),thickening of the GBM and podocyte apoptosis. However, theyall had normal blood glucose control, demonstrating that noneof these features were driven by hyperglycaemia. This suggeststhat insulin signalling to the podocyte is critically important fornormal glomerular function and may also have a role in someaspects of DN. It should be noted that the podIRKO mice onlyexhibited some features of DN and did not have enlarged kid-neys nor did they have mesangial hypercellularity or the classicnodular Kimmelstein–Wilson lesions of DN (Figure 6). A po-tential explanation for this is that in diabetes there is a loss ofinsulin sensitivity of the podocyte and this results in some of thepathological consequences associated with DN, but other aspectsof DN, for example renal hypertrophy and mesangial expansion,


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are driven by other factors. These may include pathways drivenby high glucose levels or other growth factors such as IGF-I orIGF-II acting on the cells of the glomerulus.

Our current working hypothesis is that, in both Type 1 andType 2 DM, as well as the metabolic syndrome, in those pa-tients who develop nephropathy, there is insulin resistance in thepodocyte which contributes to the development of renal damage.Type 2 DM and the metabolic syndrome are intrinsic cellularinsulin-resistant conditions, so this hypothesis would appear in-tuitively to be correct. Type 1 DM occurs due to a lack of insulin;however, there is compelling evidence that those patients withType 1 DM who develop nephropathy are more likely to alsobe insulin-resistant. Prolonged Type 1 DM causes insulin resist-ance [147], and nephropathic compared with non-nephropathicpatients with Type 1 DM require larger doses of insulin to controltheir diabetes [148], are more insulin-resistant when assessed byeuglycaemic clamps [149] and are more likely to have a strongfamily history of cellular insulin resistance [150].

There is further experimental evidence to support this hy-pothesis. Tejada et al. [95] have examined podocyte insulin re-sponses in the development of albuminuria in the db/db Type 2DM mouse model and have shown that IR and PI3K signalling islost early in the disease process. Furthermore, a recent excellentrodent study [107] examining Type 1 and Type 2 DM models hasexamined the effect of diabetes on insulin signalling in the kid-ney. Mima et al. [107] studied rats given streptozotocin inducingType 1 DM and the Zucker obese model of Type 2 DM. They al-lowed the rats to develop diabetes and then examined their insulinsignalling pathways in the glomerular and tubular compartmentsof the kidney. They found that insulin rapidly initiated PI3K andMAPK signalling in both isolated glomeruli and tubular frac-tions of control rats. However, when the rats had either Type 1 orType 2 DM they both resulted in a loss of insulin signalling viathe PI3K pathway specifically in the glomerular and not in thetubular compartment. They went on to show that high glucoseincreased ubiquitination and hence loss of IRS1 in the glomeruli,which could be a mechanistic pathway through which Type 1DM is able to directly modulate cellular insulin signalling in theglomerulus. Finally, building on previous work [151,152], theydemonstrated that inhibiting PKCβ (protein kinase Cβ) was ableto reverse high-glucose-induced insulin resistance in GEnCs viaeNOS. This adds weight to the assumption that GEnC as well aspodocyte insulin resistance may be important in the developmentof glomerular complications in systemic insulin-resistant states.

A consequence of increased insulin signalling in the podocyteis increased translocation of GLUT4 and GLUT1 to the plasmamembrane of this cell, allowing more glucose to passively dif-fuse into the cell. Previously, it has been proposed that a majorreason for cellular dysfunction in the setting of diabetes is glucosetoxicity of cells [153]. In the glomerulus there is evidence thatoverexpression of GLUTs in the mesangial cell is detrimentalto function [154]. However, this does not seem to be the casefor podocytes. An elegant study by the Brosius group [155] hasshown that increasing the glucose transporter GLUT1 specific-ally in the podocyte in a model of Type 2 DM is not detrimentalto glomerular function, but intriguingly seems to be protectiveagainst the development of some aspects of DN. GLUT1 is a

glucose transporter that is expressed at the cell surface consti-tutionally in many cells and allows basal glucose uptake, butis also found in insulin-responsive translocatable glucose trans-porter vesicular pools similar to GLUT4. One possibility whythis mouse was protected from the development of DN is thatit may have been protected from episodes of glucose deficiencywhen stimulated by insulin [155].

Our research has also shown that nephrin is crucial for theinsulin sensitivity of podocytes. Many groups have demonstratedthat nephrin is reduced early in DN [156–158]. Potentially thiscould be inducing insulin resistance in these cells resulting inpathological changes. However, it is also possible that the lossof nephrin in the development of DN is also a consequence ofpodocyte dysfunction and not a cause.

What is currently unclear is the in vivo insulin responsive-ness of the podocyte in systemic insulin-resistant states. Somegroups have found that, similar to the classically metabolic-ally insulin-responsive tissues of adipose [159], liver [160] andskeletal muscle [161], that the IR is down-regulated at the pro-tein level in the kidneys of models of Type 2 DM and systemicinsulin resistance. However, other groups have found in Type 2DM that, although adipose, liver and skeletal muscle demonstratediminished insulin binding and signalling, the kidney does not[57,162,163]. Therefore it is possible that early in the develop-ment of diabetes there is hyperstimulation of the insulin signallingaxis and not loss of insulin sensitivity in the kidney, and this mayalso be having a detrimental effect.

There is evidence that insulin can modulate the GFB, whichmay be through direct insulin ligand receptor binding. Approxim-ately 30 years ago insulin-deficient human subjects with Type 1DM were given insulin under euglycaemic clamp conditions, i.e.maintaining the blood glucose constant, and this resulted in atransient increase in albumin excretion into the urine [164]. Fur-thermore, it was shown that giving a glucose load to healthysubjects and also patients with Type 1 DM only caused an in-crease in urinary albumin excretion in the healthy subjects [165].This suggests that hyperglycaemia is not the major driver causinga loss of albumin into the urine, as this occurred in the patientswith Type 1 DM who did not develop albuminuria (and could notproduce insulin in response to the glucose challenge), but thatin healthy subjects high glucose levels stimulated insulin release(which lowers their blood glucose levels) and caused albumin toleak into the urine. These insulin effects may explain the imme-diate post-prandial proteinuria that is widely described in humansubjects and rodents [166,167].

If podocyte insulin resistance is a key factor in the develop-ment of nephropathy in diabetes then it follows that strategies toenhance podocyte cellular insulin sensitivity could be beneficialin treating this condition. There is evidence that some agentswhich have insulin-sensitizing properties, including metforminand PPARγ (peroxisome-proliferator-activated receptor γ ) ag-onists, are beneficial in preventing kidney damage in models ofDN in both Type 1 [168] and Type 2 [169,170] DM, as wellas other non-diabetic chronic kidney diseases [171,172], whichpartially supports this premise. Indeed, we have shown that thePPARγ agonist rosiglitazone is able to directly enhance insulinsensitivity of the podocyte in vitro [173].

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Figure 7 Insulin regulates the mTOR complex and is also regulated by itThis simplified diagram demonstrates how insulin is able to activate mTORC1. It is now also clear that inhibiting mTORchronically with rapamycin can induce insulin resistance in cells. This is through a loss of mTORC2, which causes decreasedAkt signalling. MEK, MAPK/ERK kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; Rictor,rapamycin-insensitive companion of mTOR; RSK, ribosomal S6 kinase.

Conversely, we have found that factors that are increasedsystemically in the insulin-resistant metabolic syndrome renderthe podocyte insulin-resistant. These include non-esterified ‘free’fatty acids such as palmitate [174]. Other groups have also ex-plored some of the molecular inhibitors of insulin signalling inthe podocyte. An elegant study demonstrated that SHIP2 (SH2-domain-containing inositol phosphatase) was able to inhibit in-sulin signalling in the podocyte and that it was up regulated inthe glomeruli in a rat model of Type 2 DM [175]. Discoveringthese molecules is important, as they could potentially be goodtherapeutic targets when inhibited.

Finally, there are findings indicating that insulin can regulatemesangial cell function in the glomerulus and when this cell isrendered resistant it potentially contributes to matrix formation[176], mesangial expansion [177] and hyperfiltration [178].

In summary, there is now evidence that all three celltypes found in the glomerulus can respond to insulin, but theyrespond in different ways. Furthermore, when insulin signallingis altered in the glomerulus it results in pathology.

mTOR, insulin and glomerular disease: anotherimportant pathwayAnother potentially important role for insulin in the glomerulusis modulating the mTOR pathway. The mTOR signalling cascadecontrols cellular protein synthesis, growth, metabolism, auto-phagy and survival in response to growth factors, stress, energyand nutrient stimuli.

mTOR is a protein kinase and the catalytic subunit of two func-tional complexes: mTORC1 (mTOR complex 1) and mTORC2(mTOR complex 2) [179]. mTORC1 is a rapamycin-sensitivecomplex in which mTOR is associated with the Raptor (regulatoryassociated protein of mTOR) and regulates a number of cellular

processes, including protein synthesis, cell growth and prolifera-tion [179,180]. Insulin is able to increase the activity of mTORC1through PI3K and ERK1/2 pathways via TSC2 (tuberous sclerosiscomplex 2). Both pathways inhibit TSC2, which then preventsit from suppressing mTORC1 expression. This results in proteintranslation, ribosomal biogenesis and autophagy [181]. Interest-ingly, it has also recently become evident that mTOR inhibitioncauses cellular insulin resistance [182] and that this action isthrough the mTORC2 complex [183]. Therefore the mTOR path-way is both controlled by, and also controls, insulin signalling(Figure 7).

Rapamycin (Sirolimus) is used in renal transplantation be-cause inhibiting mTOR inhibits the response of B- and T-cellsto IL-2 and hence prevents organ rejection. mTOR expres-sion is low or undetectable in the normal kidney but followingischaemia/reperfusion injury it increases significantly, presum-ably to enable cellular repair and regeneration to occur. In thissetting, rapamycin is detrimental as it inhibits mTOR and resultsin delayed renal repair and recovery [184].

The function of mTOR in the glomerulus remains contro-versial; however, the findings from a number of recent elegantmurine transgenic studies [185–187] have advanced the under-standing of the role of mTOR in one cell type located in thekidney, namely the podocyte. Previous work has reported thatsystemic administration of rapamycin in mouse models of bothType 1 and Type 2 DM can prevent the progression of DN [188–190], suggesting it may be clinically beneficial to inhibit mTOR inthese settings. However, it has also been shown that mTOR inhibi-tion with rapamycin can be detrimental in non-diabetic conditionsand can cause proteinuria and glomerulosclerosis in both humansand rodents [191–194]. Studies published recently by Inoki et al.[185], Godel et al. [186] and Cina et al. [187] have highlighted the



critical importance of the mTOR pathway in podocyte biology. Bymanipulating mTORC1 and mTORC2 they have shown that lossof mTORC1 is detrimental to glomerular function [186] and thisis exacerbated further when mTORC2 is also lost [186,187]. Inter-estingly increasing the activity of mTORC1 can also be harmfulto the glomerulus causing albuminuric renal disease resemblingDN [185]. Increased mTORC1 activity occurs in DN and can berescued by genetic or pharmacological inhibition of mTORC1.

In summary, insulin signalling can modify mTOR activityin the glomerulus, predominantly through mTORC1 but it canalso be modified and suppressed when mTORC2 is inhibited bylong-term rapamycin therapy.

The tubule, insulin, diabetes and the metabolicsyndromeAs discussed above, insulin is able to modulate tubular function ina number of ways. One of the most important effects seems to beits action on Na+ resorption from the tubule into the circulation.As hypertension is a prominent feature of the metabolic syndromeand DN, it is not surprising that a great deal of work has exploredthe potential role of tubular insulin sensitivity and its role in thecontrol of BP.

In 2008, Tiwari et al. [195] developed a mouse model in whichthe IR was knocked out in the renal tubules by crossing a floxedIR mouse with a ksp (kidney-specific)-cadeherin promoter-linkedcre recombinase-producing mouse. Ksp-cadeherin is only foundin the tubular epithelial cells in the kidney. It is predominantlyexpressed in the distal aspect of the tubules from the thick as-cending loop of Henle through to the collecting ducts [196]. Asdiscussed in the previous section, work based predominantly oncell culture models had suggested that a loss of tubular insulinsignalling would result in a reduction of Na+ resorption from theurinary filtrate and hence a naturesis with an associated loweringof the BP. However, intriguingly, this was not the case with thismodel. These mice had impaired urinary Na+ excretion in re-sponse to a Na+ load and were hypertensive in comparison withcontrols. The group went on to show that insulin signalling herewas able to activate local NO production and reduce BP in wild-type normal animals, presumably through a vasodilatory effect,but in tubular IR-knockout mice this did not occur. This suggestsanother role of tubular insulin signalling in NO production andBP control. It may also be clinically relevant as hypertension iscommonly associated with insulin-resistant states.

Rare renal disease associated with severe insulinresistanceIn addition to the very common conditions of DM and the meta-bolic syndrome there are a number of rare syndromic forms ofcellular insulin resistance that are associated with glomerularrenal disease. It is interesting that these do not always resultin classic DN, but rather in a spectrum of renal pathologies asfollows.

LipodystrophiesThis group of disorders is caused by a failure to deposit fatin adipose tissues. This causes abnormalities in circulatingadipokines and results in the deposition of fat in ectopic loca-

tions such as skeletal muscle and liver. These patients are oftenseverely insulin-resistant at a cellular level.

The most severe form of lipodystophy is the generalized formwhere the patients have no adipose tissue. The majority of pa-tients with congenital and acquired forms of this condition haveglomerular disease with albuminuria, but only a small subsethave the classical features of DN. The rest have a variety of renalpathologies, including FSGS and MPGN (membranoproliferativeglomerulonephritis).

Another cohort of patients suffer from acquired partiallipodystrophies. These patients lose adipose tissue from theirface, neck, upper extremities, thorax and upper abdomen, andhave immunological abnormalities with low C3 complementlevels and elevated C3 nephritic factor levels. The most com-mon renal lesion found in these patients is MPGN type 2 (densedeposit disease). It has been hypothesized, but not categoricallyproven, that this is an immunological disease; however, there maybe a contribution from insulin resistance. The renal phenotypesof extreme insulin resistance are extensively reviewed by Mussoet al. [197], which we would recommend reading.

Diseases that target the IRAuto-antibodies against the IR (type B insulin resistance).This is associated with extreme insulin resistance and DM in themajority, although paradoxically these auto-antibodies can alsocause hypoglycaemia in some situations [198]. Patients usuallyhave an underlying collagen vascular disease, most commonlysystemic lupus erythromatosis. Again, more than 50 % of patientshave albuminuria but their histology is normally that of one ofthe forms of lupus nephritis [198,199].

Mutations of the IR (type A insulin resistance). Two syn-dromes account for the majority of patients suffering from muta-tions of the IR: Donogue syndrome, previously known as Lep-rechaunism [OMIM (Online Mendelian Inheritance in Man®)147670], and Rabson–Mendenhall syndrome (OMIM 262190).These patients have extreme insulin resistance, acanthosisnigrans, hirsuitism and are generally slender. These patients com-monly, but not always, have DN [200], which may be secondaryto the cellular insulin signalling defect they experience.

FUTURE DIRECTIONS

It is now clear that the kidney is an insulin-sensitive organ, butthat different regions of the kidney respond to insulin in differentways. To elucidate the clinical relevance of insulin sensitivityof the kidney there are still some fundamental questions thatneed to be addressed and will be a focus of research in the up-coming years. These questions include the following. (i) Doesinsulin resistance in the GEnC and/or mesangial cell contributeto glomerular pathology? (ii) Do human renal cells in vivo be-come insulin-resistant in systemic insulin-resistant states? Thishas never been proven. (iii) Can we therapeutically manipulatethe insulin signalling pathway in the kidney to prevent renal dis-ease from developing? Ideally this would be kidney (cell)-specificto reduce off-target side effects, as have been experienced with

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other insulin-sensitizing drugs, such as the glitazones [201]. (iv)Why does insulin elicit different biological responses in differenttissues? This is probably due to differences in important sig-nalling nodes in the insulin and IGF pathways, but again this isnot proven.

CONCLUSIONS

It is now clear that the kidney is an insulin-responsive organ ina variety of different ways. Manipulating these responses mayhave great therapeutic potential in treating glomerular diseaseand hypertension associated with DM and other insulin-resistantstates.

ACKNOWLEDGMENT

We thank Dr Tibor Toth (Department of Pathology, Southmead Hos-pital, Bristol, U.K.) for producing Figure 6.

FUNDING

Our own work was supported by Kidney Research UK and the Med-ical Research Council.

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Received 13 July 2012/14 September 2012; accepted 26 September 2012

Published on the Internet 27 November 2012, doi: 10.1042/CS20120378


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Insulin Signalling to the Kidney in Health and Desease

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