Podocyte Growing Pains in Adaptive FSGS

FSGS is a pattern of glomerular injury caused by diverse podocyte insults. Etiologies include circulating permeability factors, genetic defects, viruses, drugs, and adaptation to elevated glomerular capillary pressures and flow rates (glomerular hypertension).¹ The clinically common adaptive form is mediated by increased filtration demand placed on a reduced number of functioning nephrons (as in renal agenesis, oligomeganephronia, or any advanced CKD) or an initially normal nephron endowment (as in morbid obesity). Glomerulomegaly and relatively mild foot process effacement are pathologic hallmarks of adaptive FSGS.²

Among the four cell types that make up the glomerular corpuscle (the glomerular endothelial cell, mesangial cell, podocyte, and parietal epithelial cell [PEC]), only the adult podocyte has limited capacity to replicate. The final common pathway in all forms of FSGS is podocyte depletion, which occurs primarily by detachment of viable cells rather than in situ apoptosis or necrosis.³ Because podocytes are terminally differentiated, sensitive to cell stressors, and incapable of self-renewal, they can be considered the weakest link in glomerular structural maintenance, such that critical reduction in podocyte number compromises glomerular integrity, leading to FSGS.

Glomerular size in the adult is determined by combined influences of body weight and nephron number (a function of nephron endowment at birth and potential loss of nephrons due to genetic defects, aging, environmental insults, and/or acquired kidney disease). Seminal works by Brenner and colleagues⁴ and Ichikawa and colleagues⁵ have shown that forms of adaptive FSGS share elevated glomerular capillary pressures, single-nephron filtration rates, and glomerular growth (hypertrophy). The resulting increased glomerular filtrate flow exerts circumferential and axial capillary wall stress, causing shear stress on podocytes.^3,6 Endlich and Endlich⁷ have estimated that the shear forces normally acting on the podocyte foot processes (8 Pa) and podocyte cell bodies (0.05 Pa) in the rat can double under hyperfiltration conditions. Elegant ultrastructural studies by Kriz and colleagues^3,6,8 have elucidated the sequence of injurious glomerular structural responses. As the glomerular tuft enlarges and its capillary loops dilate, the resulting mechanical strain on podocytes promotes hypertrophy of the podocyte cell bodies, stretching and thinning of their cytoplasm to cover a larger filtration surface, decreased podocyte density, and reduced outflow tracts from the subpodocyte space. Podocyte hypertrophy eventually reaches a breakpoint, giving rise to focal foot process detachment and podocyte loss.

Several animal models of glomerular growth have been exploited to elucidate the nature of the podocyte injury in adaptive FSGS. Most models produce a hypertrophic stimulus via renal ablation, such as 5/6 nephrectomy in the adult rat⁴ or uninephrectomy in the young growing rat.⁸ An ingenious strategy is to engineer a genetic model of impaired podocyte hypertrophy, on which hypertrophic stressors then can be applied. The AA-4E-BP1 transgenic rat, developed in the laboratory of Wiggins and colleagues,⁹ provides just such a model. Podocyte growth and hypertrophy normally are driven by growth factor and nutrient sensing through the mammalian target of rapamycin complex 1 (mTORC1) kinase, which phosphorylates and activates 4E-BP1 protein to initiate transcription. Rats expressing a podocin-AA-4E-BP1 transgene that substitutes two alanine (indicated as AA) residues for the threonine residues required for phosphorylation have defective mTORC1 signaling. Their podocytes fail to hypertrophy in response to growth stimuli. Because the transgene is expressed exclusively in podocytes under control of the podocin promoter, there is no impairment of nutrient-induced hypertrophy in any other cell type of the body. Accordingly, in this unique model, body growth and glomerular growth can be dissociated from podocyte growth.

In prior studies, Fukuda et al.⁹ showed that wild-type Fischer rats fed an ad libitum diet developed an exponential increase in glomerular tuft volume in response to increase in body weight. Podocyte volume also increased exponentially in response to body weight gain but at a lower rate, producing a mismatch between glomerular tuft volume and total podocyte volume. This mismatch was exaggerated in AA-4EBP1 transgenic rats, where podocyte hypertrophy was genetically impaired, leading to FSGS by age 7 months. The degree of FSGS and the severity of proteinuria were proportionately greater in homozygotes than heterozygotes and increased linearly with hypertrophic stressors, such as increased body weight and uninephrectomy. As seen in human adaptive FSGS, the podocyte’s interdigitating system was focally lost, giving rise to sites of podocyte depletion and glomerular basement membrane denudation that provide the nidus for FSGS.⁹

In this issue of the Journal of the American Society of Nephrology, Nishizono et al.¹⁰ introduce three strategies to reduce glomerular growth in the AA-4E-BP1 model. These include calorie intake restriction, rapamycin to inhibit mTORC1, and the angiotensin-converting enzyme inhibitor enalapril. After each intervention, they studied the relationships between the variables of body weight, kidney weight, systolic BP, urine protein-to-creatinine ratio, creatinine clearance, and FSGS lesions. A novel feature is the application of detailed podometric indices, such as podocyturia (measured as percentage change in the urine podocin-to-creatinine ratio), glomerular tuft volume, mean podocyte volume, podocyte number per tuft, and podocyte nuclear density.

In this model,¹⁰ both modest calorie restriction and rapamycin achieved a reduction in the rates of body weight gain, kidney weight gain, and glomerular hypertrophy. In turn, both strategies prevented podocyturia, podocyte depletion from glomeruli, proteinuria, development of FSGS, and reduction in GFR. By contrast, enalapril did not slow body growth. Nonetheless, enalapril did slow the rate of kidney growth and glomerular hypertrophy and did lead to reductions in podocyte detachment, proteinuria, and FSGS, while also reducing systolic BP. Thus, angiotensin-converting enzyme inhibitor therapy was the only maneuver that dissociated kidney growth from body growth, consistent with its recognized renoprotective effects. This finding suggests that normal kidney growth requires angiotensin II, whereas body growth does not, in keeping with its known role in prenatal kidney morphogenesis.¹¹ Adding modest (20%) caloric restriction to enalapril was more effective than either therapy alone.¹⁰

A conceptual question is why rapamycin, a global mammalian target of rapamycin inhibitor, does not exacerbate the glomerular phenotype by causing greater inhibition of podocyte growth. An explanation for this apparent paradox is that the mTORC1 pathway in podocytes is already impaired in this model. The protective effect of rapamycin is due to slowing the growth rate of other cells in the body to blunt body weight gain and reduce glomerular volume increase. This slowing of glomerular cell (mesangial and endothelial) growth rate reduces the rate of glomerular enlargement and thereby, prevents podocyte hypertrophic stress from reaching critical levels for FSGS development.

AA-4E-BP1 rats fed an ad libitum diet developed FSGS by 7 months, which was accelerated to 3 weeks after uninephrectomy.¹⁰ Their kidneys exhibited increased intraglomerular Ki67-positive cells, consistent with glomerular growth. The podocyte was the only cell type not found to enter cell cycle (using WT-1 and Ki67 double staining), arguing against mitotic catastrophe as a mechanism of podocyte depletion in this model.¹² All three treatments caused reduced rates of cell cycling in renal tissue compartments, consistent with reduction in kidney growth.¹⁰ Of note, PEC Ki67 staining was increased only in rats that developed FSGS, supporting the recognized capacity of PECs to replace lost podocytes.¹³

The major genes differentially expressed from the glomerular isolates of kidneys that developed FSGS versus those where FSGS was prevented by therapy were cell cycle related, including regulators of G2M checkpoint.¹⁰ Because the glomerular isolation method by sieving did not capture Bowman’s capsule, the findings support the importance of cell division by glomerular endothelial and mesangial cells (rather than PECs) in glomerular growth–induced FSGS. However, more sensitive quantitative in situ hybridization and isolated single-glomerular cell transcript analysis would be needed to determine the expression levels in specific glomerular cell types and exclude a contribution by migratory PECs.

A provocative finding is that podocyte density, but not average podocyte number, decreased in association with early-stage FSGS development, preceding detectable increases in podocyturia.¹⁰ This observation led the authors to speculate that podocytes mobilize and abandon segments destined to become sclerotic to minimize proteinuria and preserve adjacent segments. However, their methodology of averaging podocyte counts in 25 consecutive glomeruli in 3-μm histologic sections may not be sensitive enough to detect small podocyte losses in the subset of glomeruli with FSGS. Moreover, the ability of podocyte injury to induce podocyte migration has not been shown in lower organisms, such as zebrafish.¹⁴ Three-dimensional techniques should allow more precise counting of podocyte number per volume in whole glomeruli with and without FSGS lesions to test this intriguing hypothesis.¹⁵

This report convincingly shows the therapeutic value of targeting glomerular growth to preserve podocytes in adaptive FSGS. What parallels can we draw to treatment in humans? The findings reinforce the paramount importance of angiotensin II blockade as a mainstay of treatment in adaptive FSGS. They also suggest that adding modest caloric restriction, apart from low-protein diet, may be protective, even in patients who are not obese. Caloric restriction has known benefits in aging kidney, which can be considered a kind of secondary FSGS.¹⁶ Potential efficacy of rapamycin in humans is a complex issue given the reports of drug-induced FSGS in transplant recipients¹⁷ and nephrotoxicity in patients with primary FSGS treated with this agent.¹⁸ Evidence from murine models suggests that complete podocyte-specific genetic abrogation of mTORC1 activity facilitates FSGS, whereas a partial genetic reduction in mTORC1 activity or incomplete pharmacologic inhibition of mTORC1 by rapamycin may be protective.¹⁹ These data are consistent with the nonlinear dual roles of mTORC1 in podocyte maintenance and FSGS progression.¹⁹ Future clinical studies are needed to optimize combination therapies aimed at curtailing glomerular growth while stabilizing podocytes.

Disclosures

None.