Abstract
Background Primary cilia are sensory organelles that are built and maintained by intraflagellar transport (IFT) multi-protein complexes. Deletion of certain ciliary genes in Autosomal Dominant Polycystic Kidney Disease (ADPKD) mouse models markedly attenuates PKD severity, indicating that a component of cilia dysfunction may have critical therapeutic potential.
Method We have ablated the Ift-A gene, Thm1, globally in juvenile and adult mouse models of ADPKD.
Results Relative to juvenile Pkd2 conditional knock-out mice, deletion of Thm1 together with Pkd2 resulted in a complex phenotype, with reduced kidney weight/body weight (KW/BW) ratios, reduced cortical collecting duct-derived cysts, but increased proximal tubular and glomerular dilations, and similar blood urea nitrogen (BUN) levels. Additionally, primary cilia of cortical collecting duct epithelia were lengthened in Pkd2 conditional knock-out kidneys, as well as in Pkd2;Thm1 double knock-out kidneys. In contrast, Thm1 deletion in adult ADPKD mouse models markedly reduced multiple disease parameters, including KW/BW ratios, collecting duct- and loop of Henle-derived cysts, proximal tubular dilations, and BUN levels. Further, primary cilia lengths of cortical collecting duct epithelia were increased in Pkd1 and Pkd2 conditional knock-out mice, but similar to control in Pkd1;Thm1 and Pkd2;Thm1 double knock-out mice.
Conclusions These data reveal that during kidney development, Thm1 both promotes and inhibits different aspects of ADPKD renal cystogenesis in a tubule-dependent manner; however, during adult kidney homeostasis, Thm1 promotes virtually all features of ADPKD renal cyst growth. These findings suggest that differential factors between tubules and between developing versus mature renal microenvironments influence cilia dysfunction and ADPKD pathobiology.
Introduction
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is among the most common, fatal monogenetic diseases, affecting 1:500 individuals worldwide. ADPKD is characterized by the growth of large fluid-filled renal cysts, which cause injury and fibrosis and can lead to end-stage renal disease by the 6th decade of life. Tolvaptan is the only FDA-approved therapy, but has variable effectiveness1, 2. Thus, the need to discover additional underlying disease mechanisms and design new therapeutic strategies continues.
Primary cilia are small, antenna-like sensory organelles that play an important role in ADPKD pathobiology via mechanisms that remain unclear. ADPKD is caused by mutations in PKD1 (≥80% of cases) or PKD2 (≥10% of cases), which encode polycystin 1 (PC1) and polycystin 2 (PC2), respectively3, 4. PC1 and PC2 form an ion-channel receptor complex that functions at the primary cilium. While PC1 and PC2 also localize to other subcellular compartments, analyses of human ADPKD primary renal epithelial cells, of mouse models harboring human ADPKD mutations, and of an ENU-induced Pkd2 mouse mutation that causes ciliary exclusion of PC2, indicate that deficiency of PC1 or PC2 from the cilium is sufficient to cause ADPKD5-7.
Primary cilia are synthesized and maintained via intraflagellar transport (IFT), which is the bi-directional transport of protein cargo along a microtubular axoneme. Two multiprotein complexes mediate IFT. The IFT-B complex interacts with the kinesin motor and mediates anterograde IFT, while the IFT-A complex together with cytoplasmic dynein mediates retrograde IFT. IFT-A proteins are also required for ciliary import of membrane and signaling molecules8-10. In mice, deletion of Ift-A or -B genes either perinatally or in the embryonic kidney results in renal cystic disease11-13. However, these mutants differ from ADPKD models in manifesting generally smaller renal cysts and greater fibrosis relative to cyst size14, 15. Additionally, Ift-A and –B mutants differ in cilia phenotype, showing in general shortened and absent cilia, respectively, and can also show opposing signaling phenotypes, reflecting the differing functional roles of IFT-A and -B12,16-18. Intriguingly, deletion of Ift-B genes, Kif3a, Ift20, and of an IFT-A adaptor gene, Tulp3, in Pkd1 or Pkd2 conditional knock-out (cko) mice reduces severity of the PKD phenotype19-21. The mechanisms underlying this rescue remains elusive, but the impressive attenuation of PKD severity in these Pkd; cilia double knock-out (dko) mice indicates that a component of cilia dysfunction has potential critical therapeutic value.
A commonly mutated IFT gene is THM1 (TPR-containing Hedgehog modulator 1; also termed TTTC21B). Causative and modifying mutations in THM1 have been identified in 5% of patients with ciliopathies, including nephronophthisis, Bardet Biedl syndrome, Meckel syndrome and Jeune syndrome14. THM1 encodes an IFT-A component, and its deletion impairs retrograde IFT, causing accumulation of proteins in bulb-like distal tips of shortened primary cilia16. Thm1 loss also impairs cilia entry of membrane-associated proteins, delays and reduces ciliogenesis, and promotes cilia disassembly22. In mice, Thm1 deletion recapitulates many of the clinical manifestations of ciliopathies16, 23, 24. Perinatal global deletion of Thm1 results in renal cystic disease23. Deletion of Thm1 in adult mice does not result in a renal phenotype by 3 months of age, consistent with the developmental time-frame that determines whether loss of a cystogenic gene will cause rapid- or slow-progressing renal cystic disease25. Here we have examined the role of IFT-A deficiency in ADPKD by deleting Thm1 in juvenile and adult ADPKD mouse models. We observe that during postnatal kidney development, Thm1 loss both attenuates and exacerbates different features of ADPKD, while in the adult kidney, Thm1 loss markedly attenuates most aspects of ADPKD renal cystogenesis. These data reveal renal tubular- and maturation-dependent roles for IFT-A in ADPKD.
Methods
Generation of mice
Pkd1flox/flox, Pkd2flox/flox and ROSA26-Cre mice were obtained from the Jackson Laboratories (Stock numbers 010671, 017292 and 004847, respectively). Generation of Thm1 cko mice has been described previously23: Thm1aln/+; ROSA26CreERT+ male mice were mated to Thm1flox/flox females. Pkd1 floxed alleles were introduced into the colony to generate Thm1flox/flox;Pkd1flox/flox or Thm1flox/flox;Pkd1flox/+ females and Pkd1flox/flox; Thm1aln/+, ROSA26-CreERT/+ males, which were mated. Similarly, Pkd2 floxed alleles were introduced into the colony to generate Thm1flox/flox;Pkd2flox/flox or Thm1flox/flox;Pkd2flox/+ females and Pkd2flox/flox; Thm1aln/+, ROSA26-CreERT/+ males. To generate early-onset Pkd2 models, Thm1flox/flox;Pkd2flox/flox or Thm1flox/flox;Pkd2flox/+ nursing mothers mated to Pkd2flox/flox; Thm1aln/+, ROSA26-CreERT/+ males were injected intraperitoneally with tamoxifen (10mg/40g; Sigma) at postnatal day 0 (P0) to induce gene deletion. Offspring were analyzed at P21. To generate late-onset Pkd2 models, offspring from matings between Thm1flox/flox;Pkd2flox/flox or Thm1flox/flox;Pkd2flox/+ females and Pkd2flox/flox; Thm1aln/+, ROSA26-CreERT/+ males were injected intraperitoneally with tamoxifen (10mg/40g) at P28. To generate late-onset Pkd1 models, offspring from matings between Thm1flox/flox;Pkd1flox/flox or Thm1flox/flox;Pkd1flox/+ females and Pkd2flox/flox; Thm1aln/+, ROSA26-CreERT/+ males were injected intraperitoneally with tamoxifen (10mg/40g) at P35. Mice were analyzed at 6 months of age. All mouse lines were maintained on a pure C57BL6/J background (backcrossed 10 generations). All animal procedures were conducted in accordance with KUMC-IACUC and AAALAC rules and regulations.
Kidney and body weight measurements
Kidneys were dissected and weighed using a standard laboratory weighing scale. The KW/BW ratio was calculated as the total kidney weights divided by body weight for each mouse.
Western blot
Passive Lysis Buffer (Promega) containing proteinase inhibitor cocktail (Pierce) was used to generate protein extracts from frozen kidney tissue. Tissue was homogenized by using 0.5 mm zirconium oxide PINK beads (Next Advance) and a Bullet Blender Storm (Next Advance) set at Speed 10 for approximately 5 minutes. Lysates were centrifuged at 4°C at maximum speed for 1 minute and supernatant was collected. Protein concentrations were determined using the bicinchoninic acid protein (BCA) assay reagents (Pierce). Western blot was performed as described 23, using primary antibodies for P-STAT3 (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), P-ERK (Cell Signaling Technology, 4370), ERK (Cell Signaling Technology, 4696). SuperSignal West Femto Chemiluminescent Substrate (Pierce) was used to detect signal. ImageJ was used to quantify Western blot signals.
qPCR
RNA was extracted using Trizol (Life Technologies), then reverse transcribed into cDNA using Quanta Biosciences qScript cDNA mix (VWR International). qPCR for Ccl2 was performed using Quanta Biosciences Perfecta qPCR Supermix (VWR International) in a BioRad CFX Connect Real-Time PCR Detection System. Primers used were mCcl2 (Forward: 5’-AAGCTCAACCCTGACTTCTTAC-3’; Reverse: 5’-CAACGTCTGAGAACTGGAGAAA-3’). qPCR was performed in duplicate using RNA lysates from five samples per genotype.
Histology
Kidneys were bisected transversely, fixed in 10% formalin for several days, then processed in a tissue processor and embedded in paraffin. Tissue sections (7μm) were obtained with a microtome. Sections were deparaffinized, rehydrated through a series of ethanol washes, and stained with hematoxylin and eosin (H&E). Images were taken with a Nikon 80i microscope equipped with a Nikon DS-Fi1 camera. Cystic areas of H&E-stained sections were quantified using ImageJ.
Immunofluorescence
Following deparaffinization and rehydration, tissue sections were subjected to an antigen retrieval protocol, which consisted of steaming sections for 25□minutes in Sodium Citrate Buffer (10□mM Sodium Citrate, 0.05% Tween 20, pH 6.0). Sections were blocked with 1% BSA in PBS for 1 hour at room temperature, and then incubated with primary antibodies against acetylated-α tubulin (1:4000; Sigma, T7451), IFT81 (1:200; Proteintech, 11744-1-AP), αSMA (1:500; Abcam, ab5694) and PCNA (1:300; Cell Signaling Technology, 13110), DBA (1:100; Vector Laboratories, FL-1031), LTL (1:300, Vector Laboratories, FL-1321), THP (1:100; Santa Cruz Biotechnology, sc-271022) overnight at 4°C. Sections were washed three times in PBS, and then incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:500; Invitrogen, A-11001 (anti-mouse) or A-11034 (anti-rabbit)) or Alexa Fluor 594 (1:500; Invitrogen, A-11005 (anti-mouse) or A-11012 (anti-rabbit)) for 1 hour at room temperature. After three washes of PBS, sections were mounted with Fluoromount-G containing 4′,6-diamidino-2-phenylindole (DAPI) (Electron Microscopy Sciences). Staining was visualized and imaged using a Nikon 80i microscope with a photometrics camera or a Nikon Eclipse TiE attached to an A1R-SHR confocal, with an A1-DU4 detector, and LU4 laser launch.
Blood Urea Nitrogen Measurements
Mouse trunk blood was collected in Microvette CB 300 Blood Collection System tubes (Kent Scientific), and centrifuged at 1800g at room temperature for 10 minutes to collect serum. BUN was measured using the QuantiChrom Urea Assay Kit (BioAssay Systems) according to the manufacturer’s protocol.
ADPKD renal sections
Paraffin-embedded sections of de-identified normal human kidney (NHK), n=3 (K357, K402, K419), and of ADPKD, n=3 (K386, K408, K423) were obtained from the PKD Biomaterials Core. Sections were deparaffinized and rehydrated, steamed in Sodium Citrate Buffer (10□mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for antigen retrieval, and immunostained for ARL13B (1:300; Proteintech, 17711-1-AP).
Statistics
Statistical significance (P < 0.05) was determined using either one-way ANOVA followed by Tukey’s test, or using an unpaired t-test for comparing more than two groups or two groups, respectively. GraphPad Prism 8 software was used to perform these analyses.
Results
Perinatal deletion of Thm1 in Pkd2 cko mice reduces cortical cystogenesis, but does not improve kidney function
To examine the effect of IFT-A deficiency in an early-onset, rapidly progressing ADPKD mouse model, we deleted Thm1 together with Pkd2 at postnatal day (P) 0, and examined the renal phenotypes of control, Thm1 cko, Pkd2 cko and Pkd2;Thm1 dko mice at P21. At this stage, Thm1 cko kidneys appear mostly intact morphologically23, with some tubular dilations observed in the cortex and with kidney weight/body weight (KW/BW) ratios similar to control (Figures 1A and 1B). Yet, BUN levels are elevated about 2-fold (Figure 1C). In Pkd2 cko mice, renal cysts are present in both cortex and medulla, and KW/BW ratios and BUN levels are increased 5-fold and 3-fold, respectively. In Pkd2;Thm1 dko mice, renal cysts are also present in the cortex and medulla, and KW/BW ratios and BUN levels are increased 4-fold and 3-fold, respectively. Thus relative to Pkd2 cko mice, Pkd2;Thm1 dko mice have reduced KW/BW ratios, but similar kidney function. Pkd2;Thm1 dko kidneys also show decreased percent cystic index (Figure 1D, Supplemental Figure 1A), due to reduced cystogenesis in the cortex (Figure 1E, Supplemental Figure 1B), while percent cystic index in the medulla is similar (Figures 1F and S1C).
Perinatal deletion of Thm1 in Pkd2 cko mice reduces cortical collecting duct cystogenesis, but increases proximal tubular and glomerular dilations
Since cystogenesis was reduced in the cortex of Pkd2;Thm1 dko kidneys relative to Pkd2 cko kidneys, subsequent analyses focused on the cortex. At P21 in the Thm1 cko renal cortex, we observed some dilations, most of which were LTL+, marking proximal tubules, and fewer that were THP+ or DBA+, marking loop of Henle and collecting duct, respectively (Figure 2A). In Pkd2 cko renal cortex, LTL+ dilations, THP+ cysts, and multiple, large DBA+ cysts were observed. In Pkd2; Thm1 dko cortex, LTL+ dilations were increased relative to those of Pkd2 cko and Thm1 cko kidneys (Figures 2A, 2B, Supplemental Figure 1D); THP+ cysts were similar in size to those of Pkd2 cko kidneys (Figure 2C, Supplemental Figure 1E), and DBA+ cysts were decreased in size relative to those of Pkd2 cko kidneys (Figure 2D, Supplemental Figure 1F). Thus, we observed a tubular-specific effect of deleting Thm1 in juvenile Pkd2 cko mice. Thm1 deletion worsened LTL+, but attenuated cortical DBA+ cystogenesis.
Histology revealed that glomerular dilations were present across the mutant genotypes (Figure 2E). We observed a reduced number of glomeruli per cross-section in Pkd2 cko kidneys (28.3 vs 45.0; Supplemental Figure 2B), but a restored number of glomeruli per cross-section in Pkd2;Thm1 dko kidneys (46.5; Supplemental Figure 2D, Figure 2F). In Pkd2 cko kidneys, area of Bowman’s capsule/area of glomerulus and Bowman’s space were increased, suggesting presence of glomerular dilations (Figure 2G, Supplemental Figure 3B). In Pkd2; Thm1 dko kidneys, these parameters were increased to a greater extent than in Pkd2 cko kidneys, indicating that additional loss of Thm1 exacerbates the glomerular dilations caused by loss of Pkd2 (Figures 2G, 2H, Supplemental Figures 3C and 3D).
Deletion of Pkd2 increases proliferation of renal tubular epithelia
We next examined cell proliferation, a driver of ADPKD renal cystogenesis, by immunostaining for PCNA together with proximal tubule and collecting duct markers, LTL and DBA, respectively. Similar levels of PCNA staining were observed in normal LTL+ and DBA+ tubules across the various genotypes - control, Thm1 cko, Pkd2 cko and Pkd2;Thm1 dko kidneys (Figures 3A-3B). However, in Pkd2 cko kidneys, PCNA+ cells were increased in dilated LTL+ tubules relative to normal LTL+ tubules (Supplemental Figure 4), and in Pkd2 cko and Pkd2;Thm1 dko kidneys, PCNA+ cells were increased in dilated DBA+ tubules relative to normal DBA+ tubules (Figure 3B). These data support that increased proliferation is an early event in ADPKD renal cystogenesis.
Perinatal deletion of Thm1 causes fibrosis
Cyst growth compresses surrounding parenchyma, leading to injury and fibrosis in ADPKD. To assess fibrosis, we immunostained kidney sections for presence of myofibroblasts, which label with alpha smooth muscle actin (αSMA). In Thm1 cko kidneys, we observed αSMA+ cells around glomeruli and tubular dilations (Figure 3C). In Pkd2 cko kidneys, more αSMA+ labelling was observed than in Thm1 cko kidneys, and in Pkd2;Thm1 dko kidneys, levels of αSMA+ labelling were similar to those in Pkd2 cko kidneys. Thus, deletion of Thm1 alone causes fibrosis, but Thm1 deletion in Pkd2 cko mice does not exacerbate fibrosis at P21.
Perinatal deletion of Thm1 in Pkd2 cko mice increases STAT3 signaling
We have observed that perinatal deletion of Thm1 increases STAT3 activation in kidneys prior to cyst formation (data not shown). STAT3 signaling is also increased in ADPKD mouse models and pharmacological inhibition of STAT3 signaling attenuates ADPKD in mouse models26. ERK signaling is also increased during early cystic kidney disease of Thm1 cko mice (data not shown) and this pathway is elevated in ADPKD27, 28. We therefore examined these pathways using Western blot analyses. In Thm1 cko and Pkd2 cko kidneys, STAT3 activation was increased (Figures 4A, Supplemental Figure 5A, Figure 4B, Supplemental Figure 5B), and in Pkd2;Thm1 dko kidneys, STAT3 activation was further increased (Figures 4B, Supplemental Figures 5C and 5D). Additionally, in Pkd2 cko and Pkd2;Thm1 dko kidneys, there was a trend toward increased ERK activation (Figure 4C, Supplemental Figure 6). Thus, Pkd2 cystic disease causes increased STAT3 and ERK signaling consistent with previous reports29, 30, and deletion of Thm1 in Pkd2 cko mice further increases STAT3 activation.
Deletion of Pkd2 increases cilia length on renal epithelia
We examined cilia length on renal tubular epithelia by co-immunostaining for acetylated, α-tubulin together with lectins, LTL and DBA. In control kidneys, average cilia lengths were 3.0μm and 2.1μm for LTL+ and DBA+ cells, respectively (Figures 5A and 5B). We also noted qualitative differences between LTL+ and DBA+ primary cilia, with the former cilia appearing thinner and longer, and the latter being thicker and more rod-like. Cilia lengths were increased in both Pkd2 cko LTL+ and DBA+ tubules. However, relative to Pkd2 cko tubules, cilia lengths were further increased in Pkd2;Thm1 dko LTL+ tubules, but similar in Pkd2;Thm1 dko DBA+ tubules. These differences reveal tubular-specific effects on cilia length.
Deletion of Thm1 in adult Pkd2 or Pkd1 cko mice markedly attenuates ADPKD renal cystogenesis
We next examined the effect of IFT-A deficiency in late-onset, slowly progressive adult ADPKD mouse models. We deleted Thm1 together with Pkd2 at P28 and examined the renal phenotypes of control, Thm1 cko, Pkd2 cko and Pkd2;Thm1 dko mice at 6 months of age. Thm1 cko kidneys have similar morphology and BUN levels to those of control mice (Supplemental Figures 7A and 7B). Pkd2 cko mice show renal cysts mostly in the cortex, with the largest cysts being DBA+, and smaller cysts being LTL+ or THP+ (Figure 6A). In contrast, in Pkd2;Thm1 dko mice, the Pkd2 cko cystic phenotype is largely corrected morphologically. KW/BW ratios are unchanged in Pkd2 cko mice, reflecting the mild disease induced in adulthood. BUN levels show a trend toward a slight elevation in Pkd2 cko mice, but the average BUN value is still within the range of normal renal function. BUN levels of Pkd2;Thm1 dko mice were similar to those of Pkd2 cko mice. In ADPKD, pro-inflammatory cytokines, such as Ccl2, are elevated31. In Pkd2 cko kidney extracts, expression of Ccl2 showed an increasing trend, while in Pkd2; Thm1 dko extract, Ccl2 levels were similar to control, suggesting reduced inflammation (Supplemental Figure 8A).
We also deleted Thm1 together with Pkd1 at P35 and examined the renal phenotypes at 6 months of age. Thm1 cko kidneys have morphology resembling control kidneys (Supplemental Figure 7C), similar to Thm1 deletion at P28. Like Pkd2 cko adult models, Pkd1 cko renal cysts were mostly in the cortex, with the largest and most abundant cysts being DBA+. Fewer cysts were THP+, and only dilations, not cysts, were observed that were LTL+ (Figure 7A). Notably, all these features were reduced in Pkd1; Thm1 dko kidneys. KW/BW ratios were elevated in Pkd1 cko mice, and corrected in Pkd1;Thm1 dko mice (Figure 7B). Additionally, BUN levels were elevated in Pkd1 cko mice, although the average value was still within the range of normal kidney function, while BUN levels in Pkd1;Thm1 dko mice were similar to control. Further, while expression of Ccl2, and activation of STAT3 and ERK were increased in kidney extracts of Pkd1 cko mice, these parameters were normalized in kidneys of Pkd1; Thm1 dko mice, consistent with attenuation of disease severity (Supplemental Figures 8B-8D).
Cilia length is increased on cortical renal epithelia of mouse and human ADPKD kidneys
We examined cilia length on renal tubular epithelia of adult ADPKD mouse models by co-immunostaining for acetylated, α-tubulin together with DBA. Similar to juvenile ADPKD models, cilia lengths were increased in Pkd1 cko and Pkd2 cko DBA+ adult tubules. However, in contrast to juvenile models, cilia lengths were normalized in Pkd1;Thm1 and Pkd2;Thm1 dko DBA+ tubules (Figures 8A and 8B). These differences suggest maturation-dependent effects on cilia length.
Further, we examined cilia lengths on renal cortical sections of normal human kidney (NHK) and ADPKD samples. Human ADPKD sections had longer cilia than NHK sections (Figure 8C), suggesting that increased cilia length is also a feature of the human disease.
Discussion
This study demonstrates differential effects of IFT-A deficiency in early-versus late-onset ADPKD mouse models, highlighting differences in developing versus mature renal microenvironments. These data also show that deleting Thm1 in an early-onset ADPKD model has tubule-specific effects: partially protecting cortical collecting duct structure, but worsening the decline of proximal tubular structural integrity; and restoring glomerular number, but increasing glomerular dilation.
In Pkd2;Thm1 dko juvenile mice, STAT3 activation was increased. Since cortical collecting duct cystogenesis was reduced, this suggests that STAT3 signaling may contribute to other disease processes. Pkd2;Thm1 dko kidneys showed increased proximal tubular and glomerular dilations, and increased STAT3 activation could potentially drive these dilations. STAT3 signaling may also be involved in fibrosis. However, while STAT3 activation was increased in Pkd2;Thm1 dko mice, fibrosis as assessed by αSMA staining was not. In contrast to studies suggesting a pathogenic role for increased STAT3 signaling, a recent study has shown that tubular STAT3 activation restricts immune cell infiltration in Pkd1 cko mice32. Genetic deletion of Stat3 together with Pkd1 in renal tubular cells slightly reduced cystic burden, but did not ameliorate kidney function and increased interstitial inflammation. Thus, STAT3 activation in Pkd2;Thm1 dko mice could potentially serve a protective role against inflammation.
In several ADPKD mouse models, PKD1RC/RC, Pkd1 and Pkd2 cko mice, renal primary cilia are lengthened33, 34. Our data showing increased cilia length in both Pkd2 juvenile and adult mouse models and in Pkd1 adult mouse models are consistent with these studies. Additionally, the increased cilia length in ADPKD tissue sections suggest that similar ciliary mechanisms may be relevant to the human disease. We observed a range of cilia lengths within a genotype. This could result from limitations of quantifying immunostained tissue sections. Additionally, multiple factors influence renal cilia length and could also contribute to this variability. Our data suggest that in addition to genotype, cilia length varied by renal tubule and age, suggesting that factors within a tubule’s microenvironment affect cilia length. Cilia length is determined by the ratio of cilia assembly and disassembly. As well, intracellular Ca2+ and cAMP, oxidative stress, cytokines, and fluid flow influence ciliary length of renal epithelial cells35-37. These multiple factors indicate that cilia length regulation may be fine-tuned in order to maintain renal tubular structure and function. In support of this, genetic and pharmacological inhibition of cilia disassembly in Pkd1 cko mice increased renal cilia length and exacerbated ADPKD38. In the jck non-orthologous ADPKD mouse model, renal primary cilia are also lengthened, and pharmacological shortening of primary cilia in jck mutant mice was associated with an attenuation of the ADPKD phenotype19, 39. Moreover, cilia length is altered also in acute kidney injury and chronic kidney disease40-43. Thus, to understand mechanisms of renal tubule homeostasis, the connections between cilia length and cilia function, and renal disease require deeper study.
Thus far, the effects of deleting Ift-B genes, Kif3a and Ift20, and of the IFT-A adaptor, Tulp3, in ADPKD mouse models have been demonstrated. Ift-B gene deletion attenuates PKD severity in both tubular-specific juvenile and adult models of ADPKD19. In contrast, Tulp3 deletion did not rescue renal cystic disease in a tubular-specific juvenile model of ADPKD, but did in an adult model21, 44. Similarly, global deletion of Thm1 in a juvenile ADPKD model results in a complex phenotype, but in an adult model, rescues most aspects of the renal cystic disease. Perinatal loss of Thm1 results in cystic kidney disease23, indicating that Thm1 is required for kidney maturation, which might account for the lack of rescue in juvenile models. However, perinatal deletion of Kif3a and mutation of Tulp3 also causes renal cystic disease21, 45, suggesting these genes are required as well for kidney differentiation and maturation. Thus, there may be functional differences between IFT-B and IFT-A and Tulp3 that result in attenuated disease in juvenile Pkd;Ift-B dko mice, but not in Pkd;Thm1 or Pkd;Tulp3 dko mice. These differences could include differential roles in IFT, cilia length regulation, and/or ciliary-mediated signaling. For instance, Ift-B genes are required for anterograde IFT, unlike Thm1 and Tulp3. While IFT-B and IFT-A regulate cilia length, a role for Tulp3 in altering cilia length has not been reported. Further, IFT-B and IFT-A mutants have shown opposing signaling phenotypes.
We noted that in the late-onset models, BUN levels were similar between Pkd2 cko and Pkd2;Thm1 dko mice, while in contrast, BUN levels in Pkd1;Thm1 dko mice were reduced relative to those of Pkd1 cko mice, suggesting Thm1 deletion might confer greater protection in Pkd1 cko mice than in Pkd2 cko mice. We deleted Pkd2 one week earlier than Pkd1, since Pkd1 deficiency results in a more severe ADPKD phenotype than Pkd2 deficiency. Importantly, Thm1 deletion at P28 resulted in BUN levels similar to those of control mice at 6 months of age, and Thm1 deletion at either P28 or P35 resulted in kidney morphology resembling control. Thus, the BUN data may suggest a functional difference between Pkd2;Thm1 dko and Pkd1;Thm1 dko mice.
The mechanisms by which Pkd; cilia dko mice attenuate ADPKD severity are still obscure. Reducing Ccl2 signaling and altering lipid composition of the ciliary membrane have been proposed20, 21, 44. Primary cilia are designed to detect both chemical and mechanical cues in the extracellular environment. While mechanosensing by primary cilia and the polycystins has been controversial, recent studies have renewed interest in a potential mechanosensory role for the polycystins, particularly regarding tissue microenvironment stiffness46, 47. If sensing physical forces in the tissue microenvironment is essential to maintaining renal tubular function, then other mechanical cues that would change with cyst growth include shear stress and intraluminal pressure. Cilia length could then also be a possible contributing factor in PKD severity. Further, by extrapolating findings of cilia studies from the cancer field48, cilia of not only renal tubular epithelial cells, but of interstitial cells might also affect signaling and disease severity.
In summary, our data demonstrate for the first time the role of IFT-A in an ADPKD context in developing versus mature kidneys. Defining the mechanisms by which IFT-A deficiency attenuates ADPKD in adult models will be critical to identifying potential therapeutic targets.
Acknowledgements
We thank members of the KUMC Dept. of Anatomy and Cell Biology and the Jared Grantham Kidney Institute for helpful discussions. We thank Jing Huang of the KUMC Histology Core and acknowledge support of this core (Intellectual and Developmental Disabilities Research Center NIH U54 HD090216; COBRE NIH P30 GM122731). This work was also supported by a K-INBRE Summer Student Award to JTC [K-INBRE P20GM103418] and the National Institutes of Health (P20 GM14936; P30DK106912; R01DK108433 to MS; R01DK103033 to PVT).
Disclosures
The authors declare no conflict of interest.
Contributions
WW, LMS, BAA, TSP, HHW, DTJ, JTC, AC, MTP, MS, DPW, and PVT performed experiments. WW, LMS, BAA, TSP, HHW, DTJ, JTC, AC, MTP, MS, DPW, JPC and PVT analyzed and interpreted data. WW, LMS, BAA, and PVT designed research. WW, LMS, and PVT wrote the manuscript.