key: cord-0993758-7fqwtssb
authors: Dhande, Isha S.; Braun, Michael C.; Doris, Peter A.
title: Emerging Insights Into Chronic Renal Disease Pathogenesis in Hypertension From Human and Animal Genomic Studies
date: 2021-11-11
journal: Hypertension
DOI: 10.1161/hypertensionaha.121.18112
sha: d0713a05d61d3a869da0b61267715b7881021d22
doc_id: 993758
cord_uid: 7fqwtssb

The pathogenic links between elevated blood pressure and chronic kidney disease remain obscure. This article examines progress in population genetics and in animal models of hypertension and chronic kidney disease. It also provides a critique of the application of genome-wide association studies to understanding the heritability of renal function. Emerging themes identified indicate that heritable risk of chronic kidney disease in hypertension can arise from genetic variation in (1) glomerular and tubular protein handling mechanisms; (2) autoregulatory capacity of the renal vasculature; and (3) innate and adaptive immune mechanisms. Increased prevalence of hypertension-associated chronic kidney disease that occurs with aging may reflect amplification of heritable risks by normal aging processes affecting immunity and autoregulation.

T he prevalence of chronic kidney disease (CKD; stages 1-4) in US adults is 15% and increases with age, rising to 38% in people 65 and older. 1 Prevalence is greater in the non-Hispanic Black population (16%) than other ethnic groups (Hispanic [14%], non-Hispanic White [13%], and non-Hispanic Asian [12%] populations). In population-scale genomic studies, the diagnosis of CKD relies on indirect assessments including excretion of protein and serum levels of markers, such as creatinine and cystatin. Renal biopsy data is helpful from a diagnostic and prognostic standpoint but is not required for CKD staging, and there is insufficient collated biopsy information available to directly inform largescale population studies. 2 Although high blood pressure (BP) increases CKD risk, other factors also influence risk as CKD prevalence ranges from 1 in 3.6 to 4.5 in diagnosed and undiagnosed hypertensives. 3 The pathogenic mechanisms connecting hypertension to CKD are not well understood. Heritable risk has created hope that population genetic studies associating phenotypes of renal function with underlying genetic variation will point to disease mechanisms indicated by the function of genes associated with disease. Studies of genetic risk in rat models of heritable hypertensive renal diseases have also been pursued in the expectation that these studies provide an alternative path to uncover genes involved. These studies have progressed sufficiently to illustrate several themes and to examine obstacles to further progress.

Population genetics studies require evidence of heritability. The most recent population-based family studies indicate that risk of CKD is increased 3 fold in individuals with a first-degree relative affected by CKD. 4 Heritability of renal function, the proportion of trait variation attributable to genetic variation, calculated from twin-pair correlations of estimated glomerular filtration rate (eGFR) and albuminuria 5 is 77.6% and 45.2%, respectively. Heritability of GFR measured as creatinine clearance is 33% to 53%. 6 Albuminuria and eGFR are used in population-based genome-wide association studies (GWAS) because they are practical to assess in large scale. They present some limitations: urinary albumin creatinine ratio requires the independent measurement of 2 variables, each introducing error; eGFR typically uses serum creatinine and requires adjustments depending on patient variables. 7 Serum creatinine is influenced by heritable effects on creatinine metabolism, in addition to difference in GFR, leading to the recent introduction of blood urea nitrogen in GWAS studies. 8 GWAS studies test for association of renal function with allelic state at each of a very large number of genome-wide single-nucleotide polymorphisms (SNPs). The resulting multiple hypothesis testing is generally addressed in 2 ways: by elevation of the threshold for statistical significance for any single SNP/phenotype association, and; by retesting associations observed in a discovery population in a replication population. This approach requires very large subject numbers and multiple, often multi-national, populations are used to achieve these numbers. This transancestry population sample pooling may obscure genetic variation that is subpopulation specific. Another obstacle in GWAS arises from its reliance on a single class of genetic variation. New long-read sequencing technologies reveal an important role for genomic structural variation (SV) in trait divergence within populations. 9,10 SV's range upwards from 50 base pairs and include larger events, such as duplication, insertion, deletion, transposition, and inversion. Population SV is still being described, so it remains unclear to what extent SVs can be imputed from adjacent SNP genotypes. [11] [12] [13] Furthermore, some SVs can confound SNP genotyping (Figure 1 ), obstructing discovery of SNP association in regions containing SV.

An important rationale for large-scale population genetics studies was the view that genetic variants creating common polygenic disease susceptibility might be frequent in a population and shared across populations of different ancestry. This common disease:common variant hypothesis posited that the set of trait-associated variants is concise (ie, not composed of a very large number of unique variants) and allows a tractable discovery task. 14, 15 In general, this has not been the outcome. Most GWAS studies of disease susceptibility have accounted for only a portion of observed heritability. This missing heritability has been the source of much conjecture. In general, GWAS does find and confirm SNP associations, but understanding of pathogenesis has generally not emerged.

The most recent GWAS of renal function studied in excess of 750 000 subjects, with a replication population of ≈280 000 subjects. 8 Heritability in this study population of renal function was estimated at 39%. A total of 308 index SNPs were found that explained 19.6% of eGFR heritability. Thus, 308 SNP's account for ≈8% of the total (heritable and nonheritable) eGFR variation, on average 0.025% per SNP. These small effects leave 80% of the heritability missing. In the replication cohort, 264 of these SNPs were significant: even with small effect sizes, SNP associations are largely confirmed. Among replicated eGFR SNP associations, 34 SNPs were significantly associated with blood urea nitrogen. The replicated SNPs were then studied for association with CKD in the CKDGen studies (≈625 000 subjects including ≈64 000 CKD cases). This analysis found that 23 of 264 SNPs significantly associate with CKD. These studies epitomize the dichotomy of complex disease association studies: replicable SNP associations are evident but provide little insight into disease processes. There appear to be other effects that are heritable that cannot be mapped in these studies.

Several explanations for the inability of GWAS to account fully for the heritability of CKD risk have been considered. For example, heritability arises from a very large number of small effect variants, and all are discoverable with population sizes not yet achieved. This has been termed the omnigenic thesis of heritability. 16 Another possible explanation is that the genetic architecture of renal function is attributed to rare variants that have large effects but that are highly heterogenous. These may go undiscovered in GWAS because they are sufficiently rare that the SNP genotypes linked to these variants are most often linked to wild-type sequences that are not disease associated. The reliance of GWAS on genotyping arrays that address very large numbers of SNPs provides a one-dimensional view of genetic association. SNPs reflect variants largely obtained from the analysis of human genome sequencing data and provide information that is genotyped accurately and inexpensively. This sequencing used principally shortread methods that are not instructive about SV longer than the short reads. 

Elevated renal perfusion pressure results in the migration of adaptive immune cells into the kidney. The existence of this phenomenon occurring in response to elevated BP, and independent of immune signaling induced by hypertensive agents used to elevate BP, has been elegantly shown by Shimada et al. 17 Acute renal injury and associated tissue damage initiate sterile immune responses arising from danger signals conveyed by damage-associated molecular patterns. 18 Since the chronic renal disease is associated with acute injury 19 and may, in the presence of hypertension, reflect the persistent effect of injury mechanisms, the sustained activation of immune responses may play a role in CKD in hypertensives. The involvement of immune cells, notably T and B lymphocytes, in hypertension and renal injury has been extensively examined in animal models. [20] [21] [22] [23] These lymphocytes are typified by the cell surface expression of T-and B-cell receptors that are a key characteristic of these cells that drives the immunologic process by which they may contribute to renal injury. These adaptive immune receptors are highly divergent in the population and much of this divergence arises from SV. Thus,

while key receptors on immune cells are central to renal injury, the natural genetic variation in these hypervariable receptors cannot be adequately addressed by SNP arrays, and consequently, they are dramatically underrepresented on these arrays. The extent of this underrepresentation has recently been surveyed by Mikocziova et al. 24 Recent efforts to generate accurate and complete assemblies of immunoglobulin, T-cell receptor, and natural killer cell receptors in a single individual have exploited state of the art long-read and other mapping and assembly approaches. 25 They have concluded that a full accurate and complete assembly of variation in some of these regions in a single individual is a technical challenge that has not yet been overcome and will require further methodological improvements. These problematic regions of the genome, and others of perhaps similar complexity and biological importance, underscore that the notion that SNP-based GWAS is a comprehensive tool for uncovering genetic variation contributing to disease traits is one in need of re-assessment.

Disease association studies may advance when population mapping approaches are able to consider SV Left, At locus X an individual has inherited from their father a chromosomal segment (orange line) containing an SNP, while the corresponding chromosomal segment inherited from the mother lacks this SNP (C vs A). A high-throughput genotyping assay is developed that is able to call this SNP by accurately discriminating the genotyping signals that occur when an individual is homozygous for the paternal genotype (homozygous C), heterozygous, or homozygous for the maternal genotype (homozygous A). To do so, the assay must generate signals reflecting genotype that are sufficiently restricted (blue shading) to values near 0%, 50%, and 100% which are the expected values for each of the 3 Mendelian states. Right, The orange chromosomal region has been the subject of a duplication event at some point in evolution resulting in individuals that may have one or 2 copies of this segment. This duplication event occurred on a chromosome bearing the C variant. This individual has inherited an unduplicated orange segment from the maternal lineage and has obtained a duplicated paternal segment in which 2 copies of the SNP variant are present. The genotyping assay produces unexpected values for this variant because the number of copies of the C variant is double and the resulting signals are twice as strong as those arising from the A allele. As a consequence the genotyping assay for this individual (red box) produces values that fall outside of the expected distribution. Because of unexpected performance (since the duplication event is not known) the assay is eliminated from further consideration for inclusion in a high-throughput multiplex assay system. Potentially important biological variation is present. In the population there may exist all 3 alleles described here: M, P1 and P2. Not only is this genetic variation not captured by the assay because it does not fulfill expected signal ratios, but it is excluded from informing genotypephenotype associations that may arise from structural variation.

variation. SV has been predicted to have a high phenotypic impact 26 and recent studies have begun to assess this proposition directly. 10 Newer high accuracy long-read sequencing 27 supported by optical mapping techniques 28 have begun to provide a rich source of information about genomic SV. 9, [29] [30] [31] [32] The features of SV in humans have been summarized recently by Eichler 33 who points out that emerging data sets applying long-read sequencing and assembly to human genomes have begun to re-shape our view of allocation of genetic variation in the genome between the SNPs and larger-scale variation and indicates that bases with SNP variation represent only 7.6% of the total variant bases in the human genome. The SNP variation assessed in GWAS represents a small portion of the total genetic variation and may account for a similar portion of the phenotypic variation among humans.

Recently completed telomere to telomere sequencing of the human genome has revealed the 8% of the genome that was previously not represented in the data sets used to devise GWAS SNP arrays and much of this missing genome was the result of complex SV. 34, 35 Populationscale studies to assemble a deep collection of SV have begun, 9, [36] [37] [38] however, the technical approaches that allowed SNP-based association studies to sample comprehensive collections of SNPs in large populations has not yet emerged for SV.

Discovery of Black patient-specific CKD risk alleles has been made in using a modified GWAS approach called admixture mapping. 39, 40 This exploits recent admixture in American persons of African ancestry with other populations and postulates that SNP marker variation enriched in the admixed population includes genomic regions associated with CKD and this might be mapped by combining ancestral divergence in the genome with disease susceptibility mapping. A locus reflecting African ancestry was identified on chromosome 22 that was associated with focal segmental glomerulosclerosis and hypertensive renal disease. 40 In subjects homozygous for risk alleles, focal segmental glomerulosclerosis risk was increased by 5-fold and hypertensive renal disease risk was increased 2.2-fold. Variation in the APOL1 gene drives the renal disease effect. 41 APOL1 encodes an apolipoprotein providing innate immune defense against trypanosome infection. 42 Such infection is endemic in West Africa. In this locale, the human immune system and parasitic trypanosomes have engaged in a genetic arms race in which mutations in trypanosome defense mechanisms have been met by the emergence of APOL1 mutations that overcome adapted pathogen resistance. 43 Resistance to African trypanosomal diseases such as sleeping sickness likely provide a fitness benefit that has caused such variant APOL1 alleles to rise in frequency in endemic regions. 42 There are 2 variant alleles of APOL1 that confer CKD susceptibility, both lead to amino acid changes in APOL1 ( Figure 2 ). Inheritance of a single variant allele creates only a slight increased risk of nephropathy, while the presence of 2 risk alleles leads to a marked increase in risk. 44 In addition to amplifying renal disease risk in hypertension, pathogenic APOL1-mediated disease may be increased in states of innate immunity activation. Consequently, APOL1 may influence risk of CKD development in HIV 44 and coronavirus disease 2019 (COVID-19) [45] [46] [47] infection and may occur in genetically susceptible patients treated with interferons. 48 The disease-associated alleles have been recreated experimentally in both cellular and animal models (zebrafish and mouse). [49] [50] [51] The mechanism of pathogenesis remains obscure. APOL1 may participate in functional specialization of the podocyte. The minor effects of presence of a single risk allele implies a loss of function mutation, but this explanation is not fully satisfactory. The different amino acid changes in the 2 risk alleles both affect a similar region of the protein, so each allele seems likely to contribute to loss of function. However, amplification of disease in the presence of strong upregulation of expression of the APOL1 gene in an interferon-dependent manner suggests that disease results from gain of function-mediated injury. 48, 52, 53 It also suggests that disease risk arises from the combined effects of genetic variation with additional triggers, including hypertension. Thus, risk alleles are necessary but not sufficient for disease. 54 It has been observed that overexpression of even the nonrisk APOL1 allele in the podocyte can produce injury, 55 suggesting disruption of cellular functions, possibly including those involving proteins that interact with APOL1.

Most recently, a mechanism of APOL1 pathogenesis has been proposed involving interactions with APOL3 and phosphatidylinositol 4-kinase, a Golgi membranelocalized enzyme involved in vesicular trafficking and mitochondrial fusion. 56 In podocytes APOL1 resides at the Golgi in contact with APOL3. Deletion of APOL3 increases mitochondrial fission, an event recapitulated in trypanosomes that have ingested host APOL1. 57 Reduced levels of PI(4)P are observed in podocytes expressing APOL1 variants and in biopsies from patients with disease risk alleles of APOL1. 57 Variants of APOL1 have been proposed to inactivate APOL3 function, leading to reduced phosphatidylinositol 4-kinase activity, with a consequent alteration in vesicular trafficking activity in podocytes, possibly connected to alterations in the podocyte actomyosin cytoskeleton. 56 APOL3 null alleles also increase risk of nondiabetic nephropathy. 58 At this time, the mechanism by which risk alleles of APOL1 create renal injury remains an active area of investigation.

It is important to retain a suitable perspective on the population risk attributable to APOL1 variation in the Black population. Only 13% of Black individuals possess the risk genotype of APOL1. Among Black participants in the Dallas Heart Study 69.8% of individuals with nondiabetic CKD lacked the APOL1 risk genotype and 50% of Black patients with end-stage renal disease had the risk genotype. 59 Among the 50% with end-stage disease who lacked the risk genotype, it is likely that other genetic factors contribute to risk of CKD, and these unknown risk variants may be shared with populations lacking recent African ancestry.

Uromodulin UMOD (Uromodulin) encodes the most abundant urinary protein, also known as Tamm-Horsfall protein, which is produced in epithelial cells of the medullary thick ascending limb and distal convoluted tubule and secreted into urine and blood. Rare variants in uromodulin have previously been associated with renal disease, notably autosomal dominant tubulointerstitial nephropathy. 60 However, GWAS association between UMOD variation and loss of renal function has been attributed to a common variation representing the highly prevalent ancestral allele that modifies UMOD gene expression. 61, 62 The genetic and pathogenic insights into UMOD function and variation have been recently thoroughly reviewed. 63, 64 Renal functions of UMOD are complex and include a potential role as an antibacterial agent providing defense from retrograde bacterial colonization of the urinary tract to interactions with tubular mechanisms regulating sodium reabsorption. No clear mechanism of pathogenesis has yet emerged to connect genetic variation in the UMOD locus with loss of renal function.

Several groups have investigated whether GWAS associations arising outside of genes may induce effects by altering nearby gene expression. Xu et al 65 In this locus lies the APOL1 gene that contributes to innate immunity to infection with insect-borne trypanosome parasites resulting in sleeping sickness. Such infection is endemic in Central West Africa but not in Europe. Right, APOL1 infection resistance results from the action of a serum-resistance associated binding domain. Evolution of trypanosome parasite resistance to APOL1 has resulted in an arms race in which genetic variants in APOL1 that overcome this resistance are favored and increase in prevalence in endemic regions. Two such APOL1 variants have been identified that are present in some individuals with African ancestry and that restore the anti-trypanosomal effects of APOL1. When an individual inherits either of these variants (G1 and G2) in homozygosity or inherits one of each of G1 and G2 (compound heterozygosity) risk of renal disease is increased. This risk may be amplified by increased gene expression of APOL1. As a gene of innate immunity its expression has been shown to be controlled by host immune signaling. This may include signals resulting from inflammation occurring when the kidney experiences elevated blood pressure. Other inflammatory conditions such as HIV and coronavirus disease 2019 (COVID-19) infection and sickle cell disease may also amplify risk or renal disease arising from these genetic variants. their expression. Additional analysis of MANBA showed its risk allele was associated with a 50% reduction in its expression in normal human kidney. 66 Rare heterozygous loss of function mutation of MANBA is associated with loss of renal function in humans. 67 Nephrotoxic renal injury is greater in mice with monoallelic or biallelic loss of MANBA function and toxic acute tubule injury induced inflammasome activation and fibrosis in the latter. 67 MANBA may contribute to population renal disease risk by modifying adaptation to renal toxicant exposure.

In the genome, MANBA is colocalized with NFKB1, a gene also implicated by GWAS in CKD. Both NFKB1/ MANBA contain promoter sequence variation affecting regulation of their expression, 68 and this region is associated with many autoimmune diseases, including Crohn disease, ulcerative colitis, and primary biliary cholangitis. [68] [69] [70] [71] [72] Like CKD, these diseases also involve inflammation and immune cell infiltration for which NFKB1 is a key inflammatory signaling gene. Two other genes within the nuclear factor kappaB (NF-κB) signaling pathway, NFATC1 and PTPRO, have emerged in renal GWAS studies. 73 Protein tyrosine phosphatase, receptor O (PTPRO) and NF-κB are activated by T-and B-cell signaling, 74, 75 while NFATC1 is the major transcription factor responding to such signals. 76 Variants generating gene expression effects may act through more than one gene in the NFKB1/MANBA locus to drive disease risk.

Inbred rodent models of CKD harbor natural genetic variation contributing to disease pathogenesis. Full inbreeding reduces genetic complexity because each pair of maternal and paternal autosomes are identical. These models also allow confirmation of mapping results by targeted backcrossing of chromosomal segments between unaffected and affected inbred lines (congenic line creation). Once disease loci have been proven, genetic variation associated with specific genes in the congenic segment can be investigated by approaches including whole genome sequencing, targeted gene deletion or amplification, and analysis of gene expression, alternative gene splicing and protein abundance. While human population genetics studies produce statistical findings, animal models allow investigation of kidney tissue during disease progression and of the biological consequences of disease gene variation.

The capacities of such approaches are exemplified in the Munich Wistar Fromter rat, a model of BP-associated progressive renal disease. 77 Mapping studies indicated that locus on chromosome 6 was linked to renal injury. 78 A congenic line in which the chromosome 6 genomic region from the hypertensive but albuminuria resistant spontaneously hypertensive rat (SHR/Rkb) line was substituted into Munich Wistar Fromter narrowed the causative variation to a gene-rich 4.9 Mb region containing 75 protein-coding genes. 77 To identify the causal variation, genomic resequencing was performed allowing variant comparison between the lines. Five genes in the region were affected by potentially deleterious mutation, however, none appeared to be an explicit candidate gene for renal injury. Expression of genes in this region in isolated glomeruli from SHR and Munich Wistar Fromter revealed increased gene expression of Tmem63c in Munich Wistar Fromter compared with SHR and the congenic line, indicating likely cis-regulation of expression. Immunohistochemistry indicated podocyte restriction of Tmem63c expression. Tmem63c functional studies were performed in zebrafish and reduced gene function correlated with increased urinary protein filtration which was partially rescued by expression of Tmem63c. 77 These studies exemplify approaches available in animal models to identify, confirm and further investigate natural genetic variation that may drive pressure-related loss of renal function.

Roman et al have discovered genetic variation that contributes to BP-driven renal injury in the Fawn-Hooded Hypertensive (FHH) rat. FHH experiences focal segmental glomerulosclerosis and proteinuria with increasing BP as animals age. 79, 80 Their work uncovered 3 distinct pathogenic mechanisms of renal disease. Genetic variation in Shroom3 in FHH alters its interactions with actin and contributes to podocyte foot process fusion and albuminuria. 81 Shroom3 has also been mapped in human populations as containing variation that can influence renal function and Shroom3 antagonism affects podocyte structure in mice. 82 Albuminuria in FHH arises from variation in Rab38 that acts to reduce tubular reuptake of filtered protein. 83 Rab38 has been associated with diabetes-associated proteinuria in human populations. 84 Single-nucleotide variation in gamma adducin (Add3), a cytoskeletal protein, also contributes to renal injury in FHH. The Add3 variant present in FHH impairs physiological autoregulation of renal blood flow. Autoregulation occurs by intrinsic myogenic reflex contraction of renal blood vessels with increasing perfusion pressure which limits increases in blood flow and reduces transmission of elevated pressure to the glomerulus. The role of Add3 variation was uncovered by mapping of a chromosome 1 locus contributing to disease. Creation of congenic animals narrowed this region to ≈2 Mb. Genetic variation isolated in this ≈2 Mb segment affects the renovascular myogenic reflex. 85 This autoregulatory reflex requires transmembrane ion fluxes which were investigated to reveal an effect of Add3 variation to increase BK channel-opening probability. This may act to limit calcium influx into vascular smooth muscle cells resulting from pressure-induced depolarization, 86 and thereby impair development of pressure-driven myogenic tone. Interestingly, Hunt et al 6 mapped a human GFR locus to the region of the human genome containing Add3. Thus, in FHH 3 distinct mechanisms of renal injury have been identified.

Another well-established rat model of CKD is the spontaneously hypertensive rat, SHR. This strain was produced by selective breeding on the trait of elevated BP, generating animals with elevated systolic BP (180-200 mm Hg). 87 Inbreeding to fix the hypertensive genetic variation was performed in 3 parallel lineages. Among the A, but not the B and C, lineages it was observed that hypertension resulted in stroke and that stroke was preceded by the emergence of progressive proteinuric renal disease. 88 Fixation of stroke-risk alleles resulted in SHR-A3, commonly called the stroke-prone SHR, SHRSP. 88 Renal autoregulatory capacity is identical in SHR lines that differ in susceptibility to renal injury. 89, 90 Renal injury emerges when systolic BP exceeds the autoregulatory range (≈18 weeks of age) 91 and is accelerated by salt loading. 92 Before emergence of renal injury, SHR-A3 has slightly higher BP than other SHR lines. Genetic mapping in a cross between SHR-A3 and SHR-B2 revealed genetic variation on chromosome 17 responsible for higher systolic BP in SHR-A3. Congenic replacement of this locus in SHR-A3 results in systolic BP identical to SHR-B2 and partial amelioration in renal injury. 93 Genetically determined elevation of systolic BP beyond the autoregulatory range contributes to renal injury in SHR-A3.

Eng et al 94 used the 2 kidney-one clipped rat model in which hypertension is induced by reduction of blood flow in a single renal artery, resulting in elevated angiotensin II to uncover a link between hypertension and renal inflammation. The absence of injury in the clipped kidney that is partially protected from elevated BP, but experiences similar levels of angiotensin II as the unclipped kidney, indicated that injury was primarily driven by pressure. A predominant effect of pressure on renal injury was confirmed in angiotensin II or norepinephrine-induced hypertension in which one kidney was protected from increased pressure by a servo-controlled device in the renal artery. 95, 96 These studies have been extended to determine whether recruitment of leukocytes into the kidney in hypertension is driven by angiotensin or pressure and reveal that it is increased perfusion pressure that drives leukocyte infiltration. 17 Induction of injury creates renal inflammation that is accompanied by release of cytokines from damaged tissue and leukocyte recruitment. [97] [98] [99] Leukocyte infiltration in response to pressure may be relevant to APOL1-induced renal injury in humans because leukocyte infiltration will promote cytokine upregulation of APOL1 gene expression which in turn promotes glomerular injury. 48 Thus, immune mechanisms of hypertensive renal injury may be subject to genetic influences arising within immune cells recruited to the kidney.

SHR-A3 provides support for this concept. The immunoglobulin heavy chain (Igh) gene encodes B-cell receptors and secreted antibodies. Igh is extremely variable in both SNP and SV in mammals. [100] [101] [102] [103] [104] High diversity may reflect adaptations in a gene that has numerous segments (constant, joining, diversity, and variable) with substantial similarity. The expressed products of Igh are also diversified by immunoglobulin affinity maturation during B-cell development that arises by somatic hypermutation of Igh to increase antibody specificity and affinity. 105 Replacement of the Igh locus from SHR-A3 with the same locus from SHR-B2 curtails renal injury without affecting preinjury BP. 106 In SHR-A3 Igh contains variation that markedly increases serum levels of IgG2c and reduces IgG2b. 107 There is also extensive variation in the large part of the gene encoding Igh VDJ segments. 101 Immunoglobulins contribute, both directly and indirectly, to a wide range of CKD. The extensive Igh SNP and SV in humans is poorly addressed by SNP-based genotyping arrays and provides an illustration of the inability of SNP-GWAS studies to provide comprehensive genetic association studies. 100, 108, 109 A third genetic variant driving renal injury in SHR-A3 affects T-and B-lymphocyte function (Figure 3) . T-and B-cell receptor stimulation activates responses including cell proliferation and atrophy, cytokine production, and altered metabolic state that are mediated by Ca ++ signaling. Stromal interaction molecule 1 (STIM1) protein gates entry of Ca ++ into lymphocytes. [110] [111] [112] [113] In SHR-A3 Stim1 contains a premature stop codon producing a truncated protein and reducing Ca ++ signaling. 114 Replacement of the defective Stim1 allele in SHR-A3 remedies defective Ca ++ signaling and substantially reduces renal injury without effect on preinjury BP. 114 Rare genetic defects in humans producing complete loss of Stim1 function are associated with autoimmune disease attributable to disturbed antibody formation. 115, 116 Thus, SHR-A3 has accumulated 2 gene variants with important consequences for antibody formation that drive renal injury in the presence of hypertension.

CKD risk alleles coexist with nonheritable factors, such as increasing age, that compound with genetic predisposition. 117 Collectively, age-related diseases share pathogenic mechanisms including chronic sterile inflammation, called inflamm-aging. 118 Aging is associated with increased levels of cytokines and chemokines. 119 Gene expression profiling reveals age-related altered immune functions in multiple organs 119 and tissue infiltration of lymphocytes is also observed. 120 Age differences in immune function reflect altered proliferative responses to continuous antigenic inputs that may require compromises between immune cell compartment size, stemness, and differentiation. 121 This occurs alongside impairment of memory cell generation due to negative regulatory programs compromising T-cell expansion. 121 Maladaptive B-cell responses in aging are revealed by deficient antibody responses to vaccination. 122 Aged B cells have impaired interactions with T cells that may impair immunoglobulin maturation, 122 altered transcription factor profiles, 123 and altered metabolic responses to stimulation. 124 Fibroblasts increase TNF (tumor necrosis factor)-α production with aging, and this may alter healing. 125 These changes mirror some of the genetic alterations in immune function discussed earlier, yielding aging phenotypes that echo and amplify genetic predisposition to immune dysfunction.

Studies seeking to find the genetic basis of variation in longevity in human centenarians have identified genes and pathways that include extensive involvement of immune genes operating in PKC (protein kinase C) and NF-κB (nuclear factor-κB) pathways central to immune cell function. 126 Interestingly, studies in model organisms have also identified similar genes and pathways in aging traits. [127] [128] [129] Thus, interaction may occur between genetic variation that drives aging and immune system function that affects the emergence and or progression of renal disease.

The aging kidney experiences reduced glomerular filtration, 130 renal blood flow, glomerular plasma flow rate, ultrafiltration coefficient, and glomerular hydraulic permeability. 131 These are often present in aging without kidney disease and suggest that age-indexing GFR might avoid skewing the definition of CKD. 132 Age-related changes in renal function have also been examined in rats. In the normotensive outbred Sprague-Dawley rat strain 90% of 24-month-old animals experience proteinuria and glomerular IgM accumulation indicating glomerular dysfunction. 133 Afferent arteriolar resistance is decreased in aged rats; consequently, glomerular perfusion pressure increases with rising albuminuria. 134 The link between aging, hypertension, and renal injury may involve renal autoregulation. Normal autoregulation is impaired in aged mice compared with genetically Left, B-cell receptor signaling results from activation of the cell surface IgM receptor by antigen. PLCγ2 is activated to initiate inositol triphosphate (IP3) and diacyl glycerol (DAG) production. IP3 acts in the endoplasmic reticulum to allow release of stored Ca ++ via the IP3 receptor. This calcium activates PKC (protein kinase C) with resulting activation of the IKK complex leading to the generation go NF-κB (nuclear factor-κB). Depletion of the ER calcium store activates Stim1 which then gates opening of the Orai1 Ca ++ channel through which abundant extracellular calcium is able to enter the B cell. This large Ca ++ signal is sufficient to activate calcineurin, a protein phosphatase that dephosphorylates NFAT. NFAT and NF-κB enter the nucleus and drive a complex set of transcriptionally mediated phenotypic changes by altering gene expression. Right, Mutation of Stim1 in renal injury-susceptible hypertensive rat line SHR-A3, but not in injury-resistant SHR-B2, alters calcium activation in the B cell. In SHR-A3, the COOH-terminal tail of Stim1 is absent due to the presence of a premature stop codon. This impairs the gating of Orai1. When B cells are placed in low Ca ++ medium and activated by anti-IgM antibody cross linking of the B cell receptor, ER Ca ++ stores are depleted and cannot be refilled. When extracellular Ca ++ is subsequently increased to 2 mmol/L, the Orai1 mechanism in SHR-B2 is primed by Stim1 due to ER Ca ++ depletion and a large flux of Ca ++ entry ensues (store-operated calcium entry [SOCE]). In SHR-A3, the SOCE is strongly suppressed due to deficient interactions with Orai1 resulting from Stim1 mutation. Ca ++ dependent activation of lymphocyte transcriptional responses is strongly impaired in SHR-A3. matched young animals. [135] [136] [137] In aged animals increased renal artery pressure resulted in increased renal blood flow, while younger animals were able to completely autoregulate blood flow. 135 The altered myogenic response in aged animals was reflected in reduction of pressure-induced intracellular calcium in the afferent arteriole. Autoregulatory capacity is also reduced in aging humans. Hill et al 138 proposed that defective autoregulation in aging humans as indicated by morphological correlation between focal dilated renal arterioles, hypertrophic glomeruli, and subsequent focal segmental glomerulosclerosis.

Progressive renal disease in hypertension is complex with interaction between heritable and nonheritable elements. Phenotypic description of renal function in population-scale studies is constrained and likely limits GWAS success. GWAS is also limited by using SNP variants and is uninformed regarding other genomic variation, including SV, that contribute to phenotype diversity. 9, 10 Undoubtedly, better understanding of the full scope of human genetic variation and tools for SV genotyping will advance genetic studies of CKD. Animal models have contributed additional insight, amplifying the role of genes critical to glomerular barrier formation and tubular reabsorption of filtered protein and indicating that genetic variation can affect autoregulation of renal blood flow. Pressure-induced renal injury initiates a chronic sterile inflammatory and immune response. Animal models have also indicated that genetic variation affecting immune responses can determine progression and severity of disease. While experimental gene knockout is useful only for defining the capacity of a gene to induce a renal phenotype, human CKD risk alleles may ultimately be reconstituted in animal models to demonstrate their effects and their interactions with other risk variants to yield polygenic animal models of greater usefulness. Finally, heritable influences contributing to renal injury may interact with aspects of normal aging to amplify the effects of elevated BP on disease. 

This work was supported by the National Institutes of Health (Award Numbers: NIH R01DK069632 and R01DK081866) to P.A. Doris and American Heart Association postdoctoral fellowship award (AHA 17POST33660779) to I.S. Dhande.

None.

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Using de novo assembly to identify structural variation of eight complex immune system gene regions

Phenotypic impact of genomic structural variation: insights from and for human disease

Improved assembly and variant detection of a haploid human genome using singlemolecule, high-fidelity long reads

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

Characterizing the major structural variant alleles of the human genome

Genome maps across 26 human populations reveal population-specific patterns of structural variation

Multi-platform discovery of haplotype-resolved structural variation in human genomes

Structural variation detection and analysis using bionano optical mapping

Genetic variation, comparative genomics, and the diagnosis of disease

Telomere-to-telomere assembly of a complete human X chromosome

The complete sequence of a human genome

Population structure, stratification, and introgression of human structural variation

Towards populationscale long-read sequencing

Long-read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits

Family Investigation of Nephropathy and Diabetes Research Group. MYH9 is associated with nondiabetic endstage renal disease in African Americans

MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis

Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene

Evolution of the primate trypanolytic factor APOL1

The molecular arms race between African trypanosomes and humans

APOL1 and kidney disease: from genetics to biology

COVID-19-associated glomerular disease

COVAN is the new HIVAN: the re-emergence of collapsing glomerulopathy with COVID-19

AKI and Collapsing Glomerulopathy Associated with COVID-19 and APOL 1 High-Risk Genotype

Innate immunity pathways regulate the nephropathy gene Apolipoprotein L1

Transgenic expression of human APOL1 risk variants in podocytes induces kidney disease in mice

Kidney disease-associated APOL1 variants have dose-dependent, dominant toxic gain-of-function

From man to fish: what can Zebrafish tell us about ApoL1 nephropathy?

APOL1-associated collapsing focal segmental glomerulosclerosis in a patient with Stimulator of Interferon Genes (STING)-Associated Vasculopathy With Onset in Infancy (SAVI)

APOL1 renal risk variants: fertile soil for HIV-associated nephropathy

APOL1 polymorphisms and kidney disease: loss-of-function or gain-of-function?

ApoL1 overexpression drives variant-independent cytotoxicity

The mechanism of kidney disease due to APOL1 risk variants

APOL1 C-Terminal variants may trigger kidney disease through Interference with APOL3 control of Actomyosin

A null variant in the apolipoprotein L3 gene is associated with non-diabetic nephropathy

Populationbased risk assessment of APOL1 on renal disease

Genetic and clinical predictors of age of ESKD in individuals with autosomal dominant tubulointerstitial kidney disease due to UMOD mutations

Common variants in UMOD associate with urinary uromodulin levels: a meta-analysis

Uromodulin: from monogenic to multifactorial diseases

The UMOD Locus: insights into the pathogenesis and prognosis of kidney disease

Uromodulin: roles in health and disease

Molecular insights into genome-wide association studies of chronic kidney disease-defining traits

Genetic-variation-driven gene-expression changes highlight genes with important functions for kidney disease

Kidney disease genetic risk variants alter lysosomal betamannosidase (MANBA) expression and disease severity

NFKB1 and MANBA confer disease susceptibility to primary biliary cholangitis via independent putative primary functional variants

Genome-wide association study identifies TNFSF15 and POU2AF1 as susceptibility loci for primary biliary cirrhosis in the Japanese population

UK PBC Consortium

Genome-wide association study identifies 12 new susceptibility loci for primary biliary cirrhosis

Functional annotation of a novel NFKB1 promoter polymorphism that increases risk for ulcerative colitis

Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease

CHARGe-Heart Failure Group; ECHOGen Consortium. Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function

Protein tyrosine phosphatase receptor-type O truncated (PTPROt) regulates SYK phosphorylation, proximal B-cell-receptor signaling, and cellular proliferation

Roles of the NF-kappaB pathway in lymphocyte development and function

NFATc1 and NFATc2 together control both T and B cell activation and differentiation

Analysis of the genomic architecture of a complex trait locus in hypertensive rat models links Tmem63c to kidney damage

Genetic locus on MWF rat chromosome 6 affects kidney damage in response to L-NAME treatment in spontaneously hypertensive rats

Development of hypertension and proteinuria with age in fawn-hooded rats

Pathogenesis of glomerular injury in the fawn-hooded rat: early glomerular capillary hypertension predicts glomerular sclerosis

Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

SHROOM3, the gene associated with chronic kidney disease, affects the podocyte structure

Rab38 modulates proteinuria in model of hypertension-associated renal disease

Genome-wide Association Studies identify genetic loci associated with albuminuria in diabetes

Genetic basis of the impaired renal myogenic response in FHH rats

A mutation in γ-adducin impairs autoregulation of renal blood flow and promotes the development of kidney disease

Development of a strain of spontaneously hypertensive rats

Establishment of the Stroke-prone Spontaneously Hypertensive Rat (SHR)

Differential effects of salt on renal hemodynamics and potential pressure transmission in stroke-prone and stroke-resistant spontaneously hypertensive rats

Systolic blood pressure as the trigger for the renal myogenic response: protective or autoregulatory?

Critical blood pressure threshold dependence of hypertensive injury and repair in a malignant nephrosclerosis model

Renal autoregulation in health and disease

High-resolution identity by descent mapping uncovers the genetic basis for blood pressure differences between spontaneously hypertensive rat lines

Renal proliferative and phenotypic changes in rats with twokidney, one-clip Goldblatt hypertension

Pressure-induced renal injury in angiotensin II versus norepinephrine-induced hypertensive rats

Role of pressure in angiotensin II-induced renal injury: chronic servo-control of renal perfusion pressure in rats. Hypertension

Mechanistic connection between inflammation and fibrosis

Overview of factors contributing to the pathophysiology of progressive renal disease

Overview of the cellular and molecular basis of kidney fibrosis

The immunoglobulin heavy chain locus: genetic variation, missing data, and implications for human disease

Diversity in the preimmune immunoglobulin repertoire of SHR lines susceptible and resistant to end-organ injury

A comparison of immunoglobulin IGHV, IGHD and IGHJ genes in wild-derived and classical inbred mouse strains

Mosaic deletion patterns of the human antibody heavy chain gene locus shown by Bayesian haplotyping

The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains

Identification of subjectspecific immunoglobulin alleles from expressed repertoire sequencing data

Susceptibility to hypertensive renal disease in the spontaneously hypertensive rat is influenced by 2 loci affecting blood pressure and immunoglobulin repertoire

Immunoglobulin locus associates with serum IgG levels and albuminuria

Comment on "a database of human immune receptor alleles recovered from population sequencing data

Germline immunoglobulin genes: disease susceptibility genes hidden in plain sight?

CRAC channels and calcium signaling in T Cell-mediated immunity

Calcium regulation of T cell metabolism

To B, or not to B: Is calcium the answer?

BCR-Induced Ca2+ Signals dynamically tune survival, metabolic reprogramming, and proliferation of naive B Cells

Stim1 polymorphism disrupts immune signaling and creates renal injury in hypertension

STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity

Store-Operated Ca(2+) Entry in Follicular T Cells controls humoral immune responses and autoimmunity

Prevalence of CKD and comorbid illness in elderly patients in the United States: results from the Kidney Early Evaluation Program (KEEP)

Inflammaging: a new immune-metabolic viewpoint for age-related diseases

Comprehensive profiling of an aging immune system reveals Clonal GZMK+ CD8+ T Cells as conserved hallmark of inflammaging

Ageing hallmarks exhibit organ-specific temporal signatures

Successful and Maladaptive T Cell Aging

Aging induces B cell defects and decreased antibody responses to influenza infection and vaccination

Cell signaling and the aging of B cells

Age-related factors that affect B cell responses to vaccination in mice and humans

Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing

Genetic signature of human longevity in PKC and NF-κB signaling

Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila

Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH

Lifespan regulation by evolutionarily conserved genes essential for viability

Age changes in glomerular filtration rate, effective renal plasma flow, and tubular excretory capacity in adult males

Determinants of glomerular hypofiltration in aging humans

CKD: a call for an age-adapted definition

Mesangial lesions and focal glomerular sclerosis in the aging rat

Glomerular adaptations with normal aging and with long-term converting enzyme inhibition in rats

Aging impairs renal autoregulation in mice

Age-related autoregulatory dysfunction and cerebromicrovascular injury in mice with angiotensin II-induced hypertension

Aging impairs myogenic adaptation to pulsatile pressure in mouse cerebral arteries

Morphometric study of arterioles and glomeruli in the aging kidney suggests focal loss of autoregulation